Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 216402 16 pageshttpdxdoiorg1011552013216402

Research ArticleATP Is Required and Advances Cytokine-InducedGap Junction Formation in Microglia In Vitro

Pablo J Saacuteez12 Kenji F Shoji12 Mauricio A Retamal3 Paloma A Harcha12

Gigliola Ramiacuterez4 Jean X Jiang5 Rommy von Bernhardi4 and Juan C Saacuteez12

1 Departamento de Fisiologıa Pontificia Universidad Catolica de Chile Alameda 340 6513677 Santiago Chile2 Instituto Milenio Centro Interdisciplinario de Neurociencias de Valparaıso Pasaje Harrington 287 Playa Ancha2360103 Valparaıso Chile

3 Departamento de Fisiologıa Facultad de Medicina Clınica Alemana-Universidad del Desarrollo Las Condes 124387710162 Santiago Chile

4Departamento de Neurologıa Escuela deMedicina Pontificia Universidad Catolica de Chile Marcoleta 392 8330024 Santiago Chile5 Department of Biochemistry University of Texas Health Science Center 7703 Floyd Curl Drive San Antonio TX 78229 USA

Correspondence should be addressed to Pablo J Saez pjsaezuccl

Received 4 February 2013 Revised 21 March 2013 Accepted 22 March 2013

Academic Editor Diego Gomez-Nicola

Copyright copy 2013 Pablo J Saez et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Microglia are the immune cells in the central nervous systemAfter injurymicroglia release bioactivemolecules including cytokinesand ATP which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs) affecting the intercellularcommunication via extracellular and intracellular compartments respectively Here we studied the role of extracellular ATP andseveral cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniquesrespectively In microglia and the microglia cell line EOC20 ATP advanced the TNF-120572IFN-120574-induced dye coupling probablythrough the induction of IL-1120573 release Moreover TNF-120572IFN-120574 but not TNF-120572 plus ATP increased dye uptake in EOC20 cellsBlockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-120572IFN-120574 but not TNF-120572 plus ATP In addition IL-6prevented the induction of dye coupling and HC activity induced by TNF-120572IFN-120574 in EOC20 cells Our data support the notionthat extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms whichmight affect the timing of immune response under neuroinflammatory conditions

1 Introduction

Microglia are the major immune effectors in the centralnervous system (CNS) Under resting conditions surveil-lance microglia have a ramified morphology and monitortheir local microenvironment [1 2] However microglia canrapidly become activated in response to diverse stimuli anddanger signals such as ATP or bacterial lipopolysaccharide(LPS) [1ndash3] Consistently microglia are activated in neuroin-flammatory conditions and are a common hallmark in manyneurodegenerative diseases [1 2 4]

Microglial cell activation includes morphological chan-ges proliferation recruitment to the site of injury and expres-sion of specific proteins includingMHC II molecules and cell

adhesion molecules [1 2] Activated microglia also releasecytokines including TNF-120572 IL-1120573 IL-6 IFN-120574 and othersoluble molecules such as glutamate and ATP [5ndash9] Manyof these pro-inflammatory molecules act in an autocrinemanner and show synergism increasing the activation ofmicroglia [10ndash12]

Many studies have focused on ATP release mecha-nisms and the subsequent receptors activation at the CNSbecause they promote the release of other pro-inflammatorymolecules such as TNF-120572 and IL-1120573 [13] These cytokinesmediate cell communication and Ca2+ signaling amongmicroglia as well as among microglia and astrocytes [14ndash16] Microglia sense extracellular ATP through P2Y and P2Xreceptors [1] Under control conditions microglia express

2 Mediators of Inflammation

P2X7receptors which are upregulated as a required step

for microglial activation induced by amyloid-120573 peptide [1718] Moreover activation of microglia with LPS increasesthe intracellular free Ca2+ concentration ([Ca2+]

119894) and ATP

release through P2X7receptors [17 19 20] Accordingly

cytokines that increase [Ca2+]119894or a calcium ionophore induce

microglia activation [21 22] These conditions also inducegap junctional communication in primary cultures of rat ormouse microglia [23 24]

Gap junction channels (GJCs) communicate the cyto-plasm of contacting cells allowing the direct transfer of ionssecond messengers and other molecules including antigenpeptides [25] Each GJC is formed by the serial docking oftwo hemichannels (HCs) which are composed of six proteinsubunits called connexins (Cxs) [25] It is known that restingmicroglia express Cxs 32 36 43 and 45 and after microgliaactivation some of them form functional GJCs and HCs[23 24 26ndash28] Recently another family of proteins termedpannexins (Panxs) has been found to form functional GJCsand HCs [29] Like Cx HCs Panx HCs are permeable toATP and are activated by increased [Ca2+]

119894and extracellular

ATP via P2 receptors [30ndash32] Microglia express functionalPanx1 HCs that contribute to ATP-induced migration andglutamate and ATP release promoting neuronal death [33ndash35] Under inflammatory conditions gap junctional commu-nication between cultured astrocytes is reduced whereas theactivity of HCs is increased [35ndash38] However it remainsunknown if these opposite changes in GJCs and HCs alsooccur in microglia or if extracellular ATP plays a role in thischannel-based communication

In this work we studied the effect of extracellular ATPon the cytokine-induced gap junctional communication inmicroglia To achieve this goal we used primary cultures ofrat microglia and EOC20 cells treated with several cytokinesand ATP either mixed or alone We propose that TNF-120572IFN-120574 induce gap junctional communication whichmightdepend on the functional expression of HCs In addition wefound that extracellular ATP advances the onset of cytokine-induced expression of gap junctional communication aprocess that was mediated by IL-1120573 release and inhibited byIL-6

2 Materials and Methods

21 Materials Modified Eaglersquos medium (MEM) Dulbeccorsquosmodified Eaglersquos medium (DMEM) F-12 nutrient mix-ture fetal bovine serum (FBS) bovine pancreas DNAseI and trypsin-EDTA were purchased from Gibco (Auck-land NZ USA) DMSO HEPES H

2O LaCl

3(La3+)

ethidium (Etd) bromide Lucifer yellow dilithium salt (LYMW 45725Da) rhodamine-dextran (RD MW 10 kDa)adenosine 51015840-triphosphate periodate oxidized sodium salt(oATP) ATP disodium salt probenecid (Pbc) recombi-nant mouse TNF-120572 recombinant mouse IL-1120573 recombinantmouse IFN-120574 recombinant mouse IL-6 and Ponceau Sred were purchased from Sigma-Aldrich (St Louis MOUSA) Interleukin-1 receptor antagonist (IL-1ra) was from

RampD (USA) BAPTA-AM was purchased from Molecu-lar Probes (Eugene Oregon USA) Penicillin and strep-tomycin were obtained from Invitrogen (Carlsbad CAUSA) D(+)-glucose sodium hydrogen carbonate (NaHCO

3)

were purchased fromMerck (Darmstadt Germany) 10Panx1mimetic peptide (sequence WRQAAFVDSY) was purchasedfrom SBS Biotech (Beijing China) Purified rat anti-mouseCD16CD32 (mouse BD Fc-block) was purchased fromBD Pharmingen (San Jose CA USA) F(ab1015840)

2fragments

of a previously characterized polyclonal rabbit anti-Panx1serum used [39 40] The F(ab1015840)

2fragments of affinity

IgGs purified from the anti-Panx1 serum were preparedas previously described [41] Anti-Cx43 monoclonal anti-body was obtained from BD Biosciences (Minneapolis MNUSA) Cy2 conjugated goat anti-rabbit and Cy3 conjugatedgoat anti-mouse antibodies were purchased from JacksonImmunoresearch Laboratories Inc (Indianapolis IN USA)EDTA solution Halt protease inhibitor single-use cocktailand M-PER mammalian protein extraction reagent werepurchased from Thermo Scientific (Rockford IL USA)Mount solution fluoromount G was purchased from ElectronMicroscopy Sciences (Washington PA USA) Images wereexamined with a confocal laser-scanning microscope whichwas Olympus Fluoview FV1000 (Tokio Japan) Cx43(E2) isa rabbit polyclonal antibody that recognizes amino acidresidues located at the second extracellular loop of Cx43 andblocks specifically Cx43 HCs [42]

Bio-Rad protein assay was purchased from Bio-RadLaboratories (Richmond CA USA) SuperSignal kit forenhanced chemiluminescence detection and anti-rabbit anti-body conjugated to horseradish peroxidase were purchasedfrom Pierce (Rockford IL USA) EOC20 and LADMACcells were obtained fromATCC (Manassas VA USA) Tissueculture flasks (25 and 75 cm2) 60mm and 100mm tissueculture dishes were purchased from Sarstedt (Newton NCUSA) Twenty four-well plastic dishes were purchased fromNunclon (Roskilde Denmark)

22 Cell Cultures

221 Rat Microglia Primary cultures of microglia wereprepared from neocortex of newborn Sprague Dawley rats aspreviously described [23 24] Brieflymeningeswere carefullypeeled off and cortices were dissected and minced in smallpieces After incubation in Ca2+-free PBS containing trypsin(05) and EDTA (5mM) at 37∘C for 30min tissue wastriturated in presence of DNAse using a Pasteur pipetteDissociated cells were pelleted and resuspended in MEMmedium supplementedwith 10FBS 100 unitsmLpenicillinand 50120583gmL streptomycin sulfate and plated on plasticculture flasks (Sarstedt) Confluent glial cell mixed cultureswere deprived of fresh medium for two weeks to inducemicroglial cell proliferation Finallymicroglia were harvestedfrom glial cell mixed cultures by differential adhesion andseeded on glass coverslips

222 EOC20 Cells EOC20 cells are a murine microglial cellline derived fromC3HHeJmice which secrete cytokines and

Mediators of Inflammation 3

present antigens as primary microglia [43] EOC20 cells weremaintained according to ATCC recommendations usingDMEM supplemented with 10 FBS and 20 LADMACconditioned medium (see below) The medium was partiallychanged twice a week and completely changed once a weekuntil the culture reached confluence Cells were detachedwith trypsin-EDTA for 2min and mechanical stress andEOC20 cells were seeded on glass coverslips or tissue culturedishes Since rat microglia were detached by shaking usingan orbital shaker some experiments were performed withEOC20 cells detached with the same methods of purificationused for primary microglia cultures No differences wereobserved in the induction of dye coupling between EOC20cells obtained by the different purification methods (data notshown)

223 LADMAC Conditioned Medium The conditionedmedium was obtained from LADMAC cells which aremyeloid cells derived from murine bone marrow cellsLADMAC cells are nonadherent cells that secrete colony-stimulating-factor-1 (CSF-1) which stimulates cell division inEOC20 cells [43 44] LADMAC cell cultures were main-tained in culture in MEM supplemented with 10 FBSduring two weeks Fresh medium was added every two daysduplicating the previous amount ofmedium After two weeksin culture the cell suspension was centrifuged and the CSF-1-containing supernatant was filtered aliquoted and stored atminus20∘C until use

23 Dye Transfer Technique The transference of fluorescentdyes between adjacent cells has been used to monitor thefunctional state of GJCs in microglia [23 24 27] We testedthe intercellular transference of LY using RD as a negativecontrol Dyes (5wv in 150mM LiCl) were microinjectedby applying current to microglia seeded on glass coverslips(8 times 105 cellswell in a 24-multiwell dish) through glassmicroelectrodes until the impaled cells were fluorescent Cul-tures were maintained in F-12 medium supplemented withHEPES and observed with an inverted microscope equippedwith Xenon arc lamp illumination and a Nikon B filter(excitation wavelength 450ndash490 nm emission wavelengthabove 520 nm) Dye transfer was scored at 2min injectionThe incidence of dye coupling (IDC) was calculated as thepercentage of injected cells with dye transfer to one or moreneighboring cells by the total number of cellsmicroinjected ineach experiment At least 10 cells were microinjected in eachassay Since cytokine treatments induced HC activity andbecause that dye uptake from leaking microelectrodes couldaffect the measurement of fluorescent cells we use 200120583MLa3+ in the recording solution However no significantdifferences were observed compared to recording solutionwithout La3+ (data not shown)

24 Dye Uptake Ca2+ Signal Imaging and Time-Lapse Flu-orescence Imaging To evaluate dye uptake cells seeded onglass coverslips (8 times 105 cellsmL) were exposed to 5120583Methidium (Etd) bromide with Lockersquos saline solution (in mM154 NaCl 54 KCl 23 CaCl

2 1 mM MgCl

2 5mM glucose

5mM HEPES pH 742) and examined by epifluorescenceNuclei fluorescence was recorded in regions of interest con-sisting of 30 different cells per field with a water immersionOlympus 51W1I upright microscope (Melville NY USA) asdescribed [45] The calculation of slope change regressionlines was fitted to points before and after treatments usingMicrosoft (Seattle WA USA) Excel In ATP-induced dyeuptake experiments 500120583M ATP was added to recordingsolution after 5min of basal dye uptake

To evaluate Ca2+ signals EOC20 cells under controlconditions or after treatment were maintained as mentionedabove but were loaded for 30min with 5120583M Fura-2 AM inDMEM medium without serum at 37∘C Loaded cells werewashed twice with Lockersquos solution and time-measurementswere performed with an Olympus 51W1I microscope Theacquisition of 340 and 380 nm excitation wavelengths wasevery 3 s Regions of interest consisted in 30 cells per field andanalysis were performed using METAFLUOR software

25 Western Blot Confluent microglia cultures grown in60mmculture dishes (24times 106 cells) were gently rinsed twicewith cold PBS at 4∘C pH 74 and harvested by scraping witha rubber policeman in a solution containing 5mM EDTAHalt andM-PER protein extraction cocktail according to themanufacturerrsquos instructionsThe cellular suspensionwas son-icated on ice Proteinsweremeasured in aliquots of cell lysatesusing the Bio-Rad protein assay Aliquots of cell lysates (50 120583gof protein) were resuspended in Laemlirsquos sample buffer andseparated in an 8 sodium dodecyl sulfate polyacrylamidegel electrophoresis and electrotransferred to nitrocellulosesheets as previously described [24] Loading equivalenceswere confirmed by protein staining with Ponceau S red(2wv in 30 trichloroacetic acid) Nonspecific proteinbinding was blocked by incubation of nitrocellulose sheets in5 nonfat milk in PBS for 1 h at room temperature prior toovernight incubation with corresponding antibodies at 4∘CAfter several washes with PBS blots were incubated with thesecondary antibody conjugated to horseradish peroxidase for45min at room temperature Immunoreactivity was detectedby enhanced chemiluminescence using the SuperSignal kitaccording to the manufacturerrsquos instructions

26 Immunofluorescence Microglia cultured on glass coverslips were fixed with 4 formaldehyde at room temperaturefor 30min and washed twice with PBS A blocking solutioncontaining 1 IgG free BSA 50mM NH

4Cl and 005

Triton X-100 in PBS was used to permeabilize and blockunspecific reactive sites Fc receptors were masked by incu-bating samples to a solution containing Fc-Block (1 100) for45min at room temperature Panx1 and Cx43 were detectedwith a rabbit polyclonal anti-Panx1 F(ab1015840)

2fragments [40]

and an anti-Cx43 monoclonal antibody properly dilutedwith blocking solution respectively Cy2 conjugated goatanti-rabbit (1 300) and Cy3 conjugated goat anti-mouseIgGF(ab1015840)

2Igs fragments for 30min at room temperature

were used to detect bound primary antibody FluoromountG (Electron Microscopy Sciences Washington PA USA)was used as an antifade solution to mount samples Images

4 Mediators of Inflammation

were examined with a confocal laser-scanning microscope(Olympus Fluoview FV1000 Tokio Japan)

27 IL-1120573 ELISA The level of IL-1120573 present in the condi-tioned media of EOC20 cells was evaluated with the IL-1120573ELISA ReadySet-Go (e-Bioscience San Diego CA USA)for performing quantitative enzyme linked immunosorbentassays (ELISA) It has a sensitivity of 8 pgmL Standardcurve consisted of twofold serial dilutions of the recombi-nant cytokine In brief a 96-well flat bottom ELISA-plate(MICROLON Greiner Bio-One) was coated with captureantibody in coating buffer overnight at 4∘C The plate waswashed 5 times with PBS-005 Tween-20 in ELx50 Biokita 96-well bioelisa washer and Rhe plate was blocked with200120583L of assay diluent at room temperature for 1 h washedas mentioned and 100 120583L of standard IL1120573 and sampleswere incubated at 4∘C overnight Then the plate was washedand 100 120583L of detection antibody for IL-1120573 was added andincubated at room temperature by 1 h washed 5 timesincubated with 100120583L Avidin-HRP at room temperature for30min washed 7 times added 100120583L substrate solution andstopped the reaction with 50 120583L of 1M H

3PO4 The plate was

read at 450 nm with reference at 570 nm

28 Treatments Microglia were seeded 48 h before dyetransfer dye uptake or immunofluorescence experiments in24-well plastic dish containing 500 120583L of culture mediumFor Western blot experiments cells were seeded in 60mmplastic dishes in 3mL of culture medium After 48 h undercontrol conditions microglia were treated with 1mM ATPor 1 ngmL TNF-120572 IFN-120574 IL-1120573 either alone or mixedCytokines were added simultaneously and ATP was added2 h before measurement and is referred as cytokine(s) plusATP Treatment with 1 10 or 50 ngmL IL-6 20 ngmL IL-1ra 300 120583M oATP 200120583M La3+ 1 500 Cx43(E2) antibodyor 200120583M 10Panx1 was simultaneous to cytokine treat-ment We used 50120583M of 120573-GA for acute GJCs blocking(Figure S1 see Supplementary Materials available online athttpdxdoiorg1011552013216402) To avoid disruption ofcell adhesion with BAPTA the medium was replaced withculture medium of parallel cultures treated at the same timeto maintain the soluble factor released from microglia

29 Statistical Analysis Data are presented as mean plusmn SEMas percentage of the control condition 119899 represents thenumber of independent experiments For statistical analysiseach treatment was compared with its respective controland significance was determined using one-way ANOVAfollowed by Dunnrsquos test comparing all treatments against thecontrol condition To observe differences between microgliaand EOC20 cells responses we used a two-way ANOVA

3 Results

31 The Onset of the Cytokine-Induced Increase in GapJunctional Communication in Cultured Microglia Is Advancedby ATP and Delayed by IFN-120574 Calcium ionophore and pro-inflammatory molecules promote a transient expression of

functional GJCs in microglia [23 24 27] Since extracellularATP TNF-120572 and IFN-120574 play a relevant role in microglialcell responses [3 7 46] and affect the [Ca2+]

119894[47ndash49] we

decided to evaluate if these compounds affect the intercellularcommunication via GJCs in both primary cultures of ratmicroglia and EOC20 cells

After 48 h of subculture under control conditionsmicroglia were treated as indicated in Methods (Figure S1a)Both cell types presented rather homogeneousmorphologicalfeatures (Figures 1(a) and 1(b)) and very low incidence ofLucifer yellow (LY) transfer to neighboring cells (Figures1(a) and 1(b)) Under these conditions the incidence of dyecoupling (IDC) remained low for up to 12 h of culture inboth cell types (Figure 1 Supporting information Table S1)In addition intercellular transfer of rhodamine-dextran (RDsim10 kDa) which due to its high molecular weight cannotpermeate through GJCs was not observed (Figure S2a) Thisresult indicates that intercellular LY transfer ocurred viaGJCsand not through other cell-cell communication pathway suchas cytoplasmic bridges Moreover microglia treated eitherwith 1mM ATP 1 ngmL TNF-120572 1 ngmL IFN-120574 or 1 ngmLIL-1120573 showed only a slight increase in IDC which was notstatistically different from that of control cells (119875 gt 005Supporting information Table S1) However treatment withmixes of thesemolecules during different time periods causeda significant and transient increase in IDC the dye transferdata is expressed as percentage of the corresponding controlcondition (Figures 1(e) and 1(f)) In both cell types treatmentwith 1 ngmL TNF-120572 plus 1 ngmL IFN-120574 (from now andon referred as TNF-120572IFN-120574) increased the IDC reachinga maximum response at around 9 h after treatment (IDC inEOC20 cells 574 plusmn 36 of control rat microglia 552 plusmn 36of control Mean plusmn SEM 119899 = 5) as previously described[23]

We also studied if extracellular ATP affects TNF-120572IFN-120574-induced dye coupling To this end cells were treated withthese cytokines and then exposed to ATP for 2 h In bothcell types treatment with TNF-120572IFN-120574 plus ATP induced atransient increase in IDC which was maximal at around 5 h(EOC20 cells 517 plusmn 94 of control rat microglia 506 plusmn 42of control 119899 = 5) The amplitude and duration (magnitude)of the response was similar to that induced by TNF-120572IFN-120574but occurred 4 h earlier (Figure 1(e))

Since IFN-120574 potentiates TNF-120572-induced dye coupling inantigen presenting cells including dendritic cells microgliaand monocytesmacrophages [23 50 51] we tested whetherATP induces a similar effect on microglia In agreement withthis possibility cells treated with TNF-120572 plus ATP showedmaximal IDCwith similar amplitude (EOC20 cells 529plusmn12of control rat microglia 534plusmn70of control 119899 = 6 Figure 1)to that induced by TNF-120572IFN-120574 plus ATP but occurred15 h earlier (at sim35 h versus 5 h Figures 1(e) and 1(f)) Asmentioned before intercellular transfer of LY was enhancedin primarymicroglia or EOC20 cells treated with TNF-120572 plusATP (Figures 1(c) and 1(d)) However intercellular transfer ofRDwas not observed ruling out the formation of cytoplasmicbridges or vesicular mediated dye transfer in each condition(Figure S2) Microglia treated with IFN-120574 plus ATP did notincrease dye coupling at 35 h (EOC20 cells 167 plusmn 97 of

Mediators of Inflammation 5

Rat microglia

Control

lowast

(a)

EOC20

Control

lowast

(b)

Rat microglia

TNF-120572ATP

lowast

(c)

EOC20

TNF-120572ATP

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(d)

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TNF-120572ATPTNF-120572IFN-120574ATPTNF-120572IFN-120574

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ATP

IFN-120574

+

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+minus

minus

(f)

Figure 1 ATP advances the onset of TNF-120572IFN-120574-induced dye coupling (a)ndash(d) Dye transfer was evaluated 2min after Lucifer yellow (LY)microinjection in a single cell (indicated with an asterisk) Representative pictures of LY transfer in rat microglia (a) (c) or EOC20 cells (b)(d) under control condition or after TNF-120572 plus ATP (TNF-120572ATP) treatment for 35 h as indicated Phase contrasts of each micrograph areshown at the right panels Scale bar 20 120583m (e) Time course of the incidence of dye coupling (IDC) as percentage of IDC in EOC20 cellsunder control conditions (dashed line) or after treatment with TNF-120572 plus ATP (black circles) TNF-120572IFN-120574 plus ATP (white triangles) orTNF-120572IFN-120574 (black diamonds) Each point corresponds to themean of 3 independent experiments (f) Graph showing themaximum valuesof IDC after treatment with TNF-120572 plus ATP for 35 h TNF-120572IFN-120574 plus ATP for 5 h or TNF-120572IFN-120574 for 9 h lowast119875 lt 005 versus controlcondition Each bar represents the mean plusmn SEM 119899 = 6 No significant differences were observed when comparing microglia and EOC20 cellsresponses to different treatment in dye transfer assays Concentrations 1 ngmL TNF-120572 1 mM ATP 1 ngmL IFN-120574

control ratmicroglia 2108plusmn513of control) or other times(2 and 5 h data not shown)

32 The Increase of Gap Junctional Communication Inducedby TNF-120572 Plus ATP Requires an Increase of [Ca2+]

119894via

Activation of P2XReceptors and Is Prevented by IL-6 Eugenınet al (2001) described that dye coupling between microgliatreated for 9 h with TNF-120572IFN-120574 is inhibited by 120573-GAIn EOC20 cells treated with TNF-120572IFN-120574 we observeda similar acute blockade with 120573-GA (data not shown)

6 Mediators of Inflammation

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cide

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coup

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( o

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TNF-120572ATP120573-GA

IL-6oATP

BAPTA-AM

+minusminusminusminus

++minusminusminus

+

+minus

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EOC20Rat microglia

TNF-120572ATPIL-6

oATP

+minusminus

++minus

+

+minus

lowast

(b)

Figure 2 IL-6 intracellular calcium chelation and P2X inhibition prevent the induction of gap junctional communication promoted byTNF-120572 plus ATP or TNF-120572IFN-120574 (a) Graph showing the effect of acutely applied 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) or pretreatmentwith 10 ngmL interleukin-6 (IL-6) 300120583Moxidized ATP (oATP) or 10120583MBAPTA-AMon the incidence of dye coupling (IDC) of microgliatreated for 35 h with TNF-120572 plus ATP (b) Graph showing the effect of 50 ngmL IL-6 or 300120583M oATP over the IDC of microglia treated for9 h with TNF-120572IFN-120574 Data is expressed as a percentage of IDC under control conditions (dashed line) lowast119875 lt 005 versus control conditionEach bar represents the mean plusmn SEM 119899 = 5 No significant differences were observed when comparing microglia and EOC20 cells responsesto different treatment in dye transfer assays

In addition application of 50 120583M 120573-GA for 5min completelyabolished dye coupling induced by TNF-120572 plus ATP (IDC inEOC20 cells 74 plusmn 44 of control rat microglia 86 plusmn 50 ofcontrol 119899 = 5 Figure 2(a))

Since microglia treated with purinergic agonists releaseIL-6 [52] and this cytokine prevents the increase of dyecoupling induced by TNF-120572IL-1120573 in dendritic cells [50] wedecided to test if IL-6 prevents induction of dye couplingin microglia treated with TNF-120572 plus ATP In cell culturestreated simultaneously with 10 ngmL IL-6 plus TNF-120572 andthen treated with ATP for 35 h the IDC was low (EOC20cells 130 plusmn 83 of control rat microglia 162 plusmn 10 ofcontrol 119899 = 4) similar to the results obtained undercontrol conditions (Figure 2(a)) Similarly the TNF-120572IFN-120574-induced dye coupling was prevented by IL-6 (Figure 2(b))This inhibitory effect was IL-6 concentration-dependent (110 and 50 ngmL data not shown) The maximal effect wasinduced by 50 ngmL IL-6 (EOC20 180 plusmn 23 of control ratmicroglia 159 plusmn 100 of control 119899 = 4 Figure 2(b))

Since microglia express several P2X and P2Y receptors[3] the possible involvement of purinergic receptors in theTNF-120572IFN-120574-induced dye coupling in microglia treatedwith oxidized-ATP (oATP) an inhibitor of P2X receptors[53] was studied Coapplication of 300 120583M oATP preventeddye transfer induced by TNF-120572 plus ATP (IDC in EOC20cells 147plusmn41of control ratmicroglia 159plusmn100of control119899 = 5 Figure 2(a)) or by TNF-120572IFN-120574 (IDC in EOC20172 plusmn 70 of control rat microglia 176 plusmn 40 of control119899 = 5 Figure 2(b)) Moreover cells treated with TNF-120572 plus1mMADP a P2Y agonist [53] for 35 h did not show changes

in dye coupling (IDC in EOC20 cells 168 plusmn 84 of control119899 = 3) suggesting that P2Y receptors are not involved inATP-induced gap junctional communication in microglia

Since activation of P2 receptors promotes a rise in [Ca2+]119894

in microglia [54] we tested if this response was related tothe increase in dye coupling induced by TNF-120572 plus ATPCells were loaded with BAPTA a Ca2+ chelator and thenwashed and the extracellular medium was replaced withconditionedmediumof cultures treated in parallel with TNF-120572 for 90min to maintain the culture conditions as beforeloading with BAPTA In these cells treatment with TNF-120572 plus ATP did not increase dye coupling (IDC in EOC20cells 134 plusmn 51 of control rat microglia 183 plusmn 44 ofcontrol 119899 = 5 Figure 2(a)) In addition we observed thatEOC20 cells treated with TNF-120572 plus ATP present increasedCa2+ signal compared to cells under control conditions(Figure S3a) Interestingly IL-6 prevented this rise in theCa2+signal (Figure S3b) suggesting that IL-6 might regulate thepurinergic signaling in EOC20 cells

33 IL-1120573 Released by Activated Microglia Mediates the TNF-120572IFN-120574-Induced Dye Coupling in EOC20 Cells Since acti-vatedmicroglia release IL-1120573 and its natural antagonist IL-1ra[7 55] we studied possible involvement of these moleculesin the transient increase in dye coupling induced by TNF-120572plus ATP or TNF-120572IFN-120574 Coapplication of 20 ngmL IL-1rasignificantly prevented the increase in IDC induced by TNF-120572 plus ATP (in EOC20 cells 217 plusmn 36 of control 119899 = 4) orTNF-120572IFN-120574 (in EOC20 cells 241 plusmn 53 of control 119899 = 4

Mediators of Inflammation 7

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Figure 3 IL-1120573mediates gap junctional communication induced by pro-inflammatorymolecules (a) Effect of 20 ngmL IL-1 receptor agonist(IL-1ra) over LY transfer in EOC20 cells treated with TNF-120572 plus ATP for 35 h or with TNF-120572IFN-120574 for 9 h

119875 lt 005 between indicatedtreatments Each bar represents the mean plusmn SEM 119899 = 4 (b)The effect of 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) acutely applied or treatmentwith 10 ngmL interleukin-6 (IL-6) or 300120583Moxidized ATP (oATP) over LY transfer in EOC20 cells treated with TNF-120572IL-1120573 for 9 h is alsoshown Each bar represents the mean plusmn SEM 119899 = 4 and corresponds to the percentage of incidence of dye coupling under control conditions(dashed line) lowast119875 lt 005 lowastlowast119875 lt 001 versus control condition

Figure 3(a)) Moreover EOC20 cells showed an increasein IL-1120573 release after TNF-120572 plus ATP or TNF-120572IFN-120574stimulation which was partially prevented by IL-6 (FigureS4) Consistent with the involvement of IL-1120573 in the abovedye coupling response induced by both pro-inflammatorymolecules exogenous application of 1 ngmL IL-1120573 plus TNF-120572 induced a similar response than that elicited by TNF-120572 plus ATP or TNF-120572IFN-120574 (Figure 3(b)) EOC20 cellstreated with TNF-120572IL-1120573 showed a transient increase indye coupling (data not shown) reaching a maximal IDC atsim9 h of treatment (EOC20 cells 560 plusmn 43 of control 119899 =4 Figure 3(b)) The TNF-120572IL-1120573-induced dye coupling wasdrastically reduced by the acute application of 50120583M 120573-GA(IDC in EOC20 cells 192 plusmn 35 of control 119899 = 4) andprevented by 10 ngmL IL-6 (in EOC20 cells 185 plusmn 40 ofcontrol 119899 = 4) or 300 120583M oATP (in EOC20 cells 198 plusmn34 of control 119899 = 4) coapplied with the two cytokines(Figure 3(b)) However treatment with IL-1120573 did not increasedye coupling in EOC20 cells (data not shown)

34 TNF-120572IFN-120574 but Not TNF-120572 Plus ATP Increases PlasmaMembrane Permeability in EOC20 Cells Astrocytes treatedwith TNF-120572IL-1120573 for 24 h [38] and microglia treated withLPS (or TNF-120572) for 24 h showed an increased HC activity[28 35 56ndash58] Using the ethidium (Etd) uptake assay toevaluate the functional state of HCs located at the cellsurface [38 59] we studied if TNF-120572 or ATP affects themembrane permeability of microglia cells In EOC20 cellsEtd uptake evaluated with time-lapse measurements showed

no significant differences after treatment with TNF-120572 plusATP as compared to untreated cells (Figure S5) In controlconditions Etd uptake was partially blocked by 200120583M La3+(after La3+ 45 plusmn 11 of control 119899 = 5) a Cx HC blocker thatdoes not affect Panx HCs [31] and by 10 120583M carbenoxolone(Cbx) (after Cbx 36 plusmn 15 of control 119899 = 5) which atthis concentration inhibits mainly Panx HCs [60] A slightbut not statistically significant increase in Etd uptake wasrecorded after 35 h treatment with TNF-120572 plus ATP (134 plusmn25 of control 119899 = 5) and was inhibited by La3+ (afterLa3+ 47 plusmn 8 of control 119899 = 5) or Cbx (after Cbx 38 plusmn8 of control 119899 = 5) suggesting an upstream cross talkbetween Cx and Panx HCs In addition 10 ngmL IL-6 didnot affect the response induced by TNF-120572 plusATP treatmentfor 35 h (Etd uptake rate 141 plusmn 16 of control 119899 = 5Figure S5b) In contrast after treatment with TNF-120572IFN-120574for 9 h a statistically significant increase in the Etd uptake ratecompared to the control conditionwas detected (Figure 4) InEOC20 cells cultured for 9 h under control conditions the Etduptake rate remained low and was partially blocked by La3+(57 plusmn 17 of control 119899 = 5 Figures 4(a) and 4(e)) or Cbx(34plusmn4of control 119899 = 5 Figure 4(e)) However cells treatedwith TNF-120572IFN-120574 for 9 h showed a prominent increase inEtd uptake (237 plusmn 25 of control 119899 = 5) that was drasticallyreduced by La3+ (51 plusmn 12 of control 119899 = 5 Figures 4(a) and4(e)) or Cbx (76plusmn9of control 119899 = 5 Figure 4(e)) A similarincrease in dye uptake was found after treatment with TNF-120572IL-1120573 for 9 h (Etd uptake rate 197 plusmn 41 of control 119899 = 3)which was also reduced by La3+ (Etd uptake rate 105 plusmn 4

8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

esce

nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

300

400

Ethi

dium

upt

ake r

ate (

o

f con

trol

)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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of

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Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Mediators of Inflammation

P2X7receptors which are upregulated as a required step

for microglial activation induced by amyloid-120573 peptide [1718] Moreover activation of microglia with LPS increasesthe intracellular free Ca2+ concentration ([Ca2+]

119894) and ATP

release through P2X7receptors [17 19 20] Accordingly

cytokines that increase [Ca2+]119894or a calcium ionophore induce

microglia activation [21 22] These conditions also inducegap junctional communication in primary cultures of rat ormouse microglia [23 24]

Gap junction channels (GJCs) communicate the cyto-plasm of contacting cells allowing the direct transfer of ionssecond messengers and other molecules including antigenpeptides [25] Each GJC is formed by the serial docking oftwo hemichannels (HCs) which are composed of six proteinsubunits called connexins (Cxs) [25] It is known that restingmicroglia express Cxs 32 36 43 and 45 and after microgliaactivation some of them form functional GJCs and HCs[23 24 26ndash28] Recently another family of proteins termedpannexins (Panxs) has been found to form functional GJCsand HCs [29] Like Cx HCs Panx HCs are permeable toATP and are activated by increased [Ca2+]

119894and extracellular

ATP via P2 receptors [30ndash32] Microglia express functionalPanx1 HCs that contribute to ATP-induced migration andglutamate and ATP release promoting neuronal death [33ndash35] Under inflammatory conditions gap junctional commu-nication between cultured astrocytes is reduced whereas theactivity of HCs is increased [35ndash38] However it remainsunknown if these opposite changes in GJCs and HCs alsooccur in microglia or if extracellular ATP plays a role in thischannel-based communication

In this work we studied the effect of extracellular ATPon the cytokine-induced gap junctional communication inmicroglia To achieve this goal we used primary cultures ofrat microglia and EOC20 cells treated with several cytokinesand ATP either mixed or alone We propose that TNF-120572IFN-120574 induce gap junctional communication whichmightdepend on the functional expression of HCs In addition wefound that extracellular ATP advances the onset of cytokine-induced expression of gap junctional communication aprocess that was mediated by IL-1120573 release and inhibited byIL-6

2 Materials and Methods

21 Materials Modified Eaglersquos medium (MEM) Dulbeccorsquosmodified Eaglersquos medium (DMEM) F-12 nutrient mix-ture fetal bovine serum (FBS) bovine pancreas DNAseI and trypsin-EDTA were purchased from Gibco (Auck-land NZ USA) DMSO HEPES H

2O LaCl

3(La3+)

ethidium (Etd) bromide Lucifer yellow dilithium salt (LYMW 45725Da) rhodamine-dextran (RD MW 10 kDa)adenosine 51015840-triphosphate periodate oxidized sodium salt(oATP) ATP disodium salt probenecid (Pbc) recombi-nant mouse TNF-120572 recombinant mouse IL-1120573 recombinantmouse IFN-120574 recombinant mouse IL-6 and Ponceau Sred were purchased from Sigma-Aldrich (St Louis MOUSA) Interleukin-1 receptor antagonist (IL-1ra) was from

RampD (USA) BAPTA-AM was purchased from Molecu-lar Probes (Eugene Oregon USA) Penicillin and strep-tomycin were obtained from Invitrogen (Carlsbad CAUSA) D(+)-glucose sodium hydrogen carbonate (NaHCO

3)

were purchased fromMerck (Darmstadt Germany) 10Panx1mimetic peptide (sequence WRQAAFVDSY) was purchasedfrom SBS Biotech (Beijing China) Purified rat anti-mouseCD16CD32 (mouse BD Fc-block) was purchased fromBD Pharmingen (San Jose CA USA) F(ab1015840)

2fragments

of a previously characterized polyclonal rabbit anti-Panx1serum used [39 40] The F(ab1015840)

2fragments of affinity

IgGs purified from the anti-Panx1 serum were preparedas previously described [41] Anti-Cx43 monoclonal anti-body was obtained from BD Biosciences (Minneapolis MNUSA) Cy2 conjugated goat anti-rabbit and Cy3 conjugatedgoat anti-mouse antibodies were purchased from JacksonImmunoresearch Laboratories Inc (Indianapolis IN USA)EDTA solution Halt protease inhibitor single-use cocktailand M-PER mammalian protein extraction reagent werepurchased from Thermo Scientific (Rockford IL USA)Mount solution fluoromount G was purchased from ElectronMicroscopy Sciences (Washington PA USA) Images wereexamined with a confocal laser-scanning microscope whichwas Olympus Fluoview FV1000 (Tokio Japan) Cx43(E2) isa rabbit polyclonal antibody that recognizes amino acidresidues located at the second extracellular loop of Cx43 andblocks specifically Cx43 HCs [42]

Bio-Rad protein assay was purchased from Bio-RadLaboratories (Richmond CA USA) SuperSignal kit forenhanced chemiluminescence detection and anti-rabbit anti-body conjugated to horseradish peroxidase were purchasedfrom Pierce (Rockford IL USA) EOC20 and LADMACcells were obtained fromATCC (Manassas VA USA) Tissueculture flasks (25 and 75 cm2) 60mm and 100mm tissueculture dishes were purchased from Sarstedt (Newton NCUSA) Twenty four-well plastic dishes were purchased fromNunclon (Roskilde Denmark)

22 Cell Cultures

221 Rat Microglia Primary cultures of microglia wereprepared from neocortex of newborn Sprague Dawley rats aspreviously described [23 24] Brieflymeningeswere carefullypeeled off and cortices were dissected and minced in smallpieces After incubation in Ca2+-free PBS containing trypsin(05) and EDTA (5mM) at 37∘C for 30min tissue wastriturated in presence of DNAse using a Pasteur pipetteDissociated cells were pelleted and resuspended in MEMmedium supplementedwith 10FBS 100 unitsmLpenicillinand 50120583gmL streptomycin sulfate and plated on plasticculture flasks (Sarstedt) Confluent glial cell mixed cultureswere deprived of fresh medium for two weeks to inducemicroglial cell proliferation Finallymicroglia were harvestedfrom glial cell mixed cultures by differential adhesion andseeded on glass coverslips

222 EOC20 Cells EOC20 cells are a murine microglial cellline derived fromC3HHeJmice which secrete cytokines and

Mediators of Inflammation 3

present antigens as primary microglia [43] EOC20 cells weremaintained according to ATCC recommendations usingDMEM supplemented with 10 FBS and 20 LADMACconditioned medium (see below) The medium was partiallychanged twice a week and completely changed once a weekuntil the culture reached confluence Cells were detachedwith trypsin-EDTA for 2min and mechanical stress andEOC20 cells were seeded on glass coverslips or tissue culturedishes Since rat microglia were detached by shaking usingan orbital shaker some experiments were performed withEOC20 cells detached with the same methods of purificationused for primary microglia cultures No differences wereobserved in the induction of dye coupling between EOC20cells obtained by the different purification methods (data notshown)

223 LADMAC Conditioned Medium The conditionedmedium was obtained from LADMAC cells which aremyeloid cells derived from murine bone marrow cellsLADMAC cells are nonadherent cells that secrete colony-stimulating-factor-1 (CSF-1) which stimulates cell division inEOC20 cells [43 44] LADMAC cell cultures were main-tained in culture in MEM supplemented with 10 FBSduring two weeks Fresh medium was added every two daysduplicating the previous amount ofmedium After two weeksin culture the cell suspension was centrifuged and the CSF-1-containing supernatant was filtered aliquoted and stored atminus20∘C until use

23 Dye Transfer Technique The transference of fluorescentdyes between adjacent cells has been used to monitor thefunctional state of GJCs in microglia [23 24 27] We testedthe intercellular transference of LY using RD as a negativecontrol Dyes (5wv in 150mM LiCl) were microinjectedby applying current to microglia seeded on glass coverslips(8 times 105 cellswell in a 24-multiwell dish) through glassmicroelectrodes until the impaled cells were fluorescent Cul-tures were maintained in F-12 medium supplemented withHEPES and observed with an inverted microscope equippedwith Xenon arc lamp illumination and a Nikon B filter(excitation wavelength 450ndash490 nm emission wavelengthabove 520 nm) Dye transfer was scored at 2min injectionThe incidence of dye coupling (IDC) was calculated as thepercentage of injected cells with dye transfer to one or moreneighboring cells by the total number of cellsmicroinjected ineach experiment At least 10 cells were microinjected in eachassay Since cytokine treatments induced HC activity andbecause that dye uptake from leaking microelectrodes couldaffect the measurement of fluorescent cells we use 200120583MLa3+ in the recording solution However no significantdifferences were observed compared to recording solutionwithout La3+ (data not shown)

24 Dye Uptake Ca2+ Signal Imaging and Time-Lapse Flu-orescence Imaging To evaluate dye uptake cells seeded onglass coverslips (8 times 105 cellsmL) were exposed to 5120583Methidium (Etd) bromide with Lockersquos saline solution (in mM154 NaCl 54 KCl 23 CaCl

2 1 mM MgCl

2 5mM glucose

5mM HEPES pH 742) and examined by epifluorescenceNuclei fluorescence was recorded in regions of interest con-sisting of 30 different cells per field with a water immersionOlympus 51W1I upright microscope (Melville NY USA) asdescribed [45] The calculation of slope change regressionlines was fitted to points before and after treatments usingMicrosoft (Seattle WA USA) Excel In ATP-induced dyeuptake experiments 500120583M ATP was added to recordingsolution after 5min of basal dye uptake

To evaluate Ca2+ signals EOC20 cells under controlconditions or after treatment were maintained as mentionedabove but were loaded for 30min with 5120583M Fura-2 AM inDMEM medium without serum at 37∘C Loaded cells werewashed twice with Lockersquos solution and time-measurementswere performed with an Olympus 51W1I microscope Theacquisition of 340 and 380 nm excitation wavelengths wasevery 3 s Regions of interest consisted in 30 cells per field andanalysis were performed using METAFLUOR software

25 Western Blot Confluent microglia cultures grown in60mmculture dishes (24times 106 cells) were gently rinsed twicewith cold PBS at 4∘C pH 74 and harvested by scraping witha rubber policeman in a solution containing 5mM EDTAHalt andM-PER protein extraction cocktail according to themanufacturerrsquos instructionsThe cellular suspensionwas son-icated on ice Proteinsweremeasured in aliquots of cell lysatesusing the Bio-Rad protein assay Aliquots of cell lysates (50 120583gof protein) were resuspended in Laemlirsquos sample buffer andseparated in an 8 sodium dodecyl sulfate polyacrylamidegel electrophoresis and electrotransferred to nitrocellulosesheets as previously described [24] Loading equivalenceswere confirmed by protein staining with Ponceau S red(2wv in 30 trichloroacetic acid) Nonspecific proteinbinding was blocked by incubation of nitrocellulose sheets in5 nonfat milk in PBS for 1 h at room temperature prior toovernight incubation with corresponding antibodies at 4∘CAfter several washes with PBS blots were incubated with thesecondary antibody conjugated to horseradish peroxidase for45min at room temperature Immunoreactivity was detectedby enhanced chemiluminescence using the SuperSignal kitaccording to the manufacturerrsquos instructions

26 Immunofluorescence Microglia cultured on glass coverslips were fixed with 4 formaldehyde at room temperaturefor 30min and washed twice with PBS A blocking solutioncontaining 1 IgG free BSA 50mM NH

4Cl and 005

Triton X-100 in PBS was used to permeabilize and blockunspecific reactive sites Fc receptors were masked by incu-bating samples to a solution containing Fc-Block (1 100) for45min at room temperature Panx1 and Cx43 were detectedwith a rabbit polyclonal anti-Panx1 F(ab1015840)

2fragments [40]

and an anti-Cx43 monoclonal antibody properly dilutedwith blocking solution respectively Cy2 conjugated goatanti-rabbit (1 300) and Cy3 conjugated goat anti-mouseIgGF(ab1015840)

2Igs fragments for 30min at room temperature

were used to detect bound primary antibody FluoromountG (Electron Microscopy Sciences Washington PA USA)was used as an antifade solution to mount samples Images

4 Mediators of Inflammation

were examined with a confocal laser-scanning microscope(Olympus Fluoview FV1000 Tokio Japan)

27 IL-1120573 ELISA The level of IL-1120573 present in the condi-tioned media of EOC20 cells was evaluated with the IL-1120573ELISA ReadySet-Go (e-Bioscience San Diego CA USA)for performing quantitative enzyme linked immunosorbentassays (ELISA) It has a sensitivity of 8 pgmL Standardcurve consisted of twofold serial dilutions of the recombi-nant cytokine In brief a 96-well flat bottom ELISA-plate(MICROLON Greiner Bio-One) was coated with captureantibody in coating buffer overnight at 4∘C The plate waswashed 5 times with PBS-005 Tween-20 in ELx50 Biokita 96-well bioelisa washer and Rhe plate was blocked with200120583L of assay diluent at room temperature for 1 h washedas mentioned and 100 120583L of standard IL1120573 and sampleswere incubated at 4∘C overnight Then the plate was washedand 100 120583L of detection antibody for IL-1120573 was added andincubated at room temperature by 1 h washed 5 timesincubated with 100120583L Avidin-HRP at room temperature for30min washed 7 times added 100120583L substrate solution andstopped the reaction with 50 120583L of 1M H

3PO4 The plate was

read at 450 nm with reference at 570 nm

28 Treatments Microglia were seeded 48 h before dyetransfer dye uptake or immunofluorescence experiments in24-well plastic dish containing 500 120583L of culture mediumFor Western blot experiments cells were seeded in 60mmplastic dishes in 3mL of culture medium After 48 h undercontrol conditions microglia were treated with 1mM ATPor 1 ngmL TNF-120572 IFN-120574 IL-1120573 either alone or mixedCytokines were added simultaneously and ATP was added2 h before measurement and is referred as cytokine(s) plusATP Treatment with 1 10 or 50 ngmL IL-6 20 ngmL IL-1ra 300 120583M oATP 200120583M La3+ 1 500 Cx43(E2) antibodyor 200120583M 10Panx1 was simultaneous to cytokine treat-ment We used 50120583M of 120573-GA for acute GJCs blocking(Figure S1 see Supplementary Materials available online athttpdxdoiorg1011552013216402) To avoid disruption ofcell adhesion with BAPTA the medium was replaced withculture medium of parallel cultures treated at the same timeto maintain the soluble factor released from microglia

29 Statistical Analysis Data are presented as mean plusmn SEMas percentage of the control condition 119899 represents thenumber of independent experiments For statistical analysiseach treatment was compared with its respective controland significance was determined using one-way ANOVAfollowed by Dunnrsquos test comparing all treatments against thecontrol condition To observe differences between microgliaand EOC20 cells responses we used a two-way ANOVA

3 Results

31 The Onset of the Cytokine-Induced Increase in GapJunctional Communication in Cultured Microglia Is Advancedby ATP and Delayed by IFN-120574 Calcium ionophore and pro-inflammatory molecules promote a transient expression of

functional GJCs in microglia [23 24 27] Since extracellularATP TNF-120572 and IFN-120574 play a relevant role in microglialcell responses [3 7 46] and affect the [Ca2+]

119894[47ndash49] we

decided to evaluate if these compounds affect the intercellularcommunication via GJCs in both primary cultures of ratmicroglia and EOC20 cells

After 48 h of subculture under control conditionsmicroglia were treated as indicated in Methods (Figure S1a)Both cell types presented rather homogeneousmorphologicalfeatures (Figures 1(a) and 1(b)) and very low incidence ofLucifer yellow (LY) transfer to neighboring cells (Figures1(a) and 1(b)) Under these conditions the incidence of dyecoupling (IDC) remained low for up to 12 h of culture inboth cell types (Figure 1 Supporting information Table S1)In addition intercellular transfer of rhodamine-dextran (RDsim10 kDa) which due to its high molecular weight cannotpermeate through GJCs was not observed (Figure S2a) Thisresult indicates that intercellular LY transfer ocurred viaGJCsand not through other cell-cell communication pathway suchas cytoplasmic bridges Moreover microglia treated eitherwith 1mM ATP 1 ngmL TNF-120572 1 ngmL IFN-120574 or 1 ngmLIL-1120573 showed only a slight increase in IDC which was notstatistically different from that of control cells (119875 gt 005Supporting information Table S1) However treatment withmixes of thesemolecules during different time periods causeda significant and transient increase in IDC the dye transferdata is expressed as percentage of the corresponding controlcondition (Figures 1(e) and 1(f)) In both cell types treatmentwith 1 ngmL TNF-120572 plus 1 ngmL IFN-120574 (from now andon referred as TNF-120572IFN-120574) increased the IDC reachinga maximum response at around 9 h after treatment (IDC inEOC20 cells 574 plusmn 36 of control rat microglia 552 plusmn 36of control Mean plusmn SEM 119899 = 5) as previously described[23]

We also studied if extracellular ATP affects TNF-120572IFN-120574-induced dye coupling To this end cells were treated withthese cytokines and then exposed to ATP for 2 h In bothcell types treatment with TNF-120572IFN-120574 plus ATP induced atransient increase in IDC which was maximal at around 5 h(EOC20 cells 517 plusmn 94 of control rat microglia 506 plusmn 42of control 119899 = 5) The amplitude and duration (magnitude)of the response was similar to that induced by TNF-120572IFN-120574but occurred 4 h earlier (Figure 1(e))

Since IFN-120574 potentiates TNF-120572-induced dye coupling inantigen presenting cells including dendritic cells microgliaand monocytesmacrophages [23 50 51] we tested whetherATP induces a similar effect on microglia In agreement withthis possibility cells treated with TNF-120572 plus ATP showedmaximal IDCwith similar amplitude (EOC20 cells 529plusmn12of control rat microglia 534plusmn70of control 119899 = 6 Figure 1)to that induced by TNF-120572IFN-120574 plus ATP but occurred15 h earlier (at sim35 h versus 5 h Figures 1(e) and 1(f)) Asmentioned before intercellular transfer of LY was enhancedin primarymicroglia or EOC20 cells treated with TNF-120572 plusATP (Figures 1(c) and 1(d)) However intercellular transfer ofRDwas not observed ruling out the formation of cytoplasmicbridges or vesicular mediated dye transfer in each condition(Figure S2) Microglia treated with IFN-120574 plus ATP did notincrease dye coupling at 35 h (EOC20 cells 167 plusmn 97 of

Mediators of Inflammation 5

Rat microglia

Control

lowast

(a)

EOC20

Control

lowast

(b)

Rat microglia

TNF-120572ATP

lowast

(c)

EOC20

TNF-120572ATP

lowast

(d)

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Inci

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(e)

0

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800

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EOC20Rat microglia

Time (h) 35 5 9

lowastlowastlowast

TNF-120572

ATP

IFN-120574

+

+

+

+

+

+

+minus

minus

(f)

Figure 1 ATP advances the onset of TNF-120572IFN-120574-induced dye coupling (a)ndash(d) Dye transfer was evaluated 2min after Lucifer yellow (LY)microinjection in a single cell (indicated with an asterisk) Representative pictures of LY transfer in rat microglia (a) (c) or EOC20 cells (b)(d) under control condition or after TNF-120572 plus ATP (TNF-120572ATP) treatment for 35 h as indicated Phase contrasts of each micrograph areshown at the right panels Scale bar 20 120583m (e) Time course of the incidence of dye coupling (IDC) as percentage of IDC in EOC20 cellsunder control conditions (dashed line) or after treatment with TNF-120572 plus ATP (black circles) TNF-120572IFN-120574 plus ATP (white triangles) orTNF-120572IFN-120574 (black diamonds) Each point corresponds to themean of 3 independent experiments (f) Graph showing themaximum valuesof IDC after treatment with TNF-120572 plus ATP for 35 h TNF-120572IFN-120574 plus ATP for 5 h or TNF-120572IFN-120574 for 9 h lowast119875 lt 005 versus controlcondition Each bar represents the mean plusmn SEM 119899 = 6 No significant differences were observed when comparing microglia and EOC20 cellsresponses to different treatment in dye transfer assays Concentrations 1 ngmL TNF-120572 1 mM ATP 1 ngmL IFN-120574

control ratmicroglia 2108plusmn513of control) or other times(2 and 5 h data not shown)

32 The Increase of Gap Junctional Communication Inducedby TNF-120572 Plus ATP Requires an Increase of [Ca2+]

119894via

Activation of P2XReceptors and Is Prevented by IL-6 Eugenınet al (2001) described that dye coupling between microgliatreated for 9 h with TNF-120572IFN-120574 is inhibited by 120573-GAIn EOC20 cells treated with TNF-120572IFN-120574 we observeda similar acute blockade with 120573-GA (data not shown)

6 Mediators of Inflammation

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cide

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( o

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trol)

TNF-120572ATP120573-GA

IL-6oATP

BAPTA-AM

+minusminusminusminus

++minusminusminus

+

+minus

minusminus

+

+

minusminus

minus

+

+

minusminusminus

lowast

(a)

0

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Inci

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EOC20Rat microglia

TNF-120572ATPIL-6

oATP

+minusminus

++minus

+

+minus

lowast

(b)

Figure 2 IL-6 intracellular calcium chelation and P2X inhibition prevent the induction of gap junctional communication promoted byTNF-120572 plus ATP or TNF-120572IFN-120574 (a) Graph showing the effect of acutely applied 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) or pretreatmentwith 10 ngmL interleukin-6 (IL-6) 300120583Moxidized ATP (oATP) or 10120583MBAPTA-AMon the incidence of dye coupling (IDC) of microgliatreated for 35 h with TNF-120572 plus ATP (b) Graph showing the effect of 50 ngmL IL-6 or 300120583M oATP over the IDC of microglia treated for9 h with TNF-120572IFN-120574 Data is expressed as a percentage of IDC under control conditions (dashed line) lowast119875 lt 005 versus control conditionEach bar represents the mean plusmn SEM 119899 = 5 No significant differences were observed when comparing microglia and EOC20 cells responsesto different treatment in dye transfer assays

In addition application of 50 120583M 120573-GA for 5min completelyabolished dye coupling induced by TNF-120572 plus ATP (IDC inEOC20 cells 74 plusmn 44 of control rat microglia 86 plusmn 50 ofcontrol 119899 = 5 Figure 2(a))

Since microglia treated with purinergic agonists releaseIL-6 [52] and this cytokine prevents the increase of dyecoupling induced by TNF-120572IL-1120573 in dendritic cells [50] wedecided to test if IL-6 prevents induction of dye couplingin microglia treated with TNF-120572 plus ATP In cell culturestreated simultaneously with 10 ngmL IL-6 plus TNF-120572 andthen treated with ATP for 35 h the IDC was low (EOC20cells 130 plusmn 83 of control rat microglia 162 plusmn 10 ofcontrol 119899 = 4) similar to the results obtained undercontrol conditions (Figure 2(a)) Similarly the TNF-120572IFN-120574-induced dye coupling was prevented by IL-6 (Figure 2(b))This inhibitory effect was IL-6 concentration-dependent (110 and 50 ngmL data not shown) The maximal effect wasinduced by 50 ngmL IL-6 (EOC20 180 plusmn 23 of control ratmicroglia 159 plusmn 100 of control 119899 = 4 Figure 2(b))

Since microglia express several P2X and P2Y receptors[3] the possible involvement of purinergic receptors in theTNF-120572IFN-120574-induced dye coupling in microglia treatedwith oxidized-ATP (oATP) an inhibitor of P2X receptors[53] was studied Coapplication of 300 120583M oATP preventeddye transfer induced by TNF-120572 plus ATP (IDC in EOC20cells 147plusmn41of control ratmicroglia 159plusmn100of control119899 = 5 Figure 2(a)) or by TNF-120572IFN-120574 (IDC in EOC20172 plusmn 70 of control rat microglia 176 plusmn 40 of control119899 = 5 Figure 2(b)) Moreover cells treated with TNF-120572 plus1mMADP a P2Y agonist [53] for 35 h did not show changes

in dye coupling (IDC in EOC20 cells 168 plusmn 84 of control119899 = 3) suggesting that P2Y receptors are not involved inATP-induced gap junctional communication in microglia

Since activation of P2 receptors promotes a rise in [Ca2+]119894

in microglia [54] we tested if this response was related tothe increase in dye coupling induced by TNF-120572 plus ATPCells were loaded with BAPTA a Ca2+ chelator and thenwashed and the extracellular medium was replaced withconditionedmediumof cultures treated in parallel with TNF-120572 for 90min to maintain the culture conditions as beforeloading with BAPTA In these cells treatment with TNF-120572 plus ATP did not increase dye coupling (IDC in EOC20cells 134 plusmn 51 of control rat microglia 183 plusmn 44 ofcontrol 119899 = 5 Figure 2(a)) In addition we observed thatEOC20 cells treated with TNF-120572 plus ATP present increasedCa2+ signal compared to cells under control conditions(Figure S3a) Interestingly IL-6 prevented this rise in theCa2+signal (Figure S3b) suggesting that IL-6 might regulate thepurinergic signaling in EOC20 cells

33 IL-1120573 Released by Activated Microglia Mediates the TNF-120572IFN-120574-Induced Dye Coupling in EOC20 Cells Since acti-vatedmicroglia release IL-1120573 and its natural antagonist IL-1ra[7 55] we studied possible involvement of these moleculesin the transient increase in dye coupling induced by TNF-120572plus ATP or TNF-120572IFN-120574 Coapplication of 20 ngmL IL-1rasignificantly prevented the increase in IDC induced by TNF-120572 plus ATP (in EOC20 cells 217 plusmn 36 of control 119899 = 4) orTNF-120572IFN-120574 (in EOC20 cells 241 plusmn 53 of control 119899 = 4

Mediators of Inflammation 7

0

200

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800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

+minus +minusIL-1raTNF-120572ATP 35h TNF-120572IFN-120574 9h

(a)

0

200

400

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800

Inci

denc

e of d

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uplin

g (

of c

ontro

l)

lowastlowast

TNF-120572IL-1120573120573-GA

IL-6oATP

+

minus

minus

minus

+

+

minus

minus

+

+

minus

minus

+

+

minus

minus

(b)

Figure 3 IL-1120573mediates gap junctional communication induced by pro-inflammatorymolecules (a) Effect of 20 ngmL IL-1 receptor agonist(IL-1ra) over LY transfer in EOC20 cells treated with TNF-120572 plus ATP for 35 h or with TNF-120572IFN-120574 for 9 h

119875 lt 005 between indicatedtreatments Each bar represents the mean plusmn SEM 119899 = 4 (b)The effect of 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) acutely applied or treatmentwith 10 ngmL interleukin-6 (IL-6) or 300120583Moxidized ATP (oATP) over LY transfer in EOC20 cells treated with TNF-120572IL-1120573 for 9 h is alsoshown Each bar represents the mean plusmn SEM 119899 = 4 and corresponds to the percentage of incidence of dye coupling under control conditions(dashed line) lowast119875 lt 005 lowastlowast119875 lt 001 versus control condition

Figure 3(a)) Moreover EOC20 cells showed an increasein IL-1120573 release after TNF-120572 plus ATP or TNF-120572IFN-120574stimulation which was partially prevented by IL-6 (FigureS4) Consistent with the involvement of IL-1120573 in the abovedye coupling response induced by both pro-inflammatorymolecules exogenous application of 1 ngmL IL-1120573 plus TNF-120572 induced a similar response than that elicited by TNF-120572 plus ATP or TNF-120572IFN-120574 (Figure 3(b)) EOC20 cellstreated with TNF-120572IL-1120573 showed a transient increase indye coupling (data not shown) reaching a maximal IDC atsim9 h of treatment (EOC20 cells 560 plusmn 43 of control 119899 =4 Figure 3(b)) The TNF-120572IL-1120573-induced dye coupling wasdrastically reduced by the acute application of 50120583M 120573-GA(IDC in EOC20 cells 192 plusmn 35 of control 119899 = 4) andprevented by 10 ngmL IL-6 (in EOC20 cells 185 plusmn 40 ofcontrol 119899 = 4) or 300 120583M oATP (in EOC20 cells 198 plusmn34 of control 119899 = 4) coapplied with the two cytokines(Figure 3(b)) However treatment with IL-1120573 did not increasedye coupling in EOC20 cells (data not shown)

34 TNF-120572IFN-120574 but Not TNF-120572 Plus ATP Increases PlasmaMembrane Permeability in EOC20 Cells Astrocytes treatedwith TNF-120572IL-1120573 for 24 h [38] and microglia treated withLPS (or TNF-120572) for 24 h showed an increased HC activity[28 35 56ndash58] Using the ethidium (Etd) uptake assay toevaluate the functional state of HCs located at the cellsurface [38 59] we studied if TNF-120572 or ATP affects themembrane permeability of microglia cells In EOC20 cellsEtd uptake evaluated with time-lapse measurements showed

no significant differences after treatment with TNF-120572 plusATP as compared to untreated cells (Figure S5) In controlconditions Etd uptake was partially blocked by 200120583M La3+(after La3+ 45 plusmn 11 of control 119899 = 5) a Cx HC blocker thatdoes not affect Panx HCs [31] and by 10 120583M carbenoxolone(Cbx) (after Cbx 36 plusmn 15 of control 119899 = 5) which atthis concentration inhibits mainly Panx HCs [60] A slightbut not statistically significant increase in Etd uptake wasrecorded after 35 h treatment with TNF-120572 plus ATP (134 plusmn25 of control 119899 = 5) and was inhibited by La3+ (afterLa3+ 47 plusmn 8 of control 119899 = 5) or Cbx (after Cbx 38 plusmn8 of control 119899 = 5) suggesting an upstream cross talkbetween Cx and Panx HCs In addition 10 ngmL IL-6 didnot affect the response induced by TNF-120572 plusATP treatmentfor 35 h (Etd uptake rate 141 plusmn 16 of control 119899 = 5Figure S5b) In contrast after treatment with TNF-120572IFN-120574for 9 h a statistically significant increase in the Etd uptake ratecompared to the control conditionwas detected (Figure 4) InEOC20 cells cultured for 9 h under control conditions the Etduptake rate remained low and was partially blocked by La3+(57 plusmn 17 of control 119899 = 5 Figures 4(a) and 4(e)) or Cbx(34plusmn4of control 119899 = 5 Figure 4(e)) However cells treatedwith TNF-120572IFN-120574 for 9 h showed a prominent increase inEtd uptake (237 plusmn 25 of control 119899 = 5) that was drasticallyreduced by La3+ (51 plusmn 12 of control 119899 = 5 Figures 4(a) and4(e)) or Cbx (76plusmn9of control 119899 = 5 Figure 4(e)) A similarincrease in dye uptake was found after treatment with TNF-120572IL-1120573 for 9 h (Etd uptake rate 197 plusmn 41 of control 119899 = 3)which was also reduced by La3+ (Etd uptake rate 105 plusmn 4

8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

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nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

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dium

upt

ake r

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)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

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45

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Etd

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nce (

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Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

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ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

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Inci

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lowastlowast

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Cx43(E2)

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minus

minus

+

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minus +

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minus

minus

+

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Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

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Mediators of Inflammation 3

present antigens as primary microglia [43] EOC20 cells weremaintained according to ATCC recommendations usingDMEM supplemented with 10 FBS and 20 LADMACconditioned medium (see below) The medium was partiallychanged twice a week and completely changed once a weekuntil the culture reached confluence Cells were detachedwith trypsin-EDTA for 2min and mechanical stress andEOC20 cells were seeded on glass coverslips or tissue culturedishes Since rat microglia were detached by shaking usingan orbital shaker some experiments were performed withEOC20 cells detached with the same methods of purificationused for primary microglia cultures No differences wereobserved in the induction of dye coupling between EOC20cells obtained by the different purification methods (data notshown)

223 LADMAC Conditioned Medium The conditionedmedium was obtained from LADMAC cells which aremyeloid cells derived from murine bone marrow cellsLADMAC cells are nonadherent cells that secrete colony-stimulating-factor-1 (CSF-1) which stimulates cell division inEOC20 cells [43 44] LADMAC cell cultures were main-tained in culture in MEM supplemented with 10 FBSduring two weeks Fresh medium was added every two daysduplicating the previous amount ofmedium After two weeksin culture the cell suspension was centrifuged and the CSF-1-containing supernatant was filtered aliquoted and stored atminus20∘C until use

23 Dye Transfer Technique The transference of fluorescentdyes between adjacent cells has been used to monitor thefunctional state of GJCs in microglia [23 24 27] We testedthe intercellular transference of LY using RD as a negativecontrol Dyes (5wv in 150mM LiCl) were microinjectedby applying current to microglia seeded on glass coverslips(8 times 105 cellswell in a 24-multiwell dish) through glassmicroelectrodes until the impaled cells were fluorescent Cul-tures were maintained in F-12 medium supplemented withHEPES and observed with an inverted microscope equippedwith Xenon arc lamp illumination and a Nikon B filter(excitation wavelength 450ndash490 nm emission wavelengthabove 520 nm) Dye transfer was scored at 2min injectionThe incidence of dye coupling (IDC) was calculated as thepercentage of injected cells with dye transfer to one or moreneighboring cells by the total number of cellsmicroinjected ineach experiment At least 10 cells were microinjected in eachassay Since cytokine treatments induced HC activity andbecause that dye uptake from leaking microelectrodes couldaffect the measurement of fluorescent cells we use 200120583MLa3+ in the recording solution However no significantdifferences were observed compared to recording solutionwithout La3+ (data not shown)

24 Dye Uptake Ca2+ Signal Imaging and Time-Lapse Flu-orescence Imaging To evaluate dye uptake cells seeded onglass coverslips (8 times 105 cellsmL) were exposed to 5120583Methidium (Etd) bromide with Lockersquos saline solution (in mM154 NaCl 54 KCl 23 CaCl

2 1 mM MgCl

2 5mM glucose

5mM HEPES pH 742) and examined by epifluorescenceNuclei fluorescence was recorded in regions of interest con-sisting of 30 different cells per field with a water immersionOlympus 51W1I upright microscope (Melville NY USA) asdescribed [45] The calculation of slope change regressionlines was fitted to points before and after treatments usingMicrosoft (Seattle WA USA) Excel In ATP-induced dyeuptake experiments 500120583M ATP was added to recordingsolution after 5min of basal dye uptake

To evaluate Ca2+ signals EOC20 cells under controlconditions or after treatment were maintained as mentionedabove but were loaded for 30min with 5120583M Fura-2 AM inDMEM medium without serum at 37∘C Loaded cells werewashed twice with Lockersquos solution and time-measurementswere performed with an Olympus 51W1I microscope Theacquisition of 340 and 380 nm excitation wavelengths wasevery 3 s Regions of interest consisted in 30 cells per field andanalysis were performed using METAFLUOR software

25 Western Blot Confluent microglia cultures grown in60mmculture dishes (24times 106 cells) were gently rinsed twicewith cold PBS at 4∘C pH 74 and harvested by scraping witha rubber policeman in a solution containing 5mM EDTAHalt andM-PER protein extraction cocktail according to themanufacturerrsquos instructionsThe cellular suspensionwas son-icated on ice Proteinsweremeasured in aliquots of cell lysatesusing the Bio-Rad protein assay Aliquots of cell lysates (50 120583gof protein) were resuspended in Laemlirsquos sample buffer andseparated in an 8 sodium dodecyl sulfate polyacrylamidegel electrophoresis and electrotransferred to nitrocellulosesheets as previously described [24] Loading equivalenceswere confirmed by protein staining with Ponceau S red(2wv in 30 trichloroacetic acid) Nonspecific proteinbinding was blocked by incubation of nitrocellulose sheets in5 nonfat milk in PBS for 1 h at room temperature prior toovernight incubation with corresponding antibodies at 4∘CAfter several washes with PBS blots were incubated with thesecondary antibody conjugated to horseradish peroxidase for45min at room temperature Immunoreactivity was detectedby enhanced chemiluminescence using the SuperSignal kitaccording to the manufacturerrsquos instructions

26 Immunofluorescence Microglia cultured on glass coverslips were fixed with 4 formaldehyde at room temperaturefor 30min and washed twice with PBS A blocking solutioncontaining 1 IgG free BSA 50mM NH

4Cl and 005

Triton X-100 in PBS was used to permeabilize and blockunspecific reactive sites Fc receptors were masked by incu-bating samples to a solution containing Fc-Block (1 100) for45min at room temperature Panx1 and Cx43 were detectedwith a rabbit polyclonal anti-Panx1 F(ab1015840)

2fragments [40]

and an anti-Cx43 monoclonal antibody properly dilutedwith blocking solution respectively Cy2 conjugated goatanti-rabbit (1 300) and Cy3 conjugated goat anti-mouseIgGF(ab1015840)

2Igs fragments for 30min at room temperature

were used to detect bound primary antibody FluoromountG (Electron Microscopy Sciences Washington PA USA)was used as an antifade solution to mount samples Images

4 Mediators of Inflammation

were examined with a confocal laser-scanning microscope(Olympus Fluoview FV1000 Tokio Japan)

27 IL-1120573 ELISA The level of IL-1120573 present in the condi-tioned media of EOC20 cells was evaluated with the IL-1120573ELISA ReadySet-Go (e-Bioscience San Diego CA USA)for performing quantitative enzyme linked immunosorbentassays (ELISA) It has a sensitivity of 8 pgmL Standardcurve consisted of twofold serial dilutions of the recombi-nant cytokine In brief a 96-well flat bottom ELISA-plate(MICROLON Greiner Bio-One) was coated with captureantibody in coating buffer overnight at 4∘C The plate waswashed 5 times with PBS-005 Tween-20 in ELx50 Biokita 96-well bioelisa washer and Rhe plate was blocked with200120583L of assay diluent at room temperature for 1 h washedas mentioned and 100 120583L of standard IL1120573 and sampleswere incubated at 4∘C overnight Then the plate was washedand 100 120583L of detection antibody for IL-1120573 was added andincubated at room temperature by 1 h washed 5 timesincubated with 100120583L Avidin-HRP at room temperature for30min washed 7 times added 100120583L substrate solution andstopped the reaction with 50 120583L of 1M H

3PO4 The plate was

read at 450 nm with reference at 570 nm

28 Treatments Microglia were seeded 48 h before dyetransfer dye uptake or immunofluorescence experiments in24-well plastic dish containing 500 120583L of culture mediumFor Western blot experiments cells were seeded in 60mmplastic dishes in 3mL of culture medium After 48 h undercontrol conditions microglia were treated with 1mM ATPor 1 ngmL TNF-120572 IFN-120574 IL-1120573 either alone or mixedCytokines were added simultaneously and ATP was added2 h before measurement and is referred as cytokine(s) plusATP Treatment with 1 10 or 50 ngmL IL-6 20 ngmL IL-1ra 300 120583M oATP 200120583M La3+ 1 500 Cx43(E2) antibodyor 200120583M 10Panx1 was simultaneous to cytokine treat-ment We used 50120583M of 120573-GA for acute GJCs blocking(Figure S1 see Supplementary Materials available online athttpdxdoiorg1011552013216402) To avoid disruption ofcell adhesion with BAPTA the medium was replaced withculture medium of parallel cultures treated at the same timeto maintain the soluble factor released from microglia

29 Statistical Analysis Data are presented as mean plusmn SEMas percentage of the control condition 119899 represents thenumber of independent experiments For statistical analysiseach treatment was compared with its respective controland significance was determined using one-way ANOVAfollowed by Dunnrsquos test comparing all treatments against thecontrol condition To observe differences between microgliaand EOC20 cells responses we used a two-way ANOVA

3 Results

31 The Onset of the Cytokine-Induced Increase in GapJunctional Communication in Cultured Microglia Is Advancedby ATP and Delayed by IFN-120574 Calcium ionophore and pro-inflammatory molecules promote a transient expression of

functional GJCs in microglia [23 24 27] Since extracellularATP TNF-120572 and IFN-120574 play a relevant role in microglialcell responses [3 7 46] and affect the [Ca2+]

119894[47ndash49] we

decided to evaluate if these compounds affect the intercellularcommunication via GJCs in both primary cultures of ratmicroglia and EOC20 cells

After 48 h of subculture under control conditionsmicroglia were treated as indicated in Methods (Figure S1a)Both cell types presented rather homogeneousmorphologicalfeatures (Figures 1(a) and 1(b)) and very low incidence ofLucifer yellow (LY) transfer to neighboring cells (Figures1(a) and 1(b)) Under these conditions the incidence of dyecoupling (IDC) remained low for up to 12 h of culture inboth cell types (Figure 1 Supporting information Table S1)In addition intercellular transfer of rhodamine-dextran (RDsim10 kDa) which due to its high molecular weight cannotpermeate through GJCs was not observed (Figure S2a) Thisresult indicates that intercellular LY transfer ocurred viaGJCsand not through other cell-cell communication pathway suchas cytoplasmic bridges Moreover microglia treated eitherwith 1mM ATP 1 ngmL TNF-120572 1 ngmL IFN-120574 or 1 ngmLIL-1120573 showed only a slight increase in IDC which was notstatistically different from that of control cells (119875 gt 005Supporting information Table S1) However treatment withmixes of thesemolecules during different time periods causeda significant and transient increase in IDC the dye transferdata is expressed as percentage of the corresponding controlcondition (Figures 1(e) and 1(f)) In both cell types treatmentwith 1 ngmL TNF-120572 plus 1 ngmL IFN-120574 (from now andon referred as TNF-120572IFN-120574) increased the IDC reachinga maximum response at around 9 h after treatment (IDC inEOC20 cells 574 plusmn 36 of control rat microglia 552 plusmn 36of control Mean plusmn SEM 119899 = 5) as previously described[23]

We also studied if extracellular ATP affects TNF-120572IFN-120574-induced dye coupling To this end cells were treated withthese cytokines and then exposed to ATP for 2 h In bothcell types treatment with TNF-120572IFN-120574 plus ATP induced atransient increase in IDC which was maximal at around 5 h(EOC20 cells 517 plusmn 94 of control rat microglia 506 plusmn 42of control 119899 = 5) The amplitude and duration (magnitude)of the response was similar to that induced by TNF-120572IFN-120574but occurred 4 h earlier (Figure 1(e))

Since IFN-120574 potentiates TNF-120572-induced dye coupling inantigen presenting cells including dendritic cells microgliaand monocytesmacrophages [23 50 51] we tested whetherATP induces a similar effect on microglia In agreement withthis possibility cells treated with TNF-120572 plus ATP showedmaximal IDCwith similar amplitude (EOC20 cells 529plusmn12of control rat microglia 534plusmn70of control 119899 = 6 Figure 1)to that induced by TNF-120572IFN-120574 plus ATP but occurred15 h earlier (at sim35 h versus 5 h Figures 1(e) and 1(f)) Asmentioned before intercellular transfer of LY was enhancedin primarymicroglia or EOC20 cells treated with TNF-120572 plusATP (Figures 1(c) and 1(d)) However intercellular transfer ofRDwas not observed ruling out the formation of cytoplasmicbridges or vesicular mediated dye transfer in each condition(Figure S2) Microglia treated with IFN-120574 plus ATP did notincrease dye coupling at 35 h (EOC20 cells 167 plusmn 97 of

Mediators of Inflammation 5

Rat microglia

Control

lowast

(a)

EOC20

Control

lowast

(b)

Rat microglia

TNF-120572ATP

lowast

(c)

EOC20

TNF-120572ATP

lowast

(d)

0

200

400

600

800

0 2 4 6 8 10 12

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

Time (h)

TNF-120572ATPTNF-120572IFN-120574ATPTNF-120572IFN-120574

(e)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

EOC20Rat microglia

Time (h) 35 5 9

lowastlowastlowast

TNF-120572

ATP

IFN-120574

+

+

+

+

+

+

+minus

minus

(f)

Figure 1 ATP advances the onset of TNF-120572IFN-120574-induced dye coupling (a)ndash(d) Dye transfer was evaluated 2min after Lucifer yellow (LY)microinjection in a single cell (indicated with an asterisk) Representative pictures of LY transfer in rat microglia (a) (c) or EOC20 cells (b)(d) under control condition or after TNF-120572 plus ATP (TNF-120572ATP) treatment for 35 h as indicated Phase contrasts of each micrograph areshown at the right panels Scale bar 20 120583m (e) Time course of the incidence of dye coupling (IDC) as percentage of IDC in EOC20 cellsunder control conditions (dashed line) or after treatment with TNF-120572 plus ATP (black circles) TNF-120572IFN-120574 plus ATP (white triangles) orTNF-120572IFN-120574 (black diamonds) Each point corresponds to themean of 3 independent experiments (f) Graph showing themaximum valuesof IDC after treatment with TNF-120572 plus ATP for 35 h TNF-120572IFN-120574 plus ATP for 5 h or TNF-120572IFN-120574 for 9 h lowast119875 lt 005 versus controlcondition Each bar represents the mean plusmn SEM 119899 = 6 No significant differences were observed when comparing microglia and EOC20 cellsresponses to different treatment in dye transfer assays Concentrations 1 ngmL TNF-120572 1 mM ATP 1 ngmL IFN-120574

control ratmicroglia 2108plusmn513of control) or other times(2 and 5 h data not shown)

32 The Increase of Gap Junctional Communication Inducedby TNF-120572 Plus ATP Requires an Increase of [Ca2+]

119894via

Activation of P2XReceptors and Is Prevented by IL-6 Eugenınet al (2001) described that dye coupling between microgliatreated for 9 h with TNF-120572IFN-120574 is inhibited by 120573-GAIn EOC20 cells treated with TNF-120572IFN-120574 we observeda similar acute blockade with 120573-GA (data not shown)

6 Mediators of Inflammation

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

TNF-120572ATP120573-GA

IL-6oATP

BAPTA-AM

+minusminusminusminus

++minusminusminus

+

+minus

minusminus

+

+

minusminus

minus

+

+

minusminusminus

lowast

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

EOC20Rat microglia

TNF-120572ATPIL-6

oATP

+minusminus

++minus

+

+minus

lowast

(b)

Figure 2 IL-6 intracellular calcium chelation and P2X inhibition prevent the induction of gap junctional communication promoted byTNF-120572 plus ATP or TNF-120572IFN-120574 (a) Graph showing the effect of acutely applied 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) or pretreatmentwith 10 ngmL interleukin-6 (IL-6) 300120583Moxidized ATP (oATP) or 10120583MBAPTA-AMon the incidence of dye coupling (IDC) of microgliatreated for 35 h with TNF-120572 plus ATP (b) Graph showing the effect of 50 ngmL IL-6 or 300120583M oATP over the IDC of microglia treated for9 h with TNF-120572IFN-120574 Data is expressed as a percentage of IDC under control conditions (dashed line) lowast119875 lt 005 versus control conditionEach bar represents the mean plusmn SEM 119899 = 5 No significant differences were observed when comparing microglia and EOC20 cells responsesto different treatment in dye transfer assays

In addition application of 50 120583M 120573-GA for 5min completelyabolished dye coupling induced by TNF-120572 plus ATP (IDC inEOC20 cells 74 plusmn 44 of control rat microglia 86 plusmn 50 ofcontrol 119899 = 5 Figure 2(a))

Since microglia treated with purinergic agonists releaseIL-6 [52] and this cytokine prevents the increase of dyecoupling induced by TNF-120572IL-1120573 in dendritic cells [50] wedecided to test if IL-6 prevents induction of dye couplingin microglia treated with TNF-120572 plus ATP In cell culturestreated simultaneously with 10 ngmL IL-6 plus TNF-120572 andthen treated with ATP for 35 h the IDC was low (EOC20cells 130 plusmn 83 of control rat microglia 162 plusmn 10 ofcontrol 119899 = 4) similar to the results obtained undercontrol conditions (Figure 2(a)) Similarly the TNF-120572IFN-120574-induced dye coupling was prevented by IL-6 (Figure 2(b))This inhibitory effect was IL-6 concentration-dependent (110 and 50 ngmL data not shown) The maximal effect wasinduced by 50 ngmL IL-6 (EOC20 180 plusmn 23 of control ratmicroglia 159 plusmn 100 of control 119899 = 4 Figure 2(b))

Since microglia express several P2X and P2Y receptors[3] the possible involvement of purinergic receptors in theTNF-120572IFN-120574-induced dye coupling in microglia treatedwith oxidized-ATP (oATP) an inhibitor of P2X receptors[53] was studied Coapplication of 300 120583M oATP preventeddye transfer induced by TNF-120572 plus ATP (IDC in EOC20cells 147plusmn41of control ratmicroglia 159plusmn100of control119899 = 5 Figure 2(a)) or by TNF-120572IFN-120574 (IDC in EOC20172 plusmn 70 of control rat microglia 176 plusmn 40 of control119899 = 5 Figure 2(b)) Moreover cells treated with TNF-120572 plus1mMADP a P2Y agonist [53] for 35 h did not show changes

in dye coupling (IDC in EOC20 cells 168 plusmn 84 of control119899 = 3) suggesting that P2Y receptors are not involved inATP-induced gap junctional communication in microglia

Since activation of P2 receptors promotes a rise in [Ca2+]119894

in microglia [54] we tested if this response was related tothe increase in dye coupling induced by TNF-120572 plus ATPCells were loaded with BAPTA a Ca2+ chelator and thenwashed and the extracellular medium was replaced withconditionedmediumof cultures treated in parallel with TNF-120572 for 90min to maintain the culture conditions as beforeloading with BAPTA In these cells treatment with TNF-120572 plus ATP did not increase dye coupling (IDC in EOC20cells 134 plusmn 51 of control rat microglia 183 plusmn 44 ofcontrol 119899 = 5 Figure 2(a)) In addition we observed thatEOC20 cells treated with TNF-120572 plus ATP present increasedCa2+ signal compared to cells under control conditions(Figure S3a) Interestingly IL-6 prevented this rise in theCa2+signal (Figure S3b) suggesting that IL-6 might regulate thepurinergic signaling in EOC20 cells

33 IL-1120573 Released by Activated Microglia Mediates the TNF-120572IFN-120574-Induced Dye Coupling in EOC20 Cells Since acti-vatedmicroglia release IL-1120573 and its natural antagonist IL-1ra[7 55] we studied possible involvement of these moleculesin the transient increase in dye coupling induced by TNF-120572plus ATP or TNF-120572IFN-120574 Coapplication of 20 ngmL IL-1rasignificantly prevented the increase in IDC induced by TNF-120572 plus ATP (in EOC20 cells 217 plusmn 36 of control 119899 = 4) orTNF-120572IFN-120574 (in EOC20 cells 241 plusmn 53 of control 119899 = 4

Mediators of Inflammation 7

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

+minus +minusIL-1raTNF-120572ATP 35h TNF-120572IFN-120574 9h

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

lowastlowast

TNF-120572IL-1120573120573-GA

IL-6oATP

+

minus

minus

minus

+

+

minus

minus

+

+

minus

minus

+

+

minus

minus

(b)

Figure 3 IL-1120573mediates gap junctional communication induced by pro-inflammatorymolecules (a) Effect of 20 ngmL IL-1 receptor agonist(IL-1ra) over LY transfer in EOC20 cells treated with TNF-120572 plus ATP for 35 h or with TNF-120572IFN-120574 for 9 h

119875 lt 005 between indicatedtreatments Each bar represents the mean plusmn SEM 119899 = 4 (b)The effect of 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) acutely applied or treatmentwith 10 ngmL interleukin-6 (IL-6) or 300120583Moxidized ATP (oATP) over LY transfer in EOC20 cells treated with TNF-120572IL-1120573 for 9 h is alsoshown Each bar represents the mean plusmn SEM 119899 = 4 and corresponds to the percentage of incidence of dye coupling under control conditions(dashed line) lowast119875 lt 005 lowastlowast119875 lt 001 versus control condition

Figure 3(a)) Moreover EOC20 cells showed an increasein IL-1120573 release after TNF-120572 plus ATP or TNF-120572IFN-120574stimulation which was partially prevented by IL-6 (FigureS4) Consistent with the involvement of IL-1120573 in the abovedye coupling response induced by both pro-inflammatorymolecules exogenous application of 1 ngmL IL-1120573 plus TNF-120572 induced a similar response than that elicited by TNF-120572 plus ATP or TNF-120572IFN-120574 (Figure 3(b)) EOC20 cellstreated with TNF-120572IL-1120573 showed a transient increase indye coupling (data not shown) reaching a maximal IDC atsim9 h of treatment (EOC20 cells 560 plusmn 43 of control 119899 =4 Figure 3(b)) The TNF-120572IL-1120573-induced dye coupling wasdrastically reduced by the acute application of 50120583M 120573-GA(IDC in EOC20 cells 192 plusmn 35 of control 119899 = 4) andprevented by 10 ngmL IL-6 (in EOC20 cells 185 plusmn 40 ofcontrol 119899 = 4) or 300 120583M oATP (in EOC20 cells 198 plusmn34 of control 119899 = 4) coapplied with the two cytokines(Figure 3(b)) However treatment with IL-1120573 did not increasedye coupling in EOC20 cells (data not shown)

34 TNF-120572IFN-120574 but Not TNF-120572 Plus ATP Increases PlasmaMembrane Permeability in EOC20 Cells Astrocytes treatedwith TNF-120572IL-1120573 for 24 h [38] and microglia treated withLPS (or TNF-120572) for 24 h showed an increased HC activity[28 35 56ndash58] Using the ethidium (Etd) uptake assay toevaluate the functional state of HCs located at the cellsurface [38 59] we studied if TNF-120572 or ATP affects themembrane permeability of microglia cells In EOC20 cellsEtd uptake evaluated with time-lapse measurements showed

no significant differences after treatment with TNF-120572 plusATP as compared to untreated cells (Figure S5) In controlconditions Etd uptake was partially blocked by 200120583M La3+(after La3+ 45 plusmn 11 of control 119899 = 5) a Cx HC blocker thatdoes not affect Panx HCs [31] and by 10 120583M carbenoxolone(Cbx) (after Cbx 36 plusmn 15 of control 119899 = 5) which atthis concentration inhibits mainly Panx HCs [60] A slightbut not statistically significant increase in Etd uptake wasrecorded after 35 h treatment with TNF-120572 plus ATP (134 plusmn25 of control 119899 = 5) and was inhibited by La3+ (afterLa3+ 47 plusmn 8 of control 119899 = 5) or Cbx (after Cbx 38 plusmn8 of control 119899 = 5) suggesting an upstream cross talkbetween Cx and Panx HCs In addition 10 ngmL IL-6 didnot affect the response induced by TNF-120572 plusATP treatmentfor 35 h (Etd uptake rate 141 plusmn 16 of control 119899 = 5Figure S5b) In contrast after treatment with TNF-120572IFN-120574for 9 h a statistically significant increase in the Etd uptake ratecompared to the control conditionwas detected (Figure 4) InEOC20 cells cultured for 9 h under control conditions the Etduptake rate remained low and was partially blocked by La3+(57 plusmn 17 of control 119899 = 5 Figures 4(a) and 4(e)) or Cbx(34plusmn4of control 119899 = 5 Figure 4(e)) However cells treatedwith TNF-120572IFN-120574 for 9 h showed a prominent increase inEtd uptake (237 plusmn 25 of control 119899 = 5) that was drasticallyreduced by La3+ (51 plusmn 12 of control 119899 = 5 Figures 4(a) and4(e)) or Cbx (76plusmn9of control 119899 = 5 Figure 4(e)) A similarincrease in dye uptake was found after treatment with TNF-120572IL-1120573 for 9 h (Etd uptake rate 197 plusmn 41 of control 119899 = 3)which was also reduced by La3+ (Etd uptake rate 105 plusmn 4

8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

esce

nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

300

400

Ethi

dium

upt

ake r

ate (

o

f con

trol

)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Mediators of Inflammation

were examined with a confocal laser-scanning microscope(Olympus Fluoview FV1000 Tokio Japan)

27 IL-1120573 ELISA The level of IL-1120573 present in the condi-tioned media of EOC20 cells was evaluated with the IL-1120573ELISA ReadySet-Go (e-Bioscience San Diego CA USA)for performing quantitative enzyme linked immunosorbentassays (ELISA) It has a sensitivity of 8 pgmL Standardcurve consisted of twofold serial dilutions of the recombi-nant cytokine In brief a 96-well flat bottom ELISA-plate(MICROLON Greiner Bio-One) was coated with captureantibody in coating buffer overnight at 4∘C The plate waswashed 5 times with PBS-005 Tween-20 in ELx50 Biokita 96-well bioelisa washer and Rhe plate was blocked with200120583L of assay diluent at room temperature for 1 h washedas mentioned and 100 120583L of standard IL1120573 and sampleswere incubated at 4∘C overnight Then the plate was washedand 100 120583L of detection antibody for IL-1120573 was added andincubated at room temperature by 1 h washed 5 timesincubated with 100120583L Avidin-HRP at room temperature for30min washed 7 times added 100120583L substrate solution andstopped the reaction with 50 120583L of 1M H

3PO4 The plate was

read at 450 nm with reference at 570 nm

28 Treatments Microglia were seeded 48 h before dyetransfer dye uptake or immunofluorescence experiments in24-well plastic dish containing 500 120583L of culture mediumFor Western blot experiments cells were seeded in 60mmplastic dishes in 3mL of culture medium After 48 h undercontrol conditions microglia were treated with 1mM ATPor 1 ngmL TNF-120572 IFN-120574 IL-1120573 either alone or mixedCytokines were added simultaneously and ATP was added2 h before measurement and is referred as cytokine(s) plusATP Treatment with 1 10 or 50 ngmL IL-6 20 ngmL IL-1ra 300 120583M oATP 200120583M La3+ 1 500 Cx43(E2) antibodyor 200120583M 10Panx1 was simultaneous to cytokine treat-ment We used 50120583M of 120573-GA for acute GJCs blocking(Figure S1 see Supplementary Materials available online athttpdxdoiorg1011552013216402) To avoid disruption ofcell adhesion with BAPTA the medium was replaced withculture medium of parallel cultures treated at the same timeto maintain the soluble factor released from microglia

29 Statistical Analysis Data are presented as mean plusmn SEMas percentage of the control condition 119899 represents thenumber of independent experiments For statistical analysiseach treatment was compared with its respective controland significance was determined using one-way ANOVAfollowed by Dunnrsquos test comparing all treatments against thecontrol condition To observe differences between microgliaand EOC20 cells responses we used a two-way ANOVA

3 Results

31 The Onset of the Cytokine-Induced Increase in GapJunctional Communication in Cultured Microglia Is Advancedby ATP and Delayed by IFN-120574 Calcium ionophore and pro-inflammatory molecules promote a transient expression of

functional GJCs in microglia [23 24 27] Since extracellularATP TNF-120572 and IFN-120574 play a relevant role in microglialcell responses [3 7 46] and affect the [Ca2+]

119894[47ndash49] we

decided to evaluate if these compounds affect the intercellularcommunication via GJCs in both primary cultures of ratmicroglia and EOC20 cells

After 48 h of subculture under control conditionsmicroglia were treated as indicated in Methods (Figure S1a)Both cell types presented rather homogeneousmorphologicalfeatures (Figures 1(a) and 1(b)) and very low incidence ofLucifer yellow (LY) transfer to neighboring cells (Figures1(a) and 1(b)) Under these conditions the incidence of dyecoupling (IDC) remained low for up to 12 h of culture inboth cell types (Figure 1 Supporting information Table S1)In addition intercellular transfer of rhodamine-dextran (RDsim10 kDa) which due to its high molecular weight cannotpermeate through GJCs was not observed (Figure S2a) Thisresult indicates that intercellular LY transfer ocurred viaGJCsand not through other cell-cell communication pathway suchas cytoplasmic bridges Moreover microglia treated eitherwith 1mM ATP 1 ngmL TNF-120572 1 ngmL IFN-120574 or 1 ngmLIL-1120573 showed only a slight increase in IDC which was notstatistically different from that of control cells (119875 gt 005Supporting information Table S1) However treatment withmixes of thesemolecules during different time periods causeda significant and transient increase in IDC the dye transferdata is expressed as percentage of the corresponding controlcondition (Figures 1(e) and 1(f)) In both cell types treatmentwith 1 ngmL TNF-120572 plus 1 ngmL IFN-120574 (from now andon referred as TNF-120572IFN-120574) increased the IDC reachinga maximum response at around 9 h after treatment (IDC inEOC20 cells 574 plusmn 36 of control rat microglia 552 plusmn 36of control Mean plusmn SEM 119899 = 5) as previously described[23]

We also studied if extracellular ATP affects TNF-120572IFN-120574-induced dye coupling To this end cells were treated withthese cytokines and then exposed to ATP for 2 h In bothcell types treatment with TNF-120572IFN-120574 plus ATP induced atransient increase in IDC which was maximal at around 5 h(EOC20 cells 517 plusmn 94 of control rat microglia 506 plusmn 42of control 119899 = 5) The amplitude and duration (magnitude)of the response was similar to that induced by TNF-120572IFN-120574but occurred 4 h earlier (Figure 1(e))

Since IFN-120574 potentiates TNF-120572-induced dye coupling inantigen presenting cells including dendritic cells microgliaand monocytesmacrophages [23 50 51] we tested whetherATP induces a similar effect on microglia In agreement withthis possibility cells treated with TNF-120572 plus ATP showedmaximal IDCwith similar amplitude (EOC20 cells 529plusmn12of control rat microglia 534plusmn70of control 119899 = 6 Figure 1)to that induced by TNF-120572IFN-120574 plus ATP but occurred15 h earlier (at sim35 h versus 5 h Figures 1(e) and 1(f)) Asmentioned before intercellular transfer of LY was enhancedin primarymicroglia or EOC20 cells treated with TNF-120572 plusATP (Figures 1(c) and 1(d)) However intercellular transfer ofRDwas not observed ruling out the formation of cytoplasmicbridges or vesicular mediated dye transfer in each condition(Figure S2) Microglia treated with IFN-120574 plus ATP did notincrease dye coupling at 35 h (EOC20 cells 167 plusmn 97 of

Mediators of Inflammation 5

Rat microglia

Control

lowast

(a)

EOC20

Control

lowast

(b)

Rat microglia

TNF-120572ATP

lowast

(c)

EOC20

TNF-120572ATP

lowast

(d)

0

200

400

600

800

0 2 4 6 8 10 12

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

Time (h)

TNF-120572ATPTNF-120572IFN-120574ATPTNF-120572IFN-120574

(e)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

EOC20Rat microglia

Time (h) 35 5 9

lowastlowastlowast

TNF-120572

ATP

IFN-120574

+

+

+

+

+

+

+minus

minus

(f)

Figure 1 ATP advances the onset of TNF-120572IFN-120574-induced dye coupling (a)ndash(d) Dye transfer was evaluated 2min after Lucifer yellow (LY)microinjection in a single cell (indicated with an asterisk) Representative pictures of LY transfer in rat microglia (a) (c) or EOC20 cells (b)(d) under control condition or after TNF-120572 plus ATP (TNF-120572ATP) treatment for 35 h as indicated Phase contrasts of each micrograph areshown at the right panels Scale bar 20 120583m (e) Time course of the incidence of dye coupling (IDC) as percentage of IDC in EOC20 cellsunder control conditions (dashed line) or after treatment with TNF-120572 plus ATP (black circles) TNF-120572IFN-120574 plus ATP (white triangles) orTNF-120572IFN-120574 (black diamonds) Each point corresponds to themean of 3 independent experiments (f) Graph showing themaximum valuesof IDC after treatment with TNF-120572 plus ATP for 35 h TNF-120572IFN-120574 plus ATP for 5 h or TNF-120572IFN-120574 for 9 h lowast119875 lt 005 versus controlcondition Each bar represents the mean plusmn SEM 119899 = 6 No significant differences were observed when comparing microglia and EOC20 cellsresponses to different treatment in dye transfer assays Concentrations 1 ngmL TNF-120572 1 mM ATP 1 ngmL IFN-120574

control ratmicroglia 2108plusmn513of control) or other times(2 and 5 h data not shown)

32 The Increase of Gap Junctional Communication Inducedby TNF-120572 Plus ATP Requires an Increase of [Ca2+]

119894via

Activation of P2XReceptors and Is Prevented by IL-6 Eugenınet al (2001) described that dye coupling between microgliatreated for 9 h with TNF-120572IFN-120574 is inhibited by 120573-GAIn EOC20 cells treated with TNF-120572IFN-120574 we observeda similar acute blockade with 120573-GA (data not shown)

6 Mediators of Inflammation

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

TNF-120572ATP120573-GA

IL-6oATP

BAPTA-AM

+minusminusminusminus

++minusminusminus

+

+minus

minusminus

+

+

minusminus

minus

+

+

minusminusminus

lowast

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

EOC20Rat microglia

TNF-120572ATPIL-6

oATP

+minusminus

++minus

+

+minus

lowast

(b)

Figure 2 IL-6 intracellular calcium chelation and P2X inhibition prevent the induction of gap junctional communication promoted byTNF-120572 plus ATP or TNF-120572IFN-120574 (a) Graph showing the effect of acutely applied 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) or pretreatmentwith 10 ngmL interleukin-6 (IL-6) 300120583Moxidized ATP (oATP) or 10120583MBAPTA-AMon the incidence of dye coupling (IDC) of microgliatreated for 35 h with TNF-120572 plus ATP (b) Graph showing the effect of 50 ngmL IL-6 or 300120583M oATP over the IDC of microglia treated for9 h with TNF-120572IFN-120574 Data is expressed as a percentage of IDC under control conditions (dashed line) lowast119875 lt 005 versus control conditionEach bar represents the mean plusmn SEM 119899 = 5 No significant differences were observed when comparing microglia and EOC20 cells responsesto different treatment in dye transfer assays

In addition application of 50 120583M 120573-GA for 5min completelyabolished dye coupling induced by TNF-120572 plus ATP (IDC inEOC20 cells 74 plusmn 44 of control rat microglia 86 plusmn 50 ofcontrol 119899 = 5 Figure 2(a))

Since microglia treated with purinergic agonists releaseIL-6 [52] and this cytokine prevents the increase of dyecoupling induced by TNF-120572IL-1120573 in dendritic cells [50] wedecided to test if IL-6 prevents induction of dye couplingin microglia treated with TNF-120572 plus ATP In cell culturestreated simultaneously with 10 ngmL IL-6 plus TNF-120572 andthen treated with ATP for 35 h the IDC was low (EOC20cells 130 plusmn 83 of control rat microglia 162 plusmn 10 ofcontrol 119899 = 4) similar to the results obtained undercontrol conditions (Figure 2(a)) Similarly the TNF-120572IFN-120574-induced dye coupling was prevented by IL-6 (Figure 2(b))This inhibitory effect was IL-6 concentration-dependent (110 and 50 ngmL data not shown) The maximal effect wasinduced by 50 ngmL IL-6 (EOC20 180 plusmn 23 of control ratmicroglia 159 plusmn 100 of control 119899 = 4 Figure 2(b))

Since microglia express several P2X and P2Y receptors[3] the possible involvement of purinergic receptors in theTNF-120572IFN-120574-induced dye coupling in microglia treatedwith oxidized-ATP (oATP) an inhibitor of P2X receptors[53] was studied Coapplication of 300 120583M oATP preventeddye transfer induced by TNF-120572 plus ATP (IDC in EOC20cells 147plusmn41of control ratmicroglia 159plusmn100of control119899 = 5 Figure 2(a)) or by TNF-120572IFN-120574 (IDC in EOC20172 plusmn 70 of control rat microglia 176 plusmn 40 of control119899 = 5 Figure 2(b)) Moreover cells treated with TNF-120572 plus1mMADP a P2Y agonist [53] for 35 h did not show changes

in dye coupling (IDC in EOC20 cells 168 plusmn 84 of control119899 = 3) suggesting that P2Y receptors are not involved inATP-induced gap junctional communication in microglia

Since activation of P2 receptors promotes a rise in [Ca2+]119894

in microglia [54] we tested if this response was related tothe increase in dye coupling induced by TNF-120572 plus ATPCells were loaded with BAPTA a Ca2+ chelator and thenwashed and the extracellular medium was replaced withconditionedmediumof cultures treated in parallel with TNF-120572 for 90min to maintain the culture conditions as beforeloading with BAPTA In these cells treatment with TNF-120572 plus ATP did not increase dye coupling (IDC in EOC20cells 134 plusmn 51 of control rat microglia 183 plusmn 44 ofcontrol 119899 = 5 Figure 2(a)) In addition we observed thatEOC20 cells treated with TNF-120572 plus ATP present increasedCa2+ signal compared to cells under control conditions(Figure S3a) Interestingly IL-6 prevented this rise in theCa2+signal (Figure S3b) suggesting that IL-6 might regulate thepurinergic signaling in EOC20 cells

33 IL-1120573 Released by Activated Microglia Mediates the TNF-120572IFN-120574-Induced Dye Coupling in EOC20 Cells Since acti-vatedmicroglia release IL-1120573 and its natural antagonist IL-1ra[7 55] we studied possible involvement of these moleculesin the transient increase in dye coupling induced by TNF-120572plus ATP or TNF-120572IFN-120574 Coapplication of 20 ngmL IL-1rasignificantly prevented the increase in IDC induced by TNF-120572 plus ATP (in EOC20 cells 217 plusmn 36 of control 119899 = 4) orTNF-120572IFN-120574 (in EOC20 cells 241 plusmn 53 of control 119899 = 4

Mediators of Inflammation 7

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

+minus +minusIL-1raTNF-120572ATP 35h TNF-120572IFN-120574 9h

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

lowastlowast

TNF-120572IL-1120573120573-GA

IL-6oATP

+

minus

minus

minus

+

+

minus

minus

+

+

minus

minus

+

+

minus

minus

(b)

Figure 3 IL-1120573mediates gap junctional communication induced by pro-inflammatorymolecules (a) Effect of 20 ngmL IL-1 receptor agonist(IL-1ra) over LY transfer in EOC20 cells treated with TNF-120572 plus ATP for 35 h or with TNF-120572IFN-120574 for 9 h

119875 lt 005 between indicatedtreatments Each bar represents the mean plusmn SEM 119899 = 4 (b)The effect of 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) acutely applied or treatmentwith 10 ngmL interleukin-6 (IL-6) or 300120583Moxidized ATP (oATP) over LY transfer in EOC20 cells treated with TNF-120572IL-1120573 for 9 h is alsoshown Each bar represents the mean plusmn SEM 119899 = 4 and corresponds to the percentage of incidence of dye coupling under control conditions(dashed line) lowast119875 lt 005 lowastlowast119875 lt 001 versus control condition

Figure 3(a)) Moreover EOC20 cells showed an increasein IL-1120573 release after TNF-120572 plus ATP or TNF-120572IFN-120574stimulation which was partially prevented by IL-6 (FigureS4) Consistent with the involvement of IL-1120573 in the abovedye coupling response induced by both pro-inflammatorymolecules exogenous application of 1 ngmL IL-1120573 plus TNF-120572 induced a similar response than that elicited by TNF-120572 plus ATP or TNF-120572IFN-120574 (Figure 3(b)) EOC20 cellstreated with TNF-120572IL-1120573 showed a transient increase indye coupling (data not shown) reaching a maximal IDC atsim9 h of treatment (EOC20 cells 560 plusmn 43 of control 119899 =4 Figure 3(b)) The TNF-120572IL-1120573-induced dye coupling wasdrastically reduced by the acute application of 50120583M 120573-GA(IDC in EOC20 cells 192 plusmn 35 of control 119899 = 4) andprevented by 10 ngmL IL-6 (in EOC20 cells 185 plusmn 40 ofcontrol 119899 = 4) or 300 120583M oATP (in EOC20 cells 198 plusmn34 of control 119899 = 4) coapplied with the two cytokines(Figure 3(b)) However treatment with IL-1120573 did not increasedye coupling in EOC20 cells (data not shown)

34 TNF-120572IFN-120574 but Not TNF-120572 Plus ATP Increases PlasmaMembrane Permeability in EOC20 Cells Astrocytes treatedwith TNF-120572IL-1120573 for 24 h [38] and microglia treated withLPS (or TNF-120572) for 24 h showed an increased HC activity[28 35 56ndash58] Using the ethidium (Etd) uptake assay toevaluate the functional state of HCs located at the cellsurface [38 59] we studied if TNF-120572 or ATP affects themembrane permeability of microglia cells In EOC20 cellsEtd uptake evaluated with time-lapse measurements showed

no significant differences after treatment with TNF-120572 plusATP as compared to untreated cells (Figure S5) In controlconditions Etd uptake was partially blocked by 200120583M La3+(after La3+ 45 plusmn 11 of control 119899 = 5) a Cx HC blocker thatdoes not affect Panx HCs [31] and by 10 120583M carbenoxolone(Cbx) (after Cbx 36 plusmn 15 of control 119899 = 5) which atthis concentration inhibits mainly Panx HCs [60] A slightbut not statistically significant increase in Etd uptake wasrecorded after 35 h treatment with TNF-120572 plus ATP (134 plusmn25 of control 119899 = 5) and was inhibited by La3+ (afterLa3+ 47 plusmn 8 of control 119899 = 5) or Cbx (after Cbx 38 plusmn8 of control 119899 = 5) suggesting an upstream cross talkbetween Cx and Panx HCs In addition 10 ngmL IL-6 didnot affect the response induced by TNF-120572 plusATP treatmentfor 35 h (Etd uptake rate 141 plusmn 16 of control 119899 = 5Figure S5b) In contrast after treatment with TNF-120572IFN-120574for 9 h a statistically significant increase in the Etd uptake ratecompared to the control conditionwas detected (Figure 4) InEOC20 cells cultured for 9 h under control conditions the Etduptake rate remained low and was partially blocked by La3+(57 plusmn 17 of control 119899 = 5 Figures 4(a) and 4(e)) or Cbx(34plusmn4of control 119899 = 5 Figure 4(e)) However cells treatedwith TNF-120572IFN-120574 for 9 h showed a prominent increase inEtd uptake (237 plusmn 25 of control 119899 = 5) that was drasticallyreduced by La3+ (51 plusmn 12 of control 119899 = 5 Figures 4(a) and4(e)) or Cbx (76plusmn9of control 119899 = 5 Figure 4(e)) A similarincrease in dye uptake was found after treatment with TNF-120572IL-1120573 for 9 h (Etd uptake rate 197 plusmn 41 of control 119899 = 3)which was also reduced by La3+ (Etd uptake rate 105 plusmn 4

8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

esce

nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

300

400

Ethi

dium

upt

ake r

ate (

o

f con

trol

)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 5

Rat microglia

Control

lowast

(a)

EOC20

Control

lowast

(b)

Rat microglia

TNF-120572ATP

lowast

(c)

EOC20

TNF-120572ATP

lowast

(d)

0

200

400

600

800

0 2 4 6 8 10 12

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

Time (h)

TNF-120572ATPTNF-120572IFN-120574ATPTNF-120572IFN-120574

(e)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

EOC20Rat microglia

Time (h) 35 5 9

lowastlowastlowast

TNF-120572

ATP

IFN-120574

+

+

+

+

+

+

+minus

minus

(f)

Figure 1 ATP advances the onset of TNF-120572IFN-120574-induced dye coupling (a)ndash(d) Dye transfer was evaluated 2min after Lucifer yellow (LY)microinjection in a single cell (indicated with an asterisk) Representative pictures of LY transfer in rat microglia (a) (c) or EOC20 cells (b)(d) under control condition or after TNF-120572 plus ATP (TNF-120572ATP) treatment for 35 h as indicated Phase contrasts of each micrograph areshown at the right panels Scale bar 20 120583m (e) Time course of the incidence of dye coupling (IDC) as percentage of IDC in EOC20 cellsunder control conditions (dashed line) or after treatment with TNF-120572 plus ATP (black circles) TNF-120572IFN-120574 plus ATP (white triangles) orTNF-120572IFN-120574 (black diamonds) Each point corresponds to themean of 3 independent experiments (f) Graph showing themaximum valuesof IDC after treatment with TNF-120572 plus ATP for 35 h TNF-120572IFN-120574 plus ATP for 5 h or TNF-120572IFN-120574 for 9 h lowast119875 lt 005 versus controlcondition Each bar represents the mean plusmn SEM 119899 = 6 No significant differences were observed when comparing microglia and EOC20 cellsresponses to different treatment in dye transfer assays Concentrations 1 ngmL TNF-120572 1 mM ATP 1 ngmL IFN-120574

control ratmicroglia 2108plusmn513of control) or other times(2 and 5 h data not shown)

32 The Increase of Gap Junctional Communication Inducedby TNF-120572 Plus ATP Requires an Increase of [Ca2+]

119894via

Activation of P2XReceptors and Is Prevented by IL-6 Eugenınet al (2001) described that dye coupling between microgliatreated for 9 h with TNF-120572IFN-120574 is inhibited by 120573-GAIn EOC20 cells treated with TNF-120572IFN-120574 we observeda similar acute blockade with 120573-GA (data not shown)

6 Mediators of Inflammation

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

TNF-120572ATP120573-GA

IL-6oATP

BAPTA-AM

+minusminusminusminus

++minusminusminus

+

+minus

minusminus

+

+

minusminus

minus

+

+

minusminusminus

lowast

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

EOC20Rat microglia

TNF-120572ATPIL-6

oATP

+minusminus

++minus

+

+minus

lowast

(b)

Figure 2 IL-6 intracellular calcium chelation and P2X inhibition prevent the induction of gap junctional communication promoted byTNF-120572 plus ATP or TNF-120572IFN-120574 (a) Graph showing the effect of acutely applied 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) or pretreatmentwith 10 ngmL interleukin-6 (IL-6) 300120583Moxidized ATP (oATP) or 10120583MBAPTA-AMon the incidence of dye coupling (IDC) of microgliatreated for 35 h with TNF-120572 plus ATP (b) Graph showing the effect of 50 ngmL IL-6 or 300120583M oATP over the IDC of microglia treated for9 h with TNF-120572IFN-120574 Data is expressed as a percentage of IDC under control conditions (dashed line) lowast119875 lt 005 versus control conditionEach bar represents the mean plusmn SEM 119899 = 5 No significant differences were observed when comparing microglia and EOC20 cells responsesto different treatment in dye transfer assays

In addition application of 50 120583M 120573-GA for 5min completelyabolished dye coupling induced by TNF-120572 plus ATP (IDC inEOC20 cells 74 plusmn 44 of control rat microglia 86 plusmn 50 ofcontrol 119899 = 5 Figure 2(a))

Since microglia treated with purinergic agonists releaseIL-6 [52] and this cytokine prevents the increase of dyecoupling induced by TNF-120572IL-1120573 in dendritic cells [50] wedecided to test if IL-6 prevents induction of dye couplingin microglia treated with TNF-120572 plus ATP In cell culturestreated simultaneously with 10 ngmL IL-6 plus TNF-120572 andthen treated with ATP for 35 h the IDC was low (EOC20cells 130 plusmn 83 of control rat microglia 162 plusmn 10 ofcontrol 119899 = 4) similar to the results obtained undercontrol conditions (Figure 2(a)) Similarly the TNF-120572IFN-120574-induced dye coupling was prevented by IL-6 (Figure 2(b))This inhibitory effect was IL-6 concentration-dependent (110 and 50 ngmL data not shown) The maximal effect wasinduced by 50 ngmL IL-6 (EOC20 180 plusmn 23 of control ratmicroglia 159 plusmn 100 of control 119899 = 4 Figure 2(b))

Since microglia express several P2X and P2Y receptors[3] the possible involvement of purinergic receptors in theTNF-120572IFN-120574-induced dye coupling in microglia treatedwith oxidized-ATP (oATP) an inhibitor of P2X receptors[53] was studied Coapplication of 300 120583M oATP preventeddye transfer induced by TNF-120572 plus ATP (IDC in EOC20cells 147plusmn41of control ratmicroglia 159plusmn100of control119899 = 5 Figure 2(a)) or by TNF-120572IFN-120574 (IDC in EOC20172 plusmn 70 of control rat microglia 176 plusmn 40 of control119899 = 5 Figure 2(b)) Moreover cells treated with TNF-120572 plus1mMADP a P2Y agonist [53] for 35 h did not show changes

in dye coupling (IDC in EOC20 cells 168 plusmn 84 of control119899 = 3) suggesting that P2Y receptors are not involved inATP-induced gap junctional communication in microglia

Since activation of P2 receptors promotes a rise in [Ca2+]119894

in microglia [54] we tested if this response was related tothe increase in dye coupling induced by TNF-120572 plus ATPCells were loaded with BAPTA a Ca2+ chelator and thenwashed and the extracellular medium was replaced withconditionedmediumof cultures treated in parallel with TNF-120572 for 90min to maintain the culture conditions as beforeloading with BAPTA In these cells treatment with TNF-120572 plus ATP did not increase dye coupling (IDC in EOC20cells 134 plusmn 51 of control rat microglia 183 plusmn 44 ofcontrol 119899 = 5 Figure 2(a)) In addition we observed thatEOC20 cells treated with TNF-120572 plus ATP present increasedCa2+ signal compared to cells under control conditions(Figure S3a) Interestingly IL-6 prevented this rise in theCa2+signal (Figure S3b) suggesting that IL-6 might regulate thepurinergic signaling in EOC20 cells

33 IL-1120573 Released by Activated Microglia Mediates the TNF-120572IFN-120574-Induced Dye Coupling in EOC20 Cells Since acti-vatedmicroglia release IL-1120573 and its natural antagonist IL-1ra[7 55] we studied possible involvement of these moleculesin the transient increase in dye coupling induced by TNF-120572plus ATP or TNF-120572IFN-120574 Coapplication of 20 ngmL IL-1rasignificantly prevented the increase in IDC induced by TNF-120572 plus ATP (in EOC20 cells 217 plusmn 36 of control 119899 = 4) orTNF-120572IFN-120574 (in EOC20 cells 241 plusmn 53 of control 119899 = 4

Mediators of Inflammation 7

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

+minus +minusIL-1raTNF-120572ATP 35h TNF-120572IFN-120574 9h

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

lowastlowast

TNF-120572IL-1120573120573-GA

IL-6oATP

+

minus

minus

minus

+

+

minus

minus

+

+

minus

minus

+

+

minus

minus

(b)

Figure 3 IL-1120573mediates gap junctional communication induced by pro-inflammatorymolecules (a) Effect of 20 ngmL IL-1 receptor agonist(IL-1ra) over LY transfer in EOC20 cells treated with TNF-120572 plus ATP for 35 h or with TNF-120572IFN-120574 for 9 h

119875 lt 005 between indicatedtreatments Each bar represents the mean plusmn SEM 119899 = 4 (b)The effect of 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) acutely applied or treatmentwith 10 ngmL interleukin-6 (IL-6) or 300120583Moxidized ATP (oATP) over LY transfer in EOC20 cells treated with TNF-120572IL-1120573 for 9 h is alsoshown Each bar represents the mean plusmn SEM 119899 = 4 and corresponds to the percentage of incidence of dye coupling under control conditions(dashed line) lowast119875 lt 005 lowastlowast119875 lt 001 versus control condition

Figure 3(a)) Moreover EOC20 cells showed an increasein IL-1120573 release after TNF-120572 plus ATP or TNF-120572IFN-120574stimulation which was partially prevented by IL-6 (FigureS4) Consistent with the involvement of IL-1120573 in the abovedye coupling response induced by both pro-inflammatorymolecules exogenous application of 1 ngmL IL-1120573 plus TNF-120572 induced a similar response than that elicited by TNF-120572 plus ATP or TNF-120572IFN-120574 (Figure 3(b)) EOC20 cellstreated with TNF-120572IL-1120573 showed a transient increase indye coupling (data not shown) reaching a maximal IDC atsim9 h of treatment (EOC20 cells 560 plusmn 43 of control 119899 =4 Figure 3(b)) The TNF-120572IL-1120573-induced dye coupling wasdrastically reduced by the acute application of 50120583M 120573-GA(IDC in EOC20 cells 192 plusmn 35 of control 119899 = 4) andprevented by 10 ngmL IL-6 (in EOC20 cells 185 plusmn 40 ofcontrol 119899 = 4) or 300 120583M oATP (in EOC20 cells 198 plusmn34 of control 119899 = 4) coapplied with the two cytokines(Figure 3(b)) However treatment with IL-1120573 did not increasedye coupling in EOC20 cells (data not shown)

34 TNF-120572IFN-120574 but Not TNF-120572 Plus ATP Increases PlasmaMembrane Permeability in EOC20 Cells Astrocytes treatedwith TNF-120572IL-1120573 for 24 h [38] and microglia treated withLPS (or TNF-120572) for 24 h showed an increased HC activity[28 35 56ndash58] Using the ethidium (Etd) uptake assay toevaluate the functional state of HCs located at the cellsurface [38 59] we studied if TNF-120572 or ATP affects themembrane permeability of microglia cells In EOC20 cellsEtd uptake evaluated with time-lapse measurements showed

no significant differences after treatment with TNF-120572 plusATP as compared to untreated cells (Figure S5) In controlconditions Etd uptake was partially blocked by 200120583M La3+(after La3+ 45 plusmn 11 of control 119899 = 5) a Cx HC blocker thatdoes not affect Panx HCs [31] and by 10 120583M carbenoxolone(Cbx) (after Cbx 36 plusmn 15 of control 119899 = 5) which atthis concentration inhibits mainly Panx HCs [60] A slightbut not statistically significant increase in Etd uptake wasrecorded after 35 h treatment with TNF-120572 plus ATP (134 plusmn25 of control 119899 = 5) and was inhibited by La3+ (afterLa3+ 47 plusmn 8 of control 119899 = 5) or Cbx (after Cbx 38 plusmn8 of control 119899 = 5) suggesting an upstream cross talkbetween Cx and Panx HCs In addition 10 ngmL IL-6 didnot affect the response induced by TNF-120572 plusATP treatmentfor 35 h (Etd uptake rate 141 plusmn 16 of control 119899 = 5Figure S5b) In contrast after treatment with TNF-120572IFN-120574for 9 h a statistically significant increase in the Etd uptake ratecompared to the control conditionwas detected (Figure 4) InEOC20 cells cultured for 9 h under control conditions the Etduptake rate remained low and was partially blocked by La3+(57 plusmn 17 of control 119899 = 5 Figures 4(a) and 4(e)) or Cbx(34plusmn4of control 119899 = 5 Figure 4(e)) However cells treatedwith TNF-120572IFN-120574 for 9 h showed a prominent increase inEtd uptake (237 plusmn 25 of control 119899 = 5) that was drasticallyreduced by La3+ (51 plusmn 12 of control 119899 = 5 Figures 4(a) and4(e)) or Cbx (76plusmn9of control 119899 = 5 Figure 4(e)) A similarincrease in dye uptake was found after treatment with TNF-120572IL-1120573 for 9 h (Etd uptake rate 197 plusmn 41 of control 119899 = 3)which was also reduced by La3+ (Etd uptake rate 105 plusmn 4

8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

esce

nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

300

400

Ethi

dium

upt

ake r

ate (

o

f con

trol

)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Mediators of Inflammation

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

TNF-120572ATP120573-GA

IL-6oATP

BAPTA-AM

+minusminusminusminus

++minusminusminus

+

+minus

minusminus

+

+

minusminus

minus

+

+

minusminusminus

lowast

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

EOC20Rat microglia

TNF-120572ATPIL-6

oATP

+minusminus

++minus

+

+minus

lowast

(b)

Figure 2 IL-6 intracellular calcium chelation and P2X inhibition prevent the induction of gap junctional communication promoted byTNF-120572 plus ATP or TNF-120572IFN-120574 (a) Graph showing the effect of acutely applied 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) or pretreatmentwith 10 ngmL interleukin-6 (IL-6) 300120583Moxidized ATP (oATP) or 10120583MBAPTA-AMon the incidence of dye coupling (IDC) of microgliatreated for 35 h with TNF-120572 plus ATP (b) Graph showing the effect of 50 ngmL IL-6 or 300120583M oATP over the IDC of microglia treated for9 h with TNF-120572IFN-120574 Data is expressed as a percentage of IDC under control conditions (dashed line) lowast119875 lt 005 versus control conditionEach bar represents the mean plusmn SEM 119899 = 5 No significant differences were observed when comparing microglia and EOC20 cells responsesto different treatment in dye transfer assays

In addition application of 50 120583M 120573-GA for 5min completelyabolished dye coupling induced by TNF-120572 plus ATP (IDC inEOC20 cells 74 plusmn 44 of control rat microglia 86 plusmn 50 ofcontrol 119899 = 5 Figure 2(a))

Since microglia treated with purinergic agonists releaseIL-6 [52] and this cytokine prevents the increase of dyecoupling induced by TNF-120572IL-1120573 in dendritic cells [50] wedecided to test if IL-6 prevents induction of dye couplingin microglia treated with TNF-120572 plus ATP In cell culturestreated simultaneously with 10 ngmL IL-6 plus TNF-120572 andthen treated with ATP for 35 h the IDC was low (EOC20cells 130 plusmn 83 of control rat microglia 162 plusmn 10 ofcontrol 119899 = 4) similar to the results obtained undercontrol conditions (Figure 2(a)) Similarly the TNF-120572IFN-120574-induced dye coupling was prevented by IL-6 (Figure 2(b))This inhibitory effect was IL-6 concentration-dependent (110 and 50 ngmL data not shown) The maximal effect wasinduced by 50 ngmL IL-6 (EOC20 180 plusmn 23 of control ratmicroglia 159 plusmn 100 of control 119899 = 4 Figure 2(b))

Since microglia express several P2X and P2Y receptors[3] the possible involvement of purinergic receptors in theTNF-120572IFN-120574-induced dye coupling in microglia treatedwith oxidized-ATP (oATP) an inhibitor of P2X receptors[53] was studied Coapplication of 300 120583M oATP preventeddye transfer induced by TNF-120572 plus ATP (IDC in EOC20cells 147plusmn41of control ratmicroglia 159plusmn100of control119899 = 5 Figure 2(a)) or by TNF-120572IFN-120574 (IDC in EOC20172 plusmn 70 of control rat microglia 176 plusmn 40 of control119899 = 5 Figure 2(b)) Moreover cells treated with TNF-120572 plus1mMADP a P2Y agonist [53] for 35 h did not show changes

in dye coupling (IDC in EOC20 cells 168 plusmn 84 of control119899 = 3) suggesting that P2Y receptors are not involved inATP-induced gap junctional communication in microglia

Since activation of P2 receptors promotes a rise in [Ca2+]119894

in microglia [54] we tested if this response was related tothe increase in dye coupling induced by TNF-120572 plus ATPCells were loaded with BAPTA a Ca2+ chelator and thenwashed and the extracellular medium was replaced withconditionedmediumof cultures treated in parallel with TNF-120572 for 90min to maintain the culture conditions as beforeloading with BAPTA In these cells treatment with TNF-120572 plus ATP did not increase dye coupling (IDC in EOC20cells 134 plusmn 51 of control rat microglia 183 plusmn 44 ofcontrol 119899 = 5 Figure 2(a)) In addition we observed thatEOC20 cells treated with TNF-120572 plus ATP present increasedCa2+ signal compared to cells under control conditions(Figure S3a) Interestingly IL-6 prevented this rise in theCa2+signal (Figure S3b) suggesting that IL-6 might regulate thepurinergic signaling in EOC20 cells

33 IL-1120573 Released by Activated Microglia Mediates the TNF-120572IFN-120574-Induced Dye Coupling in EOC20 Cells Since acti-vatedmicroglia release IL-1120573 and its natural antagonist IL-1ra[7 55] we studied possible involvement of these moleculesin the transient increase in dye coupling induced by TNF-120572plus ATP or TNF-120572IFN-120574 Coapplication of 20 ngmL IL-1rasignificantly prevented the increase in IDC induced by TNF-120572 plus ATP (in EOC20 cells 217 plusmn 36 of control 119899 = 4) orTNF-120572IFN-120574 (in EOC20 cells 241 plusmn 53 of control 119899 = 4

Mediators of Inflammation 7

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

+minus +minusIL-1raTNF-120572ATP 35h TNF-120572IFN-120574 9h

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

lowastlowast

TNF-120572IL-1120573120573-GA

IL-6oATP

+

minus

minus

minus

+

+

minus

minus

+

+

minus

minus

+

+

minus

minus

(b)

Figure 3 IL-1120573mediates gap junctional communication induced by pro-inflammatorymolecules (a) Effect of 20 ngmL IL-1 receptor agonist(IL-1ra) over LY transfer in EOC20 cells treated with TNF-120572 plus ATP for 35 h or with TNF-120572IFN-120574 for 9 h

119875 lt 005 between indicatedtreatments Each bar represents the mean plusmn SEM 119899 = 4 (b)The effect of 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) acutely applied or treatmentwith 10 ngmL interleukin-6 (IL-6) or 300120583Moxidized ATP (oATP) over LY transfer in EOC20 cells treated with TNF-120572IL-1120573 for 9 h is alsoshown Each bar represents the mean plusmn SEM 119899 = 4 and corresponds to the percentage of incidence of dye coupling under control conditions(dashed line) lowast119875 lt 005 lowastlowast119875 lt 001 versus control condition

Figure 3(a)) Moreover EOC20 cells showed an increasein IL-1120573 release after TNF-120572 plus ATP or TNF-120572IFN-120574stimulation which was partially prevented by IL-6 (FigureS4) Consistent with the involvement of IL-1120573 in the abovedye coupling response induced by both pro-inflammatorymolecules exogenous application of 1 ngmL IL-1120573 plus TNF-120572 induced a similar response than that elicited by TNF-120572 plus ATP or TNF-120572IFN-120574 (Figure 3(b)) EOC20 cellstreated with TNF-120572IL-1120573 showed a transient increase indye coupling (data not shown) reaching a maximal IDC atsim9 h of treatment (EOC20 cells 560 plusmn 43 of control 119899 =4 Figure 3(b)) The TNF-120572IL-1120573-induced dye coupling wasdrastically reduced by the acute application of 50120583M 120573-GA(IDC in EOC20 cells 192 plusmn 35 of control 119899 = 4) andprevented by 10 ngmL IL-6 (in EOC20 cells 185 plusmn 40 ofcontrol 119899 = 4) or 300 120583M oATP (in EOC20 cells 198 plusmn34 of control 119899 = 4) coapplied with the two cytokines(Figure 3(b)) However treatment with IL-1120573 did not increasedye coupling in EOC20 cells (data not shown)

34 TNF-120572IFN-120574 but Not TNF-120572 Plus ATP Increases PlasmaMembrane Permeability in EOC20 Cells Astrocytes treatedwith TNF-120572IL-1120573 for 24 h [38] and microglia treated withLPS (or TNF-120572) for 24 h showed an increased HC activity[28 35 56ndash58] Using the ethidium (Etd) uptake assay toevaluate the functional state of HCs located at the cellsurface [38 59] we studied if TNF-120572 or ATP affects themembrane permeability of microglia cells In EOC20 cellsEtd uptake evaluated with time-lapse measurements showed

no significant differences after treatment with TNF-120572 plusATP as compared to untreated cells (Figure S5) In controlconditions Etd uptake was partially blocked by 200120583M La3+(after La3+ 45 plusmn 11 of control 119899 = 5) a Cx HC blocker thatdoes not affect Panx HCs [31] and by 10 120583M carbenoxolone(Cbx) (after Cbx 36 plusmn 15 of control 119899 = 5) which atthis concentration inhibits mainly Panx HCs [60] A slightbut not statistically significant increase in Etd uptake wasrecorded after 35 h treatment with TNF-120572 plus ATP (134 plusmn25 of control 119899 = 5) and was inhibited by La3+ (afterLa3+ 47 plusmn 8 of control 119899 = 5) or Cbx (after Cbx 38 plusmn8 of control 119899 = 5) suggesting an upstream cross talkbetween Cx and Panx HCs In addition 10 ngmL IL-6 didnot affect the response induced by TNF-120572 plusATP treatmentfor 35 h (Etd uptake rate 141 plusmn 16 of control 119899 = 5Figure S5b) In contrast after treatment with TNF-120572IFN-120574for 9 h a statistically significant increase in the Etd uptake ratecompared to the control conditionwas detected (Figure 4) InEOC20 cells cultured for 9 h under control conditions the Etduptake rate remained low and was partially blocked by La3+(57 plusmn 17 of control 119899 = 5 Figures 4(a) and 4(e)) or Cbx(34plusmn4of control 119899 = 5 Figure 4(e)) However cells treatedwith TNF-120572IFN-120574 for 9 h showed a prominent increase inEtd uptake (237 plusmn 25 of control 119899 = 5) that was drasticallyreduced by La3+ (51 plusmn 12 of control 119899 = 5 Figures 4(a) and4(e)) or Cbx (76plusmn9of control 119899 = 5 Figure 4(e)) A similarincrease in dye uptake was found after treatment with TNF-120572IL-1120573 for 9 h (Etd uptake rate 197 plusmn 41 of control 119899 = 3)which was also reduced by La3+ (Etd uptake rate 105 plusmn 4

8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

esce

nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

300

400

Ethi

dium

upt

ake r

ate (

o

f con

trol

)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 7

0

200

400

600

800In

cide

nce o

f dye

coup

ling

( o

f con

trol)

+minus +minusIL-1raTNF-120572ATP 35h TNF-120572IFN-120574 9h

(a)

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontro

l)

lowastlowast

TNF-120572IL-1120573120573-GA

IL-6oATP

+

minus

minus

minus

+

+

minus

minus

+

+

minus

minus

+

+

minus

minus

(b)

Figure 3 IL-1120573mediates gap junctional communication induced by pro-inflammatorymolecules (a) Effect of 20 ngmL IL-1 receptor agonist(IL-1ra) over LY transfer in EOC20 cells treated with TNF-120572 plus ATP for 35 h or with TNF-120572IFN-120574 for 9 h

119875 lt 005 between indicatedtreatments Each bar represents the mean plusmn SEM 119899 = 4 (b)The effect of 50 120583M 18-120573-glycyrrhetinic acid (120573-GA) acutely applied or treatmentwith 10 ngmL interleukin-6 (IL-6) or 300120583Moxidized ATP (oATP) over LY transfer in EOC20 cells treated with TNF-120572IL-1120573 for 9 h is alsoshown Each bar represents the mean plusmn SEM 119899 = 4 and corresponds to the percentage of incidence of dye coupling under control conditions(dashed line) lowast119875 lt 005 lowastlowast119875 lt 001 versus control condition

Figure 3(a)) Moreover EOC20 cells showed an increasein IL-1120573 release after TNF-120572 plus ATP or TNF-120572IFN-120574stimulation which was partially prevented by IL-6 (FigureS4) Consistent with the involvement of IL-1120573 in the abovedye coupling response induced by both pro-inflammatorymolecules exogenous application of 1 ngmL IL-1120573 plus TNF-120572 induced a similar response than that elicited by TNF-120572 plus ATP or TNF-120572IFN-120574 (Figure 3(b)) EOC20 cellstreated with TNF-120572IL-1120573 showed a transient increase indye coupling (data not shown) reaching a maximal IDC atsim9 h of treatment (EOC20 cells 560 plusmn 43 of control 119899 =4 Figure 3(b)) The TNF-120572IL-1120573-induced dye coupling wasdrastically reduced by the acute application of 50120583M 120573-GA(IDC in EOC20 cells 192 plusmn 35 of control 119899 = 4) andprevented by 10 ngmL IL-6 (in EOC20 cells 185 plusmn 40 ofcontrol 119899 = 4) or 300 120583M oATP (in EOC20 cells 198 plusmn34 of control 119899 = 4) coapplied with the two cytokines(Figure 3(b)) However treatment with IL-1120573 did not increasedye coupling in EOC20 cells (data not shown)

34 TNF-120572IFN-120574 but Not TNF-120572 Plus ATP Increases PlasmaMembrane Permeability in EOC20 Cells Astrocytes treatedwith TNF-120572IL-1120573 for 24 h [38] and microglia treated withLPS (or TNF-120572) for 24 h showed an increased HC activity[28 35 56ndash58] Using the ethidium (Etd) uptake assay toevaluate the functional state of HCs located at the cellsurface [38 59] we studied if TNF-120572 or ATP affects themembrane permeability of microglia cells In EOC20 cellsEtd uptake evaluated with time-lapse measurements showed

no significant differences after treatment with TNF-120572 plusATP as compared to untreated cells (Figure S5) In controlconditions Etd uptake was partially blocked by 200120583M La3+(after La3+ 45 plusmn 11 of control 119899 = 5) a Cx HC blocker thatdoes not affect Panx HCs [31] and by 10 120583M carbenoxolone(Cbx) (after Cbx 36 plusmn 15 of control 119899 = 5) which atthis concentration inhibits mainly Panx HCs [60] A slightbut not statistically significant increase in Etd uptake wasrecorded after 35 h treatment with TNF-120572 plus ATP (134 plusmn25 of control 119899 = 5) and was inhibited by La3+ (afterLa3+ 47 plusmn 8 of control 119899 = 5) or Cbx (after Cbx 38 plusmn8 of control 119899 = 5) suggesting an upstream cross talkbetween Cx and Panx HCs In addition 10 ngmL IL-6 didnot affect the response induced by TNF-120572 plusATP treatmentfor 35 h (Etd uptake rate 141 plusmn 16 of control 119899 = 5Figure S5b) In contrast after treatment with TNF-120572IFN-120574for 9 h a statistically significant increase in the Etd uptake ratecompared to the control conditionwas detected (Figure 4) InEOC20 cells cultured for 9 h under control conditions the Etduptake rate remained low and was partially blocked by La3+(57 plusmn 17 of control 119899 = 5 Figures 4(a) and 4(e)) or Cbx(34plusmn4of control 119899 = 5 Figure 4(e)) However cells treatedwith TNF-120572IFN-120574 for 9 h showed a prominent increase inEtd uptake (237 plusmn 25 of control 119899 = 5) that was drasticallyreduced by La3+ (51 plusmn 12 of control 119899 = 5 Figures 4(a) and4(e)) or Cbx (76plusmn9of control 119899 = 5 Figure 4(e)) A similarincrease in dye uptake was found after treatment with TNF-120572IL-1120573 for 9 h (Etd uptake rate 197 plusmn 41 of control 119899 = 3)which was also reduced by La3+ (Etd uptake rate 105 plusmn 4

8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

esce

nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

300

400

Ethi

dium

upt

ake r

ate (

o

f con

trol

)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

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8 Mediators of Inflammation

0

4

8

12

0 5 10 15 20

Etd

fuor

esce

nce (

au)

Time (min)

(a)

(b)

(c)

(d)

(e)

Control

0

100

200

300

400

Ethi

dium

upt

ake r

ate (

o

f con

trol

)

La3+

ControlTNF-120572IFN-120574IL-6TNF-120572IFN-120574

TNF-120572IFN-120574 9h

La3+

Cbx

IL-6

+

+

minus

minus

minus

+

minus

+

minus

+

+

+

+minus

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

TNF-120572IFN-120574

IL-6TNF-120572IFN-120574

lowast

Figure 4 TNF-120572IFN-120574 induces membrane permeabilization in EOC20 cells (a) Time-lapse measurements of ethidium (Etd) uptake inEOC20 cells under control conditions (white squares) or after treatment with TNF-120572IFN-120574 (black diamonds) or IL-6TNF-120572IFN-120574 (graytriangles) for 9 h Each value represents the mean plusmn SEM of 30 cells After 10min of basal uptake 200 120583M La3+ was applied to the bath(b)ndash(d) Representative fluorescence micrographs of Etd uptake after 10min of Etd uptake under indicated treatments previous to La3+application Scale bar 100 120583m (e) Graph showing the acute effect of 200120583MLa3+ 10120583Mcarbenoxolone (Cbx) or pretreatment with 50 ngmLof interleukin-6 (IL-6) Etd uptake rate expressed as percentage of control conditions (dashed line) in EOC20 cells treated with TNF-120572IFN-120574for 9 h Each bar corresponds to the mean plusmn SEM 119899 = 5 lowast119875 lt 005 versus control condition

of control 119899 = 3) Moreover coapplication of 50 ngmL IL-6 with TNF-120572IFN-120574 prevented the Etd uptake rate increasein cells treated just with TNF-120572IFN-120574 (96 plusmn 67 of control119899 = 5 Figure 4(e)) In the latter conditions the Etd uptakerate was slightly reduced by La3+ (48 plusmn 8 of control 119899 = 5)

35 Extracellular ATP Increases the PlasmaMembrane Perme-ability in EOC20 Cells Extracellular ATP in the millimolarrange induces membrane permeabilization in many cell

types including microglia [61 62] Similarly ATP permeabi-lizes macrophages in a Panx1-dependent way [31] We testedthe effect of 2mM ATP on Etd uptake in EOC20 cells aspreviously observed in macrophages and described by others[31 63] A rapid increase in Etd uptake rate (expressed as of control) was induced by the acute application of 2mMATP (529 plusmn 84 of basal uptake 119899 = 5) to cells cultured for35 h under control conditions (Figures 5(a) and 5(b)) Thisresponse was drastically blocked by 10 120583MCbx (218plusmn 81 ofbasal uptake 119899 = 5 Figure 5(a)) as well as by 50 120583M 120573-GA

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 9

0

15

30

45

60

0 5 10 15 20

Etd

fluor

esce

nce (

au)

Time (min)

Control

10min5min0min

10120583M Cbx

2mM ATP

(a)

0

300

600

900

Ethi

dium

upt

ake r

ate (

o

f bas

al u

ptak

e)

lowastlowastlowastlowastlowastlowast

ATP (2mM)Cbx

120573-GAControlTNF-120572IFN-120574 9hIL-6TNF-120572IFN-120574 9h

+

minus

minus

+

+

minus

+

+

minus

(b)

Figure 5 Extracellular ATP increases the cell membrane permeability in EOC20 cells (a) Fluorescent views of Etd uptake of EOC20 cellscultured under control conditions before (0min) or after application of 2mMATP (5 and 10min) Scale bar 25 120583mTime-lapsemeasurementAfter 5min of basal uptake 2mMATPwas added to extracellular solutionAt 15min of recording 10 120583Mof carbenoxolone (Cbx) aHCblockerwas added to the bath Black lines denote the slope at different times of Etd uptake Data represent the mean plusmn SEM of 30 cells in each of 5independent experiments (b) Graph showing the effect of acute application of extracellular ATP in EOC20 cells under control conditions orafter treatment with TNF-120572IFN-120574 or with 50 ngmL IL-6 plus TNF-120572IFN-120574 for 9 h The effect of acute blockade with 10120583M carbenoxolone(Cbx) or 50120583M 18-120573-glycyrrhetinic acid (120573-GA) is also shown Data was normalized to basal uptake in each condition (dashed line) andrepresents the mean plusmn SEM lowastlowastlowast119875 lt 0001 versus control condition

a Cx and Panx HC blocker (128 plusmn 47 of basal uptake 119899 = 5Figure 5) In cell cultures treated with TNF-120572 plus ATP for35 h acute treatment with ATP did not induce a statisticallysignificant increase in Etd uptake (173 plusmn 17 of basal uptake119899 = 5 Figure S6a) and was blocked by Cbx (85 plusmn 16 ofbasal uptake 119899 = 5) or 120573-GA (102 plusmn 63 of basal uptake119899 = 5 Figure S6b) Similarly cells treated with 10 ngmL IL-6TNF-120572 plus ATP showed a small increase in Etd uptakerate after acute application of 2mM ATP (196 plusmn 28 of basaluptake 119899 = 5 Figure S6b)This response was blocked by Cbx(85plusmn28 of basal uptake 119899 = 5) or 120573-GA (102plusmn63 of basaluptake 119899 = 5 Figure S6b)

Moreover EOC20 cells cultured for 9 h under controlconditions showed a rapid increase of Etd uptake in responseto 2mM ATP (500 plusmn 58 of basal uptake 119899 = 5) which wascompletely blocked by Cbx (136plusmn53 of basal uptake 119899 = 5)or 120573-GA (178 plusmn 28 of basal uptake 119899 = 5 Figure 5(b))EOC20 cells treated with TNF-120572IFN-120574 for 9 h exhibited asignificant increase in Etd uptake rate after ATP treatment(433 plusmn 107 of basal uptake 119899 = 5) which was blocked byCbx (186 plusmn 47 of basal uptake 119899 = 5) or 120573-GA (118 plusmn 8of basal uptake 119899 = 5) In contrast in EOC20 cells treatedfor 9 h with 50 ngmL IL-6 plus TNF-120572IFN-120574 ATP did not

increase Etd uptake (161 plusmn 11 of basal uptake 119899 = 5) andneither Cbx (104 plusmn 17 of basal uptake 119899 = 5) nor 120573-GA(141 plusmn 7 of basal uptake 119899 = 5 Figure 5(b)) affected it

In addition cultures treated for 9 h with TNF-120572IL-1120573showed increased Etd uptake rate afterATP application (510plusmn58 of basal uptake 119899 = 5 Figure S7a) which was partiallyblocked by Cbx (229plusmn32of basal uptake 119899 = 5 Figure S7a)or 120573-GA (282plusmn35 of basal uptake 119899 = 5) Interestingly theATP-induced increase in Etd uptake was almost completelyabsent in cells pretreated with 10 ngmL IL-6 plus TNF-120572IL-1120573 (243 plusmn 56 of basal uptake 119899 = 5 Figure S7a) and theactivity present was blocked by 10 120583MCbx (210plusmn71of basaluptake 119899 = 5) or 120573-GA (175 plusmn 49 of basal uptake 119899 = 5Figure S7a)

36 Blockade of Hemichannels Reduces the TNF-120572IFN-120574-Induced Dye Coupling Open HCs allow the release ofmolecules such as ATP and glutamate [35 56ndash58 64] anduptake of smallmolecules such as glucose [38] In addition inother cellular systems functional Cx46HCs stimulate forma-tion of GJCs [65] Thus we studied the possible contributionof increased HC activity on dye coupling induced by pro-inflammatorymolecules in cells incubated with HC blockers

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Mediators of Inflammation

0

200

400

600

800

Inci

denc

e of d

ye co

uplin

g (

of c

ontr

ol)

lowastlowast

lowast

TNF-120572ATP 35h TNF-120572IFN-120574 9hLa3+

Cx43(E2)

10Panx1

+

minus

minus

+

minus

minus +

minus

minus

+

minus

minus

+

minus

minus +

minus

minus

Figure 6 Blockade ofHCs prevents upregulation ofmicroglial gap junctional communication induced byTNF-120572IFN-120574 but not that inducedby TNF-120572 plus ATP Effect on the incidence of dye coupling (IDC) in EOC20 cells treated with TNF-120572 plus ATP for 35 h or TNF-120572IFN-120574for 9 h in presence of HC blockers (200 120583M La3+ 1 500 Cx43(E2) antibody 200 120583M 10Panx1) Data is expressed as percentage of IDC undercontrol conditions (dashed line) and corresponds to the mean plusmn SEM 119899 = 4 lowast119875 lt 005 versus control condition

Treatment with 200120583M La3+ prevented the TNF-120572IFN-120574-induced dye coupling recorded as IDC (134plusmn45 of control119899 = 4 Figure 6) A similar inhibitory effect was induced bythe application of 1 500 Cx43(E2) antibody (117 plusmn 41 ofcontrol 119899 = 4) a specific Cx43 HC blocker [66] or 200120583M10Panx1 (IDC in EOC20 cells 109 plusmn 55 of control 119899 =4 Figure 6) However neither irrelevant IgG nor scramble10Panx1 peptide prevented the TNF-120572IFN-120574-induced dyecoupling (data not shown) On the other hand treatmentwith La3+ (484 plusmn 34 of control 119899 = 4) Cx43(E2) antibody(5408 plusmn 30 of control 119899 = 4) or 10Panx1 (474 plusmn 43 ofcontrol 119899 = 4) did not change the dye coupling induced byTNF-120572 plus ATP (Figure 6)

37 Pro-Inflammatory Molecules Regulate Cx43 and Panx1Levels and Distribution in Microglia Cx32 Cx36 and Cx43have been detected in cultured microglia [23 24 26ndash28]However Cx43 seems to be the main contributor involvedin cytokine-induced gap junctional communication becausemicroglia from Cx43deldel mice do not express functionalGJCs in response to TNF-120572IFN-120574 [23] In addition expres-sion of Panx1 by microglia has been reported recently [35]Thus the distribution and levels of Cx43 and Panx1 duringtreatments that affect GJC and HC activity were evaluated byimmunofluorescence and Western blot analyses

Under control conditions rat microglia presented lowand heterogeneous Cx43 and Panx1 reactivity (Figure 7(a))After treatment with TNF-120572 plus ATP (35 h) or TNF-120572IFN-120574 (9 h) Cx43 and Panx1 reactivity were higher than incontrol conditions (Figure 7(a)) However treatment with

IL-6 (10 ngmL)TNF-120572 plus ATP or IL-6 (50 ngmL)TNF-120572IFN-120574 did not affect the reactivity of Cx43 and Panx1(Figure 7(a)) Moreover in cultures treated with IL-6 plusTNF-120572ATP a redistribution of Cx43 and Panx1 wasobserved these proteins were segregated providing a ldquocellpolarizationrdquo appearance whichwas quantified (Figure 7(b))Under control conditions rat microglia exhibited little or nosegregation (polarized 19 plusmn 6 119899 = 5) although somecells showed more Cx43 or Panx1 reactivity Segregation ofthese proteins was not significantly affected by TNF-120572 plusATP for 35 h (polarized 8 plusmn 4 119899 = 5) but the numberof cells with segregation was increased by the simultaneoustreatment with IL-6 and TNF-120572 plus ATP (polarized 61plusmn1119899 = 5) However treatmentwith TNF-120572IFN-120574 for 9 h did notaffect the resting distribution (polarized 21 plusmn 6 119899 = 5) andremained unchanged in cells simultaneously treated with IL-6TNF-120572IFN-120574 (polarized 15 plusmn 4 119899 = 5) Similar resultswere found in EOC20 cells treated with TNF-120572IL-1120573 for 9 h(Figure S7b)

Protein levels were evaluated in EOC20 cells by Westernblot analyses Total levels of Cx43 and Panx1 increased aftertreatments with TNF-120572 plus ATP TNF-120572IFN-120574 or TNF-120572IL-1120573 which caused the maximal effect on gap junctionalcommunication (Figure 7(c)) Only the increase in total Cx43levels was prevented by IL-6 in the same conditions thatprevented the induction of dye coupling Even when IL-6prevented the increase in total Panx1 levels after treatmentwith TNF-120572IFN-120574 or TNF-120572IL-1120573 coapplication of IL-6failed to prevent the increase observed after TNF-120572 plus ATPtreatment (Figure 7(c))

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 11

(a)

(b)

(c)

0

20

40

60

80

100

Cell

s (

)

NonpolarizedPolarized

0

05

1

15

0

05

1

15

2

Cx43 Panx1

1 2 3 4 5 6 C 1 2 3 4 5 6 C

Cx43 Panx1 MergeIL

-6IL

-6C

ontro

l

TNF-120572

ATP

TNF-120572

IFN

-120574

TNF-120572ATPIL-6

IFN-120574

+

+

minus

minusminus

minus

minus

minus

+

++

minus

+

+

+

minus

+

+

minusminus

Ratio

Pan

x1

120573-a

ctin

(au

)

Ratio

Cx4

3120573

-act

in (a

u)

P1P2

120573-act

Figure 7 Pro-inflammatory treatments upregulate Cx43 and Panx1 protein levels in microglia (a) Confocal images show immunoreactivityfor Cx43 (red) and Panx1 (green) in primary rat microglia under control conditions of after treatment with TNF-120572 plus ATP for 35 h or withTNF-120572IFN-120574 for 9 h in absence or presence of IL-6 (10 or 50 ngmL respectively) Arrows show microglia with segregation of Cx43 andPanx1 Scale bar 10 120583m (b) Quantification of nonpolarized (black bars) versus polarized (dashed white bars) rat microglia under controlconditions or after treatments shown in (a) Data are expressed as a percentage of the total number of cells per field 119899 = 5 (up to 100 cells perfield) lowast119875 lt 005 versus control condition (c) Representative Western blots from 3 independent experiments showing total protein levels ofCx43 and Panx1 Cell lysates were obtained from EOC20 cells under control conditions (lane C) or after the following treatments TNF-120572 plusATP (lane 1) IL-6TNF-120572 plus ATP (lane 2) TNF-120572IFN-120574 (lane 3) IL-6TNF-120572IFN-120574 (lane 4) TNF-120572IL-1120573 (lane 5) and IL-6TNF-120572IL-1120573(lane 6) Quantitation of Cx43 and Panx1 is shown 120573-actin was used as a loading control for densitometric analysis

4 Discussion

In this study we demonstrated that extracellular ATP isrequired and advances the TNF-120572IFN-120574-induced dye cou-pling in cultured microglia in an IL-1120573-dependent mannerTNF-120572IFN-120574 but not TNF-120572 plus ATP enhances the basaland ATP-induced membrane permeability mediated by HCsThe increase in dye coupling induced by TNF-120572IFN-120574or TNF-120572 plus ATP was blocked by IL-6 Furthermoreinhibition of HCs prevents the pro-inflammatory molecules-induced upregulation of GJCs

The ATP effects on the TNF-120572IFN-120574-induced dye cou-pling could be explained by activation of P2X receptors viaATP release because theTNF-120572IFN-120574-induced dye couplingwas prevented by oATP a P2X receptor blocker Activationof P2X receptors in microglia rises the [Ca2+]

119894[1] which

is known to induce gap junctional communication betweencultured microglia in a PKC-dependent manner [24] Inagreement with the latter BAPTA loaded microglia did notpresent dye coupling after treatment with TNF-120572 plus ATPThus it is suggested that rises in [Ca2+]

119894together with other

downstream pathways contribute to up-regulate Cx43 levelsand formation of HCs and GJCs as observed in other celltypes [45 67] In HeLa cells expressing Cx43 rises in [Ca2+]

119894

enhance the cell surface levels of Cx43 HCs [45] a responsethat is directly associated to ATP release [68] Thus rises in[Ca2+]

119894might contribute to increase the number ofHCs in the

plasmamembrane ofmicrogliaThe increase in [Ca2+]119894could

be initially mediated by activation of P2X receptors but lateron HCs might also contribute to increase their own activityfavoring the Ca2+ influx because they are permeable to Ca2+[69ndash71]

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Mediators of Inflammation

Low HC activity no GJC communication

(1)

Extracellular ATPuarrPanx1 HCs activity

(4)TNF-120572IFN-120574(3)

TNF-120572ATP(2)

uarrGJCs and HCscommunicationIL-6

(5)

uarrGJCscommunication

P2X receptor

Cx HC Panx1 HC

Cx GJC

Figure 8 Cytokine-induced activation and the effect on gap junctional communication and HCs activity in cultured microglia (1) Underresting condition microglia express P2X receptors Cx43 and Panx1 which have a low activity Furthermore no gap junction channel(GJC) communication is observed (2) After TNF-120572 plus ATP exposition activated microglia exhibit gap junctional communication butnot intercellular communication mediated by hemichannels (HCs) (3) However treatment with TNF-120572IFN-120574 increased both GJC and HCfunctional state (4) Extracellular ATP increases the Panx1 HC activity in both resting or TNF-120572IFN-120574-activated microglia (5) IL-1120573 releasefrom activated microglia favors gap junctional communication (6) IL-6 prevents IL-1120573 release and the increase in GJC and HC functionalstate

The cytokine-dependent induction of gap junctionalcommunication between microglial cells was transient aspreviously observed in dendritic cells and monocytesma-crophages [50 51 72] The transient response might beexplained by the production and release of anti-inflammatorycytokines such as IL-6 IL-10 and TGF-120573 by activatedmicroglia [1] Accordingly IL-6 drastically reduces thecytokine-induced dye coupling between microglia treatedwith TNF-120572 plus ATP or TNF-120572IFN-120574 as it also occursin dendritic cells treated with TNF-120572IL-1120573 [50] SinceIL-6 reduces cell adhesion in breast cancer cells [73] asimilar mechanism might affect the stability of cellularcontacts between microglia impairing gap junctional com-munication In addition IL-6 was found to prevent therise in [Ca2+]

119894 This might explain the inhibition of TNF-

120572 plus ATP because IL-6 did not prevent the increase inPanx1 levels Although IFN-120574 signaling positively regulatespurinergic receptors in microglia [11 74] this might notexplain the increase in dye coupling induced by TNF-120572IFN-120574 because we found that IFN-120574 delayed the appearance ofdye coupling induced by TNF-120572 plus ATP Further studiesare required to unveil the mechanism underlying this cellularresponse

We also found that in addition to TNF-120572IFN-120574 extracel-lular ATP and IL-1120573 also positivelymodulate the formation of

GJCs inmicrogliaThe link between purinergic signaling andIL-1120573 release has been well established in microglia [75] andhere it was corroborated in EOC20 cells using IL-1ra whichprevented IL-1120573 release and establishment of dye couplingupon treatment with TNF-120572 plus ATP or TNF-120572IFN-120574Interestingly pro-inflammatory-like conditions (TNF-120572IL-1120573 or supernatant of microglia pretreated with LPS) increaseHC activity but decrease gap junctional communicationin primary astrocytes cultures [38] However we observedthat TNF-120572IFN-120574 increases both HC and GJC activity inmicroglia indicating that different mechanisms control thefunctional expression of these channels in astrocytes andmicroglia

As shown in this work the activity of microglial Cxand Panx HCs was increased by TNF-120572IFN-120574 InterestinglyPanx1 HCs and several Cx HCs are pathways of ATP releaseto the extracellular space in several cell types includingastrocytes and microglia [25 35 37 76 77] Thereforeenhanced HC opening may control ATP release from acti-vated microglia maintaining a higher [Ca2+]

119894compared with

resting microglia [78] Extracellular ATP could open Panx1HCs which are also activated after TNF-120572IFN-120574 leadingto release of IL-1120573 [31] Because the HC activity remainslow after treatment with TNF-120572 plus ATP even after acuteapplication of ATP we propose that under these conditions

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 13

ATP released bymicroglia viaHCswas not required to induceIL-1120573 release The latter is consistent with the preventionof TNF-120572IFN-120574- but not TNF-120572 plus ATP-induced dyecoupling in EOC20 cells treated with 10Panx1 a Panx1 HCblocker In addition we speculate that after treatment withTNF-120572 plus ATP P2X receptors also contribute in a Panx1HC-independent way as it has been proposed to occurduring microglial proliferation [79] The role of Cx43 HCsin TNF-120572IFN-120574ndashinduced dye coupling was confirmed usingCx43(E2) antibody a specific Cx43 HC blocker However thisconclusion should be taken cautiously because it was recentlyshown that several hours after Cx43(E2) antibody appli-cation gap junctional communication is partially reduced[42]

Under control conditions microglial cells express lowlevels of Cxs [23 24 26ndash28] Accordingly in this study wedetected low levels of Cx43 and also Panx1 However braindamage or cytokine exposure promotesmicroglial activationand under this condition they present elevated levels of Cx43and become coupled through GJCs [23 24 27 28] Here wefound that TNF-120572 in presence of IFN-120574 upregulates Cx43GJCs in microglia as it was previously demonstrated [23 28]In addition and similar to dendritic cells [50] TNF-120572IL-1120573increased Cx43 levels in microglia On the other hand IL-6prevents the formation ofGJCs induced by pro-inflammatorycytokines in dendritic cells [50] Accordingly we found thatIL-6 efficiently prevented the pro-inflammatory molecules-induced increase in GJC and HC activity in microglia Thiseffect could be explained at least in part by prevention ofCx43 and Panx1 upregulation by IL-6 and prevention of IL-1120573release

So far Panx1 has been demonstrated to form GJCs onlyin exogenous expression systems [71] Together with theevidence that microglia from Cx43deldel mice do not expressfunctional GJCs [23] and that Cx43(E2) antibody preventedthe pro-inflammatory-induced dye coupling it is suggestedthat dye coupling induced by TNF-120572 plus ATP or TNF-120572IFN-120574 could be due to Cx43 GJCs To recapitulate wepropose that in presence of extracellular ATP Panx1 HCactivity is enhanced andmicrogliamigrate toward the injuredsite and release cytokines as reported previously [33] ATPcould act in an autocrine and paracrine manner allowing IL-1120573 release and providing a pro-inflammatory microenviron-ment which promotes an early up-regulation of Cx43 andPanx1 favoring the formation of HCs andGJCs in a stimulus-dependent manner (Figure 8) Later on anti-inflammatorycytokines are produced and released to the extracellularmilieu leading to reduction in intercellular communicationmediated by HCs and GJCs similar to that of restingconditions The latter is relevant because downregulationprevents a massive andor prolonged ATPglutamate releasefrommicroglia which in turn can induce neurodegeneration[35 56] Thus understanding the regulation of microglialpurinergic receptors and intercellular communication viaHCs and GJCs might contribute to modulate the timing ofneuroinflammatory responses and led us to the identificationof new therapeutic targets for neurodegenerative diseases[80]

Acknowledgments

This work was partially supported by Grants CONICYT24100062 (to P J Saez) FONDECYT 1111033 FONDEFDO7I1086 and ANILLO ACT-71 (to J C Saez) FONDECYT1090353 (R von Bernhardi) Welch Foundation Grant AQ-1507 (to J X Jiang) All the authors declare no conflict ofinterests The data of this work was presented by Pablo JSaez as partial fulfillment of the requirements to obtain thedegree of Bachelor of Sciences in Biology at the PontificiaUniversidad Catolica de Chile

References

[1] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 pp461ndash553 2011

[2] RMRansohoff andVH Perry ldquoMicroglial physiology uniquestimuli specialized responsesrdquo Annual Review of Immunologyvol 27 pp 119ndash145 2009

[3] K Inoue ldquoPurinergic systems inmicrogliardquoCellular andMolec-ular Life Sciences vol 65 no 19 pp 3074ndash3080 2008

[4] R Von Bernhardi J E Tichauer and J Eugenın ldquoAging-dependent changes of microglial cells and their relevance forneurodegenerative disordersrdquo Journal of Neurochemistry vol112 no 5 pp 1099ndash1114 2010

[5] S W Barger and A S Basile ldquoActivation of microglia bysecreted amyloid precursor protein evokes release of glutamateby cystine exchange and attenuates synaptic functionrdquo Journalof Neurochemistry vol 76 no 3 pp 846ndash854 2001

[6] R De Simone G Levi and F Aloisi ldquoInterferon 120574 geneexpression in rat central nervous system glial cellsrdquo Cytokinevol 10 no 6 pp 418ndash422 1998

[7] U K Hanisch ldquoMicroglia as a source and target of cytokinesrdquoGlia vol 40 no 2 pp 140ndash155 2002

[8] Y K Soo H M Ju G L Hwan U K Seung and B L YongldquoATP released from 120573-amyloid-stimulated microglia inducesreactive oxygen species production in an autocrine fashionrdquoExperimental and Molecular Medicine vol 39 no 6 pp 820ndash827 2007

[9] D Piani and A Fontana ldquoInvolvement of the cystine transportsystem xc- in the macrophage-induced glutamate-dependentcytotoxicity to neuronsrdquo Journal of Immunology vol 152 no 7pp 3578ndash3585 1994

[10] C C Chao S Hu L Ehrlich and P K Peterson ldquoInterleukin-1and tumor necrosis factor-120572 synergistically mediate neurotox-icity involvement of nitric oxide and of N-methyl-D-aspartatereceptorsrdquo Brain Behavior and Immunity vol 9 no 4 pp 355ndash365 1995

[11] F P Gendron M Chalimoniuk J Strosznajder et al ldquoP2X7nucleotide receptor activation enhances IFN120574-induced type IInitric oxide synthase activity in BV-2 microglial cellsrdquo Journalof Neurochemistry vol 87 no 2 pp 344ndash352 2003

[12] R Kuno J Wang J Kawanokuchi H Takeuchi T Mizunoand A Suzumura ldquoAutocrine activation of microglia by tumornecrosis factor-120572rdquo Journal of Neuroimmunology vol 162 no 1-2pp 89ndash96 2005

[13] K Inoue ldquoMicroglial activation by purines and pyrimidinesrdquoGlia vol 40 no 2 pp 156ndash163 2002

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Mediators of Inflammation

[14] F Bianco E Pravettoni A Colombo et al ldquoAstrocyte-derivedATP induces vesicle shedding and IL-1120573 release frommicrogliardquoJournal of Immunology vol 174 no 11 pp 7268ndash7277 2005

[15] C G Schipke C Boucsein C Ohlemeyer F Kirchhoff andH Kettenmann ldquoAstrocyte Ca2+ waves trigger responses inmicroglial cells in brain slicesrdquo The FASEB Journal vol 16 no2 pp 255ndash257 2002

[16] C Verderio and M Matteoli ldquoATP mediates calcium signalingbetween astrocytes and microglial cells modulation by IFN-120574rdquoJournal of Immunology vol 166 no 10 pp 6383ndash6391 2001

[17] J M Sanz P Chiozzi D Ferrari et al ldquoActivation of microgliaby amyloid 120573 requires P2X

7receptor expressionrdquo Journal of

Immunology vol 182 no 7 pp 4378ndash4385 2009[18] S D Skaper P Debetto and P Giusti ldquoThe P2X

7purinergic

receptor from physiology to neurological disordersrdquo FASEBJournal vol 24 no 2 pp 337ndash345 2010

[19] D Ferrari P Chiozzi S Falzoni S Hanau and F Di Vir-gilio ldquoPurinergic modulation of interleukin-1120573 release frommicroglial cells stimulated with bacterial endotoxinrdquo Journal ofExperimental Medicine vol 185 no 3 pp 579ndash582 1997

[20] D R Seo K Y Kim and Y B Lee ldquoInterleukin-10 expressionin lipopolysaccharide-activated microglia is mediated by extra-cellular ATP in an autocrine fashionrdquo NeuroReport vol 15 no7 pp 1157ndash1161 2004

[21] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[22] A Hoffmann O Kann C Ohlemeyer U K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquo Journal of Neuroscience vol 23no 11 pp 4410ndash4419 2003

[23] E A Eugenın D Eckardt M Theis K Willecke M V LBennett and J C Saez ldquoMicroglia at brain stab woundsexpress connexin 43 and in vitro form functional gap junctionsafter treatment with interferon-120574 and tumor necrosis factor-120572rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 7 pp 4190ndash4195 2001

[24] A D Martınez E A Eugenın M C Branes M V Bennettand J C Saez ldquoIdentification of second messengers that induceexpression of functional gap junctions in microglia culturedfrom newborn ratsrdquo Brain Research vol 943 pp 191ndash201 2002

[25] J A Orellana P J Saez K F Shoji et al ldquoModulation of brainhemichannels and gap junction channels by pro-inflammatoryagents and their possible role in neurodegenerationrdquo Antioxi-dants and Redox Signaling vol 11 no 2 pp 369ndash399 2009

[26] K Dobrenis H Y Chang M H Pina-Benabou et al ldquoHumanand mouse microglia express connexin36 and functional gapjunctions are formed between rodent microglia and neuronsrdquoJournal of Neuroscience Research vol 82 no 3 pp 306ndash3152005

[27] S Garg M M Syed and T Kielian ldquoStaphylococcus aureus-derived peptidoglycan induces Cx43 expression and functionalgap junction intercellular communication inmicrogliardquo Journalof Neurochemistry vol 95 no 2 pp 475ndash483 2005

[28] S B Shaikh B Uy A Perera and L F Nicholson ldquoAGEs-RAGEmediated up-regulation of connexin43 in activated humanmicroglial CHME-5 cellsrdquo Neurochemistry International vol60 no 6 pp 640ndash651 2012

[29] Y V Panchin ldquoEvolution of gap junction proteinsmdashthe pan-nexin alternativerdquo Journal of Experimental Biology vol 208 no8 pp 1415ndash1419 2005

[30] S Locovei J Wang and G Dahl ldquoActivation of pannexin 1channels by ATP through P2Y receptors and by cytoplasmiccalciumrdquo FEBS Letters vol 580 no 1 pp 239ndash244 2006

[31] P Pelegrin and A Surprenant ldquoPannexin-1 mediates large poreformation and interleukin-1120573 release by the ATP-gated P2X

7

receptorrdquo EMBO Journal vol 25 no 21 pp 5071ndash5082 2006[32] L Bao S Locovei and G Dahl ldquoPannexin membrane channels

are mechanosensitive conduits for ATPrdquo FEBS Letters vol 572no 1ndash3 pp 65ndash68 2004

[33] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[34] A M Fontainhas M Wang K J Liang et al ldquoMicroglialmorphology and dynamic behavior is regulated by ionotropicglutamatergic and GABAergic neurotransmissionrdquo PLoS ONEvol 6 no 1 article e15973 2011

[35] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoJournal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[36] N Froger J A Orellana C F Calvo et al ldquoInhibition ofcytokine-induced connexin43 hemichannel activity in astro-cytes is neuroprotectiverdquo Molecular and Cellular Neurosciencevol 45 no 1 pp 37ndash46 2010

[37] J A Orellana N Froger P Ezan et al ldquoATP and glutamatereleased via astroglial connexin 43 hemichannels mediate neu-ronal death through activation of pannexin 1 hemichannelsrdquoJournal of Neurochemistry vol 118 pp 826ndash840 2011

[38] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo Journal of Neuroscience vol 27 no50 pp 13781ndash13792 2007

[39] S Buvinic G Almarza M Bustamante et al ldquoATP released byelectrical stimuli elicits calcium transients and gene expressionin skeletal musclerdquo Journal of Biological Chemistry vol 284 no50 pp 34490ndash34505 2009

[40] L A Cea M A Riquelme B A Cisterna et al ldquoConnexin- andpannexin-based channels in normal skeletal muscles and theirpossible role in muscle atrophyrdquo Journal of Membrane Biologyvol 245 pp 423ndash436 2012

[41] M C Branes J E Contreras and J C Saez ldquoActivation ofhuman polymorphonuclear cells induces formation of func-tional gap junctions and expression of connexinsrdquo MedicalScience Monitor vol 8 pp BR313ndashBR323 2002

[42] B Bao J Jiang T Yanase Y Nishi and J R MorganldquoConnexon-mediated cell adhesion drives microtissue self-assemblyrdquo FASEB Journal vol 25 no 1 pp 255ndash264 2011

[43] W S Walker J Gatewood E Olivas D Askew and C EG Havenith ldquoMouse microglial cell lines differing in consti-tutive and interferon-120574-inducible antigen-presenting activitiesfor naive and memory CD4+ and CD8+ T cellsrdquo Journal ofNeuroimmunology vol 63 no 2 pp 163ndash174 1995

[44] M D Sklar A Tereba B D M Chen and W S WalkerldquoTransformation of mouse bone marrow cells by transfectionwith a human oncogene related to c-myc is associated withthe endogenous production of macrophage colony stimulatingfactor 1rdquo Journal of Cellular Physiology vol 125 no 3 pp 403ndash412 1985

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 15

[45] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]

119894responsesrdquo Molecular Biology of the Cell vol

19 no 8 pp 3501ndash3513 2008[46] T Nagano Y Kawasaki A BabaM Takemura and TMatsuda

ldquoUp-regulation of Na+-Ca2+ exchange activity by interferon-120574in cultured ratmicrogliardquo Journal of Neurochemistry vol 90 no4 pp 784ndash791 2004

[47] S Franciosi H B Choi S U Kim and J G McLarnonldquoInterferon-120574 acutely induces calcium influx in humanmicrogliardquo Journal of Neuroscience Research vol 69 no 5 pp607ndash613 2002

[48] T Moller ldquoCalcium signaling in microglial cellsrdquo Glia vol 40no 2 pp 184ndash194 2002

[49] J G McLarnon S Franciosi X Wang J H Bae H B Choiand S U Kim ldquoAcute actions of tumor necrosis factor-120572on intracellular Ca2+ and K+ currents in human microgliardquoNeuroscience vol 104 no 4 pp 1175ndash1184 2001

[50] L A Corvalan R Araya M C Branes et al ldquoInjury of skeletalmuscle and specific cytokines induce the expression of gapjunction channels in mouse dendritic cellsrdquo Journal of CellularPhysiology vol 211 pp 649ndash660 2007

[51] E A EugenınM C Branes JW Berman and J C Saez ldquoTNF-120572 plus IFN-120574 induce connexin43 expression and formation ofgap junctions between human monocytesmacrophages thatenhance physiological responsesrdquo Journal of Immunology vol170 no 3 pp 1320ndash1328 2003

[52] Y Shigemoto-Mogami S Koizumi M Tsuda K Ohsawa SKohsaka and K Inoue ldquoMechanisms underlying extracellularATP-evoked interleukin-6 release in mouse microglial cell lineMG-5rdquo Journal of Neurochemistry vol 78 no 6 pp 1339ndash13492001

[53] R A North ldquoMolecular physiology of P2X receptorsrdquo Physio-logical Reviews vol 82 no 4 pp 1013ndash1067 2002

[54] J G McLarnon ldquoPurinergic mediated changes in Ca2+ mobi-lization and functional responses in microglia effects of lowlevels of ATPrdquo Journal of Neuroscience Research vol 81 no 3pp 349ndash356 2005

[55] F Pousset K Palin D Verrier et al ldquoProduction of interleukin-1 receptor antagonist isoforms by microglia in mixed ratglial cells stimulated by lipopolysacchariderdquo European CytokineNetwork vol 11 no 4 pp 682ndash689 2000

[56] H Takeuchi S Jin J Wang et al ldquoTumor necrosis factor-120572induces neurotoxicity via glutamate release from hemichannelsof activated microglia in an autocrine mannerrdquo Journal ofBiological Chemistry vol 281 no 30 pp 21362ndash21368 2006

[57] H Takeuchi H Mizoguchi Y Doi et al ldquoBlockade of gapjunction hemichannel suppresses disease progression in mousemodels of amyotrophic lateral sclerosis and Alzheimerrsquos dis-easerdquo PLoS ONE vol 6 no 6 article e21108 2011

[58] I Yawata H Takeuchi Y Doi J Liang T Mizuno and ASuzumura ldquoMacrophage-induced neurotoxicity is mediated byglutamate and attenuated by glutaminase inhibitors and gapjunction inhibitorsrdquo Life Sciences vol 82 no 21-22 pp 1111ndash11162008

[59] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[60] R Bruzzone M T Barbe N J Jakob and H MonyerldquoPharmacological properties of homomeric and heteromericpannexin hemichannels expressed in Xenopus oocytesrdquo Journalof Neurochemistry vol 92 no 5 pp 1033ndash1043 2005

[61] D Ferrari M Villalba P Chiozzi S Falzoni P Ricciardi-Castagnoli and F Di Virgilio ldquoMouse microglial cells expressa plasmamembrane pore gated by extracellular ATPrdquo Journal ofImmunology vol 156 no 4 pp 1531ndash1539 1996

[62] M Monif C A Reid K L Powell M L Smart and DA Williams ldquoThe P2X

7receptor drives microglial activation

and proliferation a trophic role for P2X7R porerdquo Journal ofNeuroscience vol 29 no 12 pp 3781ndash3791 2009

[63] R Bartlett J J Yerbury and R Sluyter ldquoP2X7receptor activa-

cion induces reactive oxygen species formation and cell death inmurine EOC13microgliardquoMediators of Inflammation vol 2013Article ID 271813 18 pages 2013

[64] J Kang N Kang D Lovatt et al ldquoConnexin 43 hemichannelsare permeable to ATPrdquo Journal of Neuroscience vol 28 no 18pp 4702ndash4711 2008

[65] D L Beahm and J E Hall ldquoOpening hemichannels innonjunctional membrane stimulates gap junction formationrdquoBiophysical Journal vol 86 no 2 pp 781ndash796 2004

[66] A J Siller-Jackson S Burra S Gu et al ldquoAdaptation ofconnexin 43-hemichannel prostaglandin release to mechanicalloadingrdquo Journal of Biological Chemistry vol 283 no 39 pp26374ndash26382 2008

[67] E Decrock M Vinken M Bol et al ldquoCalcium and connexin-based intercellular communication a deadly catchrdquo Cell Cal-cium vol 50 pp 310ndash321 2011

[68] E De Vuyst N Wang E Decrock et al ldquoCa2+ regulation ofconnexin 43 hemichannels in C6 glioma and glial cellsrdquo CellCalcium vol 46 no 3 pp 176ndash187 2009

[69] H A Sanchez G Mese M Srinivas T W White and VK Verselis ldquoDifferentially altered Ca2+ regulation and Ca2+permeability in Cx26 hemichannels formed by the A40Vand G45E mutations that cause keratitis ichthyosis deafnesssyndromerdquo Journal of General Physiology vol 136 no 1 pp 47ndash62 2010

[70] K A Schalper H A Sanchez S C Lee G A AltenbergM H Nathanson and J C Saez ldquoConnexin 43 hemichannelsmediate the Ca2+ influx induced by extracellular alkalinizationrdquoAmerican Journal of Physiology vol 299 no 6 pp C1504ndashC15152010

[71] F Vanden Abeele G Bidaux D Gordienko et al ldquoFunctionalimplications of calcium permeability of the channel formed bypannexin 1rdquo Journal of Cell Biology vol 174 no 4 pp 535ndash5462006

[72] A Mendoza-Naranjo P J Saez C C Johansson et al ldquoFunc-tional gap junctions facilitate melanoma antigen transfer andcross-presentation between human dendritic cellsrdquo Journal ofImmunology vol 178 no 11 pp 6949ndash6957 2007

[73] K S Asgeirsson K Olafsdottir J G Jonasson and H MOgmundsdottir ldquoThe effects of IL-6 on cell adhesion and E-cadherin expression in breast cancerrdquo Cytokine vol 10 no 9pp 720ndash728 1998

[74] M Tsuda T Masuda J Kitano H Shimoyama H Tozaki-Saitoh and K Inoue ldquoIFN-120574 receptor signaling mediates spinalmicroglia activation driving neuropathic painrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 19 pp 8032ndash8037 2009

[75] T Takenouchi S Sugama Y Iwamaru M Hashimoto and HKitani ldquoModulation of the ATP-lnduced release and processing

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

16 Mediators of Inflammation

of IL-1Β inmicroglial cellsrdquoCritical Reviews in Immunology vol29 no 4 pp 335ndash345 2009

[76] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[77] S Iwabuchi and K Kawahara ldquoFunctional significance of thenegative-feedback regulation of ATP release via pannexin-1hemichannels under ischemic stress in astrocytesrdquoNeurochem-istry International vol 58 no 3 pp 376ndash384 2011

[78] D Sieger C Moritz T Ziegenhals S Prykhozhij and FPeri ldquoLong-range Ca2+ waves transmit brain-damage signals tomicrogliardquo Developmental Cell vol 22 pp 1138ndash1148 2012

[79] C Rigato N Swinnen R Buckinx et al ldquoMicroglia pro-liferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cordinvasionrdquo The Journal of Neuroscience vol 32 pp 11559ndash115732012

[80] R A Quintanilla J A Orellana and R von BernhardildquoUnderstanding risk factors for Alzheimerrsquos disease interplayof neuroinflammation connexin-based communication andoxidative stressrdquo Archives of Medical Research vol 43 pp 632ndash644 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom