Diversified serum IgG response involving non-myelin CNS proteins during experimental autoimmune encephalomyelitis

gy 179 (2006) 53–64www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolo

Diversified serum IgG response involving non-myelin CNS proteinsduring experimental autoimmune encephalomyelitis

Helene Zephir a,b,⁎,1, Lionel Almeras a,1, Mohamed El Behi a, Patricia Dussart a,Jerome de Seze b, Jerome Steibel c, Elizabeth Trifilieff d, Sylvain Dubucquoi a,

Jean-Paul Dessaint a, Patrick Vermersch b, Lionel Prin a, Didier Lefranc a

a Laboratoire d'immunologie EA2686, Faculté de Médecine, 1, Place de Verdun, 59045 Lille Cedex, Franceb Service de Neurologie D, Hôpital Roger Salengro, 59037 Lille Cedex, France

c Institut de physique biologique (UPRES-A ULP-CNRS), Faculté de Médecine, F-67085 Strasbourg, Franced Laboratoire de Chimie Organique des Substances Naturelles, Unité Mixte de Recherche 7509, CNRS, Université Louis Pasteur, Strasbourg, France

Received 9 February 2006; received in revised form 17 May 2006; accepted 19 June 2006

Abstract

We sequentially analyzed the serum IgG response against normal mouse brain during experimental autoimmune encephalomyelitis inSJL/J mice injected with CFA, Bordetella pertussis toxin (BPT) and proteolipid protein 139–151 peptide, compared with mice that receivedCFA and BPT or were uninjected. Dynamic changes were observed from day 0 to day 28 in the 3 groups. Six highly discriminant antigenicbands (κ=0.974) were identified. Three non-myelin proteins were characterized (mitochondrial aconitase hydratase 2, phosphoglyceratemutase 1, brain specific pyruvate deshydrogenase). The IgG response against two of them was less frequent in EAE whereas it was associatedwith multiple sclerosis in our previous work.© 2006 Elsevier B.V. All rights reserved.

Keywords: EAE; Autoimmunity; Neuroimmunology; Multiple sclerosis

1. Introduction

Multiple sclerosis (MS) is a chronic inflammatorydemyelinating disease of the central nervous system(CNS), which is thought to be caused by autoaggressiveimmune responses against various myelin proteins, includ-ing myelin basic protein (MBP), proteolipid protein (PLP)

Abbreviations: BPT, Bordetella pertussis toxin; H, healthy; LDA, lineardiscriminant analysis; MBP, myelin basic protein; MOG, myelin oligoden-drocyte glycoprotein; MS, multiple sclerosis; MW, molecular weight; PLP,proteolipid protein of the myelin; 2-D, two dimensional.⁎ Corresponding author. Laboratoire d'immunologie EA2686, Faculté de

Médecine, 1, Place de Verdun, 59045 Lille Cedex, France. Tel.: +33 3 20 6269 77; fax: +33 3 20 62 68 93.

E-mail address: [email protected] (H. Zephir).1 These authors contributed equally.

0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2006.06.016

and myelin oligodendrocyte glycoprotein (MOG). Myelinprotein reactive T-cell responses appear to participateactively in the progression of pathogenic events (Rajan etal., 1996; Hellings et al., 2002; Bischof et al., 2004; Croxfordet al., 2005; McMahon et al., 2005). However, such specificT-cells are found at similar frequencies in healthy subjectsand MS patients (Lovett-Racke et al., 1998; Viglietta et al.,2004). Thus, other immunological mechanisms must beinvolved in the initiation and progression of the disease(Lassmann and Ransohoff, 2004). Autoantibodies also seemto play a role, sometimes with harmful and sometimes withbeneficial properties (Lyons et al., 1999; Archelos et al.,2000; Ziemssen and Ziemsen, 2005). In MS, the elevatedlevels of intrathecally synthesized IgG in the cerebrospinalfluid (CSF) are an important feature for the diagnosis even iftheir precise role is unknown. In addition, accumulation of Band plasma cells in the CSF (Colombo et al., 2000) and IgG

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http://dx.doi.org/10.1016/j.jneuroim.2006.06.016

Table 1Number of mice at each stage of the protocol

Mice group Day 0 Day 7 Day 14 Day 21 Day 28

H 19 (3 a) 16 (2 b) 14 (2 b) 12 (2 b) 10 (2 c)CFA/BPT 18 18 (3 b) 15 (3 b) 12 (2 b) 10 (3 b)EAE 37 37 (3 b) (7 c) 27 (2 b) (5 c) 20 (3 b) 17 (2 b)a Number of mice sacrificed to obtain normal brain homogenates.b Number of mice sacrificed for neuropathological examinations.c Number of mice that died between days 7 and 14 and between days 14

and 21.

54 H. Zephir et al. / Journal of Neuroimmunology 179 (2006) 53–64

deposition have been described in MS lesions (Genain et al.,1999). The antigenic specificity of antibodies in oligoclonalimmunoglobulin (Ig) bands in the CSF is quite hetero-geneous (Avasarala et al., 2001; Hemmer et al., 2002). InCSF and sera from MS patients, antibody reactivities againstrelevant targets such as myelin proteins have been found, butthey constitute only a small proportion of the oligoclonalbands and their significance is controversial (Berger et al.,2003; Lampasona et al., 2004; Lim et al., 2005). Inexperimental autoimmune encephalomyelitis (EAE), syner-gic interactions between autoimmune T and B cells related todemyelination processes have been noted (Iglesias et al.,2001). Several studies argue for a causative role ofantibodies in EAE models, such as the common marmosetmonkey, where antibodies to MOG are critical for EAEinduction (Genain et al., 1995; Genain and Hauser, 1997).IgG autoreactive response to MOG rather than to PLP andMBP is also found in rodent EAE models (Lassmann et al.,1988; Bourquin et al., 2003; Bischof et al., 2004).

All these data prompt reconsideration of the role of thehumoral response in MS and in EAE, where a variety of Bcell functions have been postulated (Ziemssen and Ziemsen,2005). To avoid a restricted analysis of humoral immunityagainst pre-selected antigenic targets and to define moreprecisely the effect of the humoral response on theprogression of the disease, we previously evaluated theglobal IgG antibody repertoire against human brain antigensin the serum from different forms of MS (Lefranc et al.,2004). Linear discriminant analysis (LDA) enabled us todistinguish the control subjects from the MS patients, andalso to categorize the different clinical forms of MSaccording to their autoreactive profile (Lefranc et al.,2004). The patients with definite MS could be tested onlyonce. The cluster of discriminant antigenic bands thusappears to be a potent hallmark of disease activity at a giventime point but its significance in terms of the disease coursehas yet to be determined. We developed a sequential analysisof the self-reactive IgG antibody response after induction ofEAE in SJL/J mice. As was the case in MS patients, our datarevealed a diversified specific IgG antibody repertoire inEAE, with the identification of discriminant targets related tonon-myelin CNS proteins.

2. Materials and methods

2.1. Mice

Syngeneic female SJL/J (H-2s) mice, 5–6 weeks old,were purchased from Harlan (Harlan France, Gannat,France) and bred under specific-pathogen-free conditions.Mice were randomized for all experiments and wereimmunized at 9–11 weeks of age. Three groups of micewere studied: an EAE group, a CFA group receiving onlyCFA and pertussis toxin without PLP139–151, and a controlgroup of healthy uninjected mice (H group). All animalswere maintained on standard laboratory food and water ad

libitum. Symptomatic mice were given ready access to foodand water. All experimental procedures were approved bythe Experimental Animal Committee of the University ofLille (France).

Each mouse was followed up, clinically scored daily(Greer et al., 2001), and blood sampled at weekly intervalsfor 28 days. Several mice were sacrificed at weekly intervalsfor pathology analyses. Before immunization or sampling(of blood or nervous tissues) the mice were anesthetizedusing ketamine (Imalgène Merial, France) and xylasine(Rompun, Bayer, Germany). The number of mice in each ofthe three groups, at each time-point of the protocol, is shownin Table 1.

2.2. Immunization protocol and humoral IgG response

PLP139–151 (HCLGKWLGHPDKF), used as an encepha-litic antigen, was produced by the laboratory of organicchemistry (E. Trifilief, UMR 7509 CNRS, Strasbourg,France). Peptides were dissolved at a concentration of 5 mg/ml in 0.1 M acetic acid and diluted with distilled water to theappropriate concentration. EAE was induced as previouslydescribed (Greer et al., 2001). Clinical and neuropathologicalsigns and anti-PLP139–151 IgG Ab specificities were onlyobserved following immunization with PLP139–151. Substan-tial weight loss (>10%) preceded the neurological deficits. Thefirst attack (at 13±1 days [mean±S.D.], with a meanmaximum clinical score of 2.7±1.6) was lethal in 32.3% ofmice (Table 1), in agreement with previous reports (McRae etal., 1992). In all the EAE-induced mice examined weobserved dense perivascular encephalitis (by hematoxylin–eosin staining) with demyelination (Luxol fast blue staining)14 days after immunization. Mice in the CFA group alsopresented, 14 days post immunization, minor infiltration ofmononuclear cells but without perivascular encephalitis ordemyelination (Table 1).

The serum IgG level was compared weekly in the threegroups of mice, using an ELISA kit allowing detection of allIgG subclasses (Alpha Diagnostic, San Antonio, CA, USA).From day 7, raised levels of serum IgG were observed in allgroups, but direct binding and competitive ELISA proce-dures as used by Potter and Lees (Potter and Lees, 1988)showed that IgG anti-PLP139–151 Ab specificities appearedonly in the EAE group, as expected (data not shown).

55H. Zephir et al. / Journal of Neuroimmunology 179 (2006) 53–64

2.3. Preparation of tissue homogenates

Brain samples were taken from healthy, uninjected miceand homogenized in a Tris buffer containing 5% SDS toobtain a 10 mg/ml protein solution before sonication(3×30 s) (Laemmli, 1970). The homogenates were thenreduced with 10 mM dithiothreitol (Sigma, St Louis, MI,USA) and boiled for 5 min. Three hundred microliters of thislysate was loaded per well onto a 10–20% gradient

Fig. 1. Healthy mouse brain homogenates as antigenic targets. (A and B) IgG reactivhuman serum (A) and a representative mouse at baseline (B). (C) Comparison ofuninjected). (D) Inter-individual natural mouse IgG antibody against a healthy mcommon protein bands, indicated by similarly shaped arrows.

polyacrylamide gel in parallel with a standard MW marker(Amersham Pharmacia Biotech, Uppsala, Sweden). Mouseheart and liver tissues were prepared in the same way.

2.4. Blotting and analysis procedure

Immunoblotting was performed using a standardized assayas previously described (Lefranc et al., 2004). Briefly, serawere diluted 1:100 for overnight incubation at 4 °C. Mouse

e patterns against brain samples from two healthy mice, tested with a healthymouse self IgG reactive patterns against distinct tissue targets at day 0 (i.e.ouse brain homogenate. Detailed analysis revealed the presence of some


IgG was revealed with a goat polyclonal anti-mouse Fcγhorseradish peroxidase (HRP) conjugated antibody 1:5000(Sigma, St Louis,MI, USA) and human IgGwas revealedwitha polyclonal anti-humanFcγHRP conjugated antibody 1:5000(Sigma, St Louis,MI, USA). Immune profiles were consideredfor analysis when two independent assays had producedidentical patterns. Image analysis was performed using a GS-800 calibrated densitometer (Biorad, Hercules, CA, USA) tolocalize and compare the IgG patterns. The Diversity Databasefingerprinting software package (version 2.2) (Biorad, Her-cules, CA, USA) was used for detection and alignment ofantibody reactivities. Immune profiles from the 12 EAE micethat died as a result of the EAE process (7 died between day 7and day 14 and 5 between day 14 and day 21) were alsoconsidered and analyzed at days 0, 7 and 14.However, they didnot allow a sequential analysis until day 28.

2.5. Bidimensional electrophoresis and MALDI-TOF massspectrometry

Bidimensional electrophoresis procedures were performedusing a standardized protocol (Almeras et al., 2004). As apositive control, the membrane was probed with an antihumanenolase goat Ab (Santa Cruz Biotechnology, Santa Cruz, CA,USA) as this protein is well-characterized on our CCB-stainedgel. Immune profiles were analyzed when two independentassays had produced identical patterns. The two-dimensionalprotein patterns in the gels and autoradiographs were analyzedwith PDQuest software (Bio-Rad). MALDI-TOF massspectrometry was used to obtain mass fingerprinting forproteins using a Voyager DESIR instrument (AppliedBiosystems, Framingham, MA, USA) (Almeras et al., 2004).

2.6. Database search based on peptide mass fingerprintspectra

The obtained peptide mass fingerprint spectra wereanalyzed by searching the National Center for Biotechnol-ogy Information non-redundant protein database with theProFound (http://prowl.rockfeller.edu/cgi-bin/ProFound),version 3.2, and Peptident programs.

2.7. Statistical analysis

Comparisons of the number of antigenic bands in eachgroup of mice and their changes were expressed in binary

Table 2Number of antigenic bands recognized by IgG antibodies on healthy brain tissue

Group Day 0 Day 7 Day 14

H 6.2±3.6 b (n=19 c) 7±3.9 (n=16) 7.3±3.9 (n=1CFA/BPT 5±3.4 (n=18) 4.8±2.9 (n=18) 5.9±2.7 (n=1

EAE 5.9±3.9 (n=37) 5.7±3.9 (n=37) 7.2±3.9 (n=2

a Mean increase in the number of bands between day 0 and day 28.b Number of bands per strip (mean±S.D.) for the mice of each group.c Number of strips analyzed.

mode (0, absence of antigenic band; 1, presence of antigenicband) so as to subject IgG Ab patterns to analysis usingeither the Chi square or Fisher exact test. This approachenabled us to select the most relevant antigens that supportqualitatively different immune recognition, between thethree groups of mice. In a second stage, we used LDA to findthe discriminant antigens between the three groups of mice,as previously described (Marshall and Baron, 2000; Lefrancet al., 2004). The Kappa test was performed according to thefinal score determined using the LDA with regard to thegroup of membership.

3. Results

3.1. Validity of the Western blot approach

Healthy mouse brain lysates were used as a source ofpotentially relevant antigenic targets. To assess the qualityof IgG patterns and to calibrate and define the alignmentof antibody reactivities obtained with each mouse serum,one healthy human serum was used (because of the smallquantity of blood available for each mouse) as a referencein all tests (Fig. 1A). We verified that a given mouseserum always revealed the same pattern of self IgGreactivity whatever the healthy mouse brain tested (Fig.1B). To ensure that the patterns of IgG self response werespecific to brain homogenates, mouse IgG self reactivitywas also tested against mouse heart and liver tissueextracts. Quite different patterns were then obtained (Fig.1C). Natural IgG auto reactivity against healthy mousebrain tissue extracts was evaluated at day 0 for all miceand revealed variable patterns with a few conserved sets ofIgG reactivities (Fig. 1D).

3.2. Sequential analysis of self IgG reactivities

Mapping and alignment of the 282 patterns obtainedduring the 28-day follow-up with all mice sera (Table 1)identified a total of 61 distinct antigenic bands, rangingfrom 13 to 142 kDa, but only 0 to 16 bands per individualstrip (Table 2). Changes in the sequential self IgG patterns,involving the appearance or disappearance of proteinbands, were observed in all 3 groups. A representativeindividual of each group is shown in Fig. 2. When wecompared changes in the number of bands from day 0 today 28 between each group of mice, a similar, progressive

Day 21 Day 28 Day 0–Day 28 a

4) 7.6±4 (n=12) 8.5±3.5 (n=10) 2.3±2.55) 7.2±2.2 (n=12) 7.4±3 (n=10) 2.4±2.37) 7.8±4.2 (n=20) 8.1±3.9 (n=17) 2.1±2

http://prowl.rockfeller.edu/cgiin/ProFound


increase was found in the 3 groups (Table 2). If eachchange in terms of appearance or disappearance of a givenprotein band counts for 1, similar changes were also notedin all groups (5.1±2.7 in H, n=10; 7.9±3.3 in CFA/BPT,n=10; 7.6±2.7, in EAE, n=17; mean±S.D.). However, 44protein bands were affected by such changes in the EAEgroup, more than in the CFA/BPT group (32 bands) andthe H group (31 bands).

Fig. 2. Sequential analysis of serum self IgG reactivities. Sequential Western blotmouse of each experimental group (H, CFA/BPT, and EAE groups) revealed either spoints as indicated.

3.3. Sequential analysis reveals distinct IgG repertoire inthe 3 groups of mice

Of the 61 distinct antigenic bands, 27 bands weredetected as being differentially represented at day 28between the 3 groups of mice (each at p<0.3). Fig. 3Ashows the frequency of each of these selected antigenicbands in each group of mice at day 28. Single antigenic

ting and densitometric profiles of serum IgG reactivities of a representativetable (gray arrows) or changing IgG reactivity (black arrows) at different time

Fig. 3. Identification of discriminant protein bands by self IgG reactivities. (A) Frequencies of 27 protein bands (numbered by decreasing MW) identified in eachgroup of mice (□H group, ▧CFA/BPT group, ■EAE group), selected after Chi square and Fisher analyses (all p<0.3, ✸p<0.2, ✸✸p<0.1) to perform LDA.LDA, coupled with stepwise logistic regression found 6 discriminant antigenic bands, indicated by ( ). (B) Illustrative evolution of densitometric profiles ofserum IgG reactivities in a representative individual of each group of mice. The discriminant autoreactivities, at day 28, are numbered and indicated by blackarrows.


bands with p<0.3 can only indicate a trend, but acombination of antigenic bands clustered by the LDAenabled us to differentiate more accurately the differentmice groups. LDA coupled to stepwise logistic regressionfound 6 discriminant antigenic bands (antigenic bandsnumbered 5, 11, 20, 51 and 54 by decreasing molecularweight [MW]). Antigenic bands 20 and 51 were

respectively identified with ∼20% of sera from the Hgroup and with ∼30% of sera from the CFA/BPT group,as illustrated in Fig. 3A. The evolution of self IgGrepertoires in each group of representative mice, illustratedin Fig. 3B, pointed out the following antigenic bandnumbers: 11 for the H group, 44, and 54 for the CFA/BPTgroup, and 5 for the EAE group. Coefficient values,


assigned by LDA for the 6 discriminant antigenic bandsidentified at day 28 (Fig. 4A), allowed us to calculategraphic coordinates for each mouse and draw a two-axisgraph projection which showed that, at day 28 but notearlier, the three groups of mice were clearly discriminated(Fig. 4B) with a high degree of concordance with theclinical data (κ=0.974). Dead EAE mice provided a shortsequential analysis. They were indistinguishable from theother mice at days 0, 7 and 14. We projected the lastimmune profile of each of these mice (at day 7 or day 14)on the graph projection system at day 28 (Fig. 4B).

Fig. 4. Graphic extrapolation of data obtained in the three groups of mice. (A) Schemand their coefficient given by LDA. (B) Coefficient values associated with the prcoordinates for each mouse. At day 28, the 3 groups of mice were clearly discrCalculated graph projections of the last-obtained immune profile of the 12 dead EAother mice group area at day 28.

3.4. Characterization of the 3 major discriminant antigensusing a serological proteomic approach (SERPA)

Among 282 mice sera, 6 were selected for SERPAbecause their immune reactivity was directed against at leastone of the 6 discriminant antigenic bands (2 sera from theEAE group, 3 sera from the H group, and 1 serum from theCFA/BPT group). The 6 mice sera and 1 MS serum weresubsequently used in 2-D immunoblotting, revealing thepresence of multiple antigenic spots, as illustrated in Fig. 5for 3 mice sera.

atic illustrative Western blot strip showing the 6 discriminant brain antigensesence or absence of each discriminant antigen led us to calculate graphiciminated with a high degree of concordance with clinical data (κ=0.974).E mice subgroup (at day 7 or day 14) illustrated on the graph representing the

Fig. 5. Identification of discriminant brain antigenic spots by 2-D immunoblots. Three selected H mouse sera (A, B, C), 1 CFA/BPT mouse serum (D) and 2 EAEmouse sera (E, F) were successively used as probes to identify discriminant antigenic spots on healthy mouse brain. Q99K10, Q9D051 and Q9DBJ1 wererespectively detected by H mouse sera 38 and 39, H mouse serum 50, CFA/BPT mouse serum 18 and then further characterized by mass spectrometry. Spotsrelated to the antigenic band number 5 (P111) identified by two EAE mouse sera (36 and 43) could unfortunately not be characterized using mass spectrometry.Our reference MS serum, known to recognize antigenic band number 11, reveals similar antigenic spots (B′), to those obtained with H serum 38 (Q99K10). Anti-enolase monoclonal antibody enabled us to identify (D′) spots related to enolase 1 (P17182) used as an internal landmark for spot matching between the CCBstained 2-D electrophoresis and 2-D immunoblot. IEF: isoelectrofocusing.


After the use of enolase 1 as an internal landmark (Fig.5B, and B′) the superimposition of protein antigenic spotsenabled to select the proteins for further in-gel digestion andmatrix-assisted laser desorption ionization-time of flight(MALDI-TOF) analysis. This approach enabled us toidentify discriminant antigens using the ProFound andEXPASy ALDente databases (Table 3). Discriminant anti-genic band number 11 was resolved in 3 spots when revealed

Table 3Discriminant antigenic proteins from healthy mouse brain

Discriminant antigenic proteins from healthy mouse brain

Antigenicbandnumber

Swiss-Prot orTrEMBLreferences

Abbr. name Protein name

11 Q99K10 ACON_Mouse Aconitase 2 mitochondrial44 Q9D051 PDHB_Mouse Pyruvate dehydrogenase brain specific54 Q9DBJ1 PGAM1_Mouse Phosphoglycerate mutase 1

by 2-D immunoblot with the selected mice sera (Fig. 5A).These 3 spots were further characterized as the isoforms ofmouse mitochondrial aconitate hydratase 2 (ACON_Mouse:Q99K10). These 3 spots were also detected using an MSreference serum (Fig. 5A′). The antigenic spot correspond-ing to immune reactive band number 44 (Fig. 5B) wascharacterized as brain-specific pyruvate dehydrogenase(PDHB_Mouse: Q9D051). Two spots related to antigenic

MW Isoelectric point MALDI-MSpeptides

Sequencecoverage(%)

Score

Theor. Observ. Theor. Observ. Matching Total

86,190 90,000 8.4 8.3–8.7 9 21 17 2.2535,160 38,000 5.6 5.5 6 20 26 2.0528,930 30,000 6.7 6.7–7.1 8 30 43 2.32


band number 54 (Fig. 5B) were identified as phosphogly-cerate mutase 1 (PGAM1_Mouse: Q9DBJ1), a brain isoformthat is also recognized by MS sera (Almeras et al., 2004;Lefranc et al., 2004). We failed to characterize antigenicbands numbered 5, 20 and 51. Proteic spots corresponding toantigenic bands numbered 5, 20 and 51 remained unchar-acterized by mass spectrometry despite repeated assays.

4. Discussion

In this longitudinal study we compared serum antibodychanges in 3 groups of SJL/J mice: healthy, CFA/BPT, andEAEmice. Self IgG responses directed against healthymousebrain antigens, formerly considered as sequestered antigens,were evaluated. Six discriminant antigenic molecules wereidentified after a follow-up of 28 days and three of them werecharacterized. Although the role of autoreactive T cells iscrucial, the role of the humoral response system in MS and inits animal model EAE cannot be ignored (Genain et al., 1999;Colombo et al., 2000; Ziemssen and Ziemsen, 2005). Thepresent choice of testing serum IgG is not irrelevant, becausea breakdown of the blood–brain barrier (BBB), an early eventduring inflammation, might give the autoantibodies andcomplement access to the CNS and allow them to initiate oraggravate lesions (Berger et al., 2003; Uccelli et al., 2005). Inaddition, peripheral B cells can enter the CNS and induce anintrathecal antibody response detectable in serum even whenthe BBB is functionally intact (Knopf et al., 1998). At day 0,IgG autoreactivity was found against healthy brain antigens.Natural autoantibodies in normal serum, of both the IgM andIgG isotypes, have already been described against brain andother tissue targets (Ronda et al., 1994; Mouthon et al., 1995;Hurez et al., 1997; Stahl et al., 2000). As expected, patterns ofnatural IgG autoreactivity obtained with individual micediffered when we compared brain and other tissue extracts(Mouthon et al., 1995; Lefranc et al., 2004).

Non-specific interference with the self IgG reactivitiescannot be ruled out, since whole serum was tested (Hurez etal., 1997). Nevertheless, previous studies, also using immu-noblots and LDA, have shown it is possible to characterizemouse strains through an analysis of their homogeneousnatural IgM antibody reactivities (Vasconcellos et al., 1998)and have emphasized MHC control of natural antibodyrepertoires (Paisansinup et al., 2002). Other studies havedemonstrated the significance of heterogenous self IgGpatterns in other neurological disorders, such as Guillain–Barré syndrome (Dziewas et al., 2001). In our approach,studying natural IgG antibody repertoires in the SJL/J strain,only a few conserved sets of protein bands were found whenwe compared all mice at day 0. Inter-individual differences innatural IgG antibody reactivities to brain antigens wereinsufficient to distinguish subgroups among SJL/J mice usingLDA at day 0.

Sequential analysis revealed a progressive elevation ofserum IgG levels in all groups of mice, but a specific anti-PLP IgG antibody response was only found in EAE mice, as

detailed in Materials and methods. Increasing IgG levelswere associated with diversified IgG autoreactivities, inWestern blots. All mice were kept in the same environmentand were subjected to the same procedures of investigationand stress. In this respect, weekly blood sampling can createlymphopenic conditions which can lead to T-cell homeo-static proliferation-induced autoimmunity (King et al., 2004)impacting on B-cells and class-switch recombination. Thesignificance of these dynamic changes, observed in all 3groups of mice, remains to be determined. Natural IgGresponses against healthy brain antigens, at day 0, mightreflect physiological immunosurveillance of the CNS, withprogressive induced disturbances detectable in the CFA/BPTand EAE mice. It is interesting to note that in both the H andCFA/BPT mice a peculiar distortion of the self IgG responsewas found in the absence of clinical signs and with no (in Hmice) or only minor (in CFA/BPT mice) histologicalchanges. Healthy brains with an intact BBB, or leakage ofbrain-derived molecules through an altered BBB induced byCFA and BPT, were associated with detectable changes inserum IgG self-response, but were unrelated to pathogenicevents and/or were related to neuroprotective events vis-à-vis an altered BBB. In contrast, brain damage with markedclinical signs was associated, in EAE mice, with significantchanges in IgG reactivities at day 28 in addition to the PLP-specific immune response. The analysis of a subgroup ofpotentially more severely affected EAE mice, namely thosethat died between days 13 and 16, did not allow us todistinguish them from the others at days 7 or 14. However,when their last immune profile was projected onto the graphat day 28, they appeared to be early grouped mainly on theEAE area. The small number of mice that died between days7 and 14, and between days 14 and 21, makes it difficult todraw any conclusions regarding potential factors that mightpredict the severity of the disease.

The bands detected on healthy brain by serum IgG from Hmice and/or CFA/BPT mice but not detected in EAE micecould reflect: (i) a defect of response against endogenousantigens required for the maintenance of self tolerance andneuroprotection (Garza et al., 2000), (ii) in EAE, the loss ofcirculating IgG antibodies that have bound to altered CNStissue (Genain et al., 1999). Conversely, bands preferentiallyor exclusively detected by serum IgG from EAE mice mightbe linked to normally cryptic/hidden self antigens that induceantibody responses after brain damage (McRae et al., 1995;Vanderlugt and Miller, 1996; Yu et al., 1996a). Thepreparative treatment of healthy brain tissues for proteinextraction could make these antigenic targets available fordetection by Western blot. Previous reports have demon-strated intra- and/or inter-molecular spreading associatedwith the emergence of encephalitogenic T cells (King et al.,2004; Yu et al., 1996a,b). The diversity of antigenic targetsrecognized by self-IgG antibodies in EAE could rather reflectinter-molecular epitope spreading first triggered byPLP139–151 (Disis et al., 2004). Concerning T cells, epitopespreading with new self-reactivities appears to follow a well-


ordered sequencewith predictable patterns in PLP- andMOG-induced EAE in SJL/J mice (McRae et al., 1995; Yu et al.,1996b; Zhang et al., 2004), but this sequential cascade is notfound for IgG antibody responses, as shown here. Likewise, inhumans we have found discriminant stable patterns in MSpatients after 1 year of follow up (Dubucquoi et al., 2002).

To characterize the molecular targets involved, the role ofmyelin proteins was first evaluated. In our EAE protocol, theIgG response against MBP, evaluated using specific mono-clonal antibodies, was not discriminant (data not shown), asalso found in MS (Lefranc et al., 2004). The absence ofdetection of PLP and MOG by Western blots and 2-Dimmunoblots, can be related either to the high hydrophobicityof the protein (PLP), or to denaturing conditions used toextract protein from the brain material, preventing recogni-tion of MOG conformation-dependent epitopes (Bourquin etal., 2003). Previous data have shown that antibodies directedagainst linear MOG peptides are not pathogenic (Brehm etal., 1999; Von Budingen et al., 2002). B-cell reactivity inPLP139–151-induced EAE was previously reported to bedirected only against the disease-inducing peptide(PLP139–151) and not directed against MOG and MBPpeptides, but the targeting of non-myelin peptides was notevaluated (Bischof et al., 2004). In our study the discrimina-tion between H, CFA/BPT, and EAE mice was due to non-myelin antigens. Among 27 selected bands, 6 were found tobe discriminant, and 3 of them could be characterized(mitochondrial aconitase hydratase 2, phosphoglyceratemutase 1 and brain specific pyruvate dehydrogenase). Twoof these antigens (mitochondrial aconitase hydratase 2 andphosphoglycerate mutase 1) have already been shown to behighly discriminant targets in MS (Lefranc et al., 2004), buttheir physiopathological significance has not yet been clearlydefined. In the present study, however, the IgG antibodyresponse against mitochondrial aconitase 2 and phosphogly-cerate mutase 1 was less frequent in the EAE group, whereasin a previous study it was positively associated withMSwhenMS patients were compared to control groups (healthyhumans and a Gougerot Sjögren group) (Lefranc et al., 2004).Thus, a comparison of the data obtained in humans and inmice reveals conflicting results. Antigens were preferentiallyrecognized in the CFA/BPTor H groups (for bands numbered11, 44, 54). We speculate that during the short period ofinvestigation at the acute phase of EAE, B cells and relevantIgG autoantibodies could still be bound to inflamed CNStissue, limiting their detection in serum in the EAE mice. Anextended follow-up study would help to resolve this issue.The conflicting results, in humans and mice could also berelated to the experimental conditions used for the inductionof EAE with only one PLP peptide in syngeneic mice,whereas MS is a multifactorial disease in geneticallyheterogeneous patients. An association between MS andHLA class II alleles, such as HLA DR2, is well established(Dyment et al., 2004). A previous study demonstrated thatHLA class II influences the immune response and antibodydiversification in humans (Paisansinup et al., 2002).

Accordingly in mice, this genetic control could explain thesingular autoreactive patterns obtained in a given (H-2s)strain (Vasconcellos et al., 1998).

The 3 antigenic proteins found in our study are ofparticular interest because of their suggested role in the brain.Indeed, aconitase hydratase is a key enzyme in Krebs cycle,present both in mitochondria and cytosol, and might beinvolved in neuroprotection (Kim et al., 2005). PGAM1 is abrain isoform involved in glucose metabolism and is of vitalimportance in the brain, notably for synaptic functions(Ikemoto and Ueda, 2003). PDHB deficiency is known to bea major cause of early childhood neurological dysfunctionand neurodegenerative diseases (Head et al., 2005). Thetargeting of cytoplasmic/mitochondrial proteins associatedwith neurodegenerative or neuroprotective processes mightbe related to the recognition of apoptotic bodies that containpotential self antigens (Bellone, 2000).

Large-scale screening, without a priori assumptions, hasprovided evidence that the combination of IgG antibodyresponses against a cluster of antigens is more effective thanindividual responses against single antigens as a means ofdiscriminating both MS patients and EAE mice from theirrespective control groups (Hemmer et al., 2002; Cross andStark, 2005). In other organ-specific autoimmune diseases,ubiquitous proteins are also targets of diversified IgGresponses detectable in serum (Pratesi et al., 2000; Ochi etal., 2002; Saulot et al., 2002; Singh et al., 2004; Fujii et al.,2005; Matsushita et al., 2005). Taken together, such resultssupport the hypothesis that autoimmune disease might be adeviation from normal autoreactivity. In addition, alterationsin serum self IgG antibody repertoire leading to antibodyclustering scored on brain antigens might be a useful meansof evaluating susceptibility to a pathogenic response indiseases involving the CNS.

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Diversified serum IgG response involving non-myelin CNS proteins during experimental autoimmune encephalomyelitis