Cytoadhesion of Plasmodium falciparum-infected erythrocytes … · 2006. 12. 13. · Globally, malaria is the most widespread human parasitic disease, affecting 300 to 500 million

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Braz J Med Biol Res 39(12) 2006

Plasmodium falciparum cytoadhesion in the placenta

Cytoadhesion of Plasmodiumfalciparum-infected erythrocytesand the infected placenta: a two-waypathway

1Departamento de Microbiologia e Imunologia,2Departamento de Parasitologia, Instituto de Biologia,Universidade Estadual de Campinas, Campinas, SP, Brasil3Unité de Parasitologie Expérimentale, URA Institut Pasteur,Université de la Méditerranée, Marseille, France4Centro de Pesquisa em Medicina Tropical, Porto Velho, RO, Brasil

F.T.M. Costa1,2, M. Avril3,P.A. Nogueira4

and J. Gysin3

Abstract

Malaria is undoubtedly the world’s most devastating parasitic disease,affecting 300 to 500 million people every year. Some cases of Plasmo-dium falciparum infection progress to the deadly forms of the diseaseresponsible for 1 to 3 million deaths annually. P. falciparum-infectederythrocytes adhere to host receptors in the deep microvasculature ofseveral organs. The cytoadhesion of infected erythrocytes to placentalsyncytiotrophoblast receptors leads to pregnancy-associated malaria(PAM). This specific maternal-fetal syndrome causes maternal ane-mia, low birth weight and the death of 62,000 to 363,000 infants peryear in sub-Saharan Africa, and thus has a poor outcome for bothmother and fetus. However, PAM and non-PAM parasites have beenshown to differ antigenically and genetically. After multiple pregnan-cies, women from different geographical areas develop adhesion-blocking antibodies that protect against placental parasitemia andclinical symptoms of PAM. The recent description of a new parasiteligand encoded by the var2CSA gene as the only gene up-regulated inPAM parasites renders the development of an anti-PAM vaccine morefeasible. The search for a vaccine to prevent P. falciparum sequestra-tion in the placenta by eliciting adhesion-blocking antibodies and acellular immune response, and the development of new methods forevaluating such antibodies should be key priorities in mother-childhealth programs in areas of endemic malaria. This review summarizesthe main molecular, immunological and physiopathological aspectsof PAM, including findings related to new targets in the P. falciparumvar gene family. Finally, we focus on a new methodology for mimick-ing cytoadhesion under blood flow conditions in human placentaltissue.

CorrespondenceF.T.M. Costa

Departamento de Microbiologia

e Imunologia

Instituto de Biologia, UNICAMP

13083-862 Campinas, SP

Brasil

Fax: +55-19-3788-6276

E-mail: [email protected]

F.T.M. Costa is supported by FAPESP

and CNPq-Foundation.

Received January 18, 2006

Accepted August 18, 2006

Key words• Plasmodium falciparum• Cytoadhesion• Pregnancy-associated

malaria• var2CSA gene

Brazilian Journal of Medical and Biological Research (2006) 39: 1525-1536ISSN 0100-879X Review

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Introduction

Globally, malaria is the most widespreadhuman parasitic disease, affecting 300 to500 million people per year. Four species ofPlasmodium can infect humans: P. falcipa-rum, P. vivax, P. malariae, and P. ovale. Nocomplications are observed in most malariacases, but some P. falciparum infectionsdevelop into severe forms of the disease,such as cerebral malaria and pregnancy-as-sociated malaria (PAM), which cause morethan two million deaths annually. It is esti-mated that 2.4 billion people, almost half theworld’s population, are at risk of contractingmalaria. In subtropical regions, and sub-Saharan African countries in particular, thisdisease limits economic development. Thecontrol of this disease has been hampered bythe alarming spread of drug-resistant para-sites, insecticide-resistant mosquitoes, andthe lack of an effective vaccine.

The situation has been aggravated by thedeterioration of socioeconomic conditionsin rural areas and disordered human migra-tion in countries in which malaria is en-demic. These factors have contributed to there-emergence of malaria. As a result, muchof the current research into malaria contin-ues to focus on attempts to develop a vaccinecapable of controlling parasite transmission.Some promising results have been obtained,but it seems unlikely that a vaccine confer-ring significant levels of immune protection,particularly against severe infection, will bedeveloped in the near future.

Severe malaria and Plasmodiumfalciparum cytoadhesion

Severe malaria is a multifactorial phe-nomenon involving the sequestration of P.falciparum-infected erythrocytes (IE) in deepvascular beds and the production of inflam-matory cytokines, such as TNF-α and IFN-γ(1). IE adhere directly to various host endo-thelial receptors, including CD36, intracel-

lular adhesion molecule-1 (ICAM-1), vas-cular cellular adhesion molecule-1 (VCAM-1), E-selectin, P-selectin, hyaluronic acid(HA), and chondroitin sulfate-A (CSA), orto other IE. They may also form rosettes byadhering to non-infected erythrocytes (Fig-ure 1B) (2). It has been suggested that adhe-sion to host receptors expressed on the sur-face of endothelial cells enables IE to avoidspleen-mediated filtration and host immuneattack, potentially implicating cytoadhesionin parasite survival (3). In addition to directparasite adhesion to host receptors, plateletscan act as a bridge between IE and endothe-lial cells, providing additional CD36 recep-tors for cytoadhesion (Figure 1A).

Following infection, P. falciparum pro-teins are exported to the erythrocyte surface,altering host cell conformation and generat-ing electron-dense structures. These struc-tures are known as knobs (Figure 1), andcorrespond to IE-binding sites adhering tothe host endothelium. Knobs are composedof several polypeptides, including P. falci-parum erythrocyte membrane protein 1(PfEMP-1; Figure 2A). PfEMP-1 is a vari-able protein 200 to 350 kDa in size, encodedby the members of the var multigene family,which are present as about 60 distinct copiesper haploid parasite genome. These proteinsmediate both antigenic variation and cyto-adhesion (2,4), and therefore play an impor-tant role in parasite virulence. It has beensuggested that var gene diversity is largelybased on the clustering of these genes at theends of several chromosomes, creating a“hot-spot” recombination zone facilitatingthe alignment of homologous sequences lo-cated on heterologous chromosomes. De-spite this variability, only one antigenic vari-ant is expressed on the surface of the IE at agiven time (5). PfEMP-1 contains a trans-membrane and an extracellular region, whichhas been implicated in binding to the cytoad-hesion-binding site and as a target for thehost immune response (Figure 2A). Thisextracellular region has a succession of bind-

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Figure 1. Sequestration mechan-isms involved in Plasmodium fal-ciparum infections. P. falcipa-rum-infected erythrocytes (IE)adhere directly to different re-ceptors on the host endotheliumvia knobs (A); to other IE byauto-agglutination (B); to non-in-fected erythrocytes (nIE), form-ing rosettes (C); to platelets,which act as a bridge in IEcytoadhesion via the CD36 re-ceptor (D). All these phenomenaare thought to contribute to bloodflow occlusion (E) and produc-tion of the inflammatory cyto-kines, TNF-α and IFN-γ (F); thusleading to the poor clinical out-comes observed in severe ma-laria.

Figure 2. Schematic structure ofPlasmodium falciparum erythro-cyte membrane protein-1 (PfEPM-1). The intracellular and the im-munogenic extracellular regionsof a PfEMP-1 are represented.A, One PfEMP-1 encoded by avar gene contains several Duffybinding-like (DBL) domains in-tercalated by cysteine-rich inter-domain region domains (CIDR),responsible for mediating para-site cytoadhesion to different re-ceptors directly on the host en-dothelium, multiadhesion, or ad-hesion to non-infected erythro-cytes, forming rosettes. NTS =N-terminal segment; TM = trans-membrane domain; ATS = acidicteminal segment; CR1 = com-plement receptor 1; ICAM-1 =intracellular adhesion molecule1; PECAM-1 = platelet endothe-lial cell adhesion molecule; CSA= chondroitin sulfate A. B, ThePfEMP-1 encoded by the var2CSA

gene 3D7gDNA-PFL0030cvarcontains DBL domains capableof CSA-binding and inter-do-main regions (ID).

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ing sites arranged in tandem at the N-termi-nal end of the molecule (Figure 2A). Thesemotifs are known as Duffy binding-like(DBL) domains, as they were first identifiedin P. vivax Duffy binding protein, interca-lated by cysteine-rich interdomain regiondomains (CIDR). Both CIDR and DBL re-gions can be identified on the basis of theiramino-acid sequences (2,4).

Different PfEMP-1 molecules have bind-ing sites for adhesion to different host recep-tors (Figure 2A), such as CD36, ICAM-1,VCAM-1, E-selectin, P-selectin, CSA, andothers dependent on multiple functional bind-ing domains within PfEMP-1. For example,adhesion to CD36, ICAM-1 and CSA ismediated by different PfEMP-1 variants, asvar genes are expressed in a mutually exclu-sive manner, with only one PfEMP-1 ex-pressed on the surface of an IE at a giventime (5). Thus, placental parasites can bindCSA, but not the CD36 receptor (6). Thisdichotomous behavior may result from dif-ferences in gene location and transcriptionorientations between CSA-binding and non-binding parasites (7-8).

General aspects of PAM

After years of exposure to the parasite,individuals living in areas of endemic ma-laria acquire high levels of immunity, limit-ing parasitemia and attenuating the clinicaloutcome of malaria. However, pregnantwomen remain susceptible, especially in theirfirst pregnancy, in which case the risk ofcontracting malaria is two to ten times higherthan that in non-pregnant women living inthe same area. Until recently, it was thoughtthat this particular susceptibility of womento malaria during pregnancy was due to preg-nancy-related immune suppression and hor-monal alterations. However, it has been re-cently shown that the placenta provides anideal environment for the development of asubpopulation of malaria parasites that ad-here to receptors in the placental syncy-

tiotrophoblast. In most cases, the parasitesremain on the maternal side of the placenta,but this maternal-fetal syndrome, known asPAM, has adverse effects on both motherand unborn child, causing maternal anemiaand low-birth weight (LBW) babies (9). PAMis thought to be responsible for 62,000 to363,000 infant deaths in sub-Saharan Africaannually (10). Unfortunately, these figuresare probably underestimates since peripher-al parasitemia is not always observed andthe symptoms are not well characterized insome cases.

In PAM, parasite adhesion to CSA, HAand other placental receptors may trigger aninflammatory process involving cytokine-secreting mononuclear cells. The inflamma-tory component, which may appear afterparasite accumulation in the placenta, is as-sociated with the immune-pathological con-sequences of PAM, such as cytotrophoblastproliferation and cytotrophoblast membranethickening (10). This inflammatory process,characterized by massive cell deposition, isthought to alter local blood flow, disruptingmetabolic pathways and hindering IgG trans-fer across the placenta and the exchange ofnutrients between mother and fetus, result-ing in placental lesions and LBW (10,11).However, the clinical outcomes of PAM,such as fetal growth restriction and pretermdelivery, the strict association with LBW atterm and placental parasitemia, have beenobserved in primigravidae (9). It is alsoknown that infants born to infected Cam-eroonian mothers are significantly more sus-ceptible to plasmodial infections, especiallyin the first two years of life (11). A recentepidemiological analysis in Tanzania, in-cluding twice as many infants as the Cam-eroon study (11), also showed that childrenborn to women with placental malaria pre-sented parasitemia earlier in life than thoseborn to non-infected peers (12). Surpris-ingly, this study also showed that parityplayed a role, as the offspring of primi-gravidae had a significantly lower risk of

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parasitemia than infants born to multi-gravidae (12). The precise reasons for thisremain unclear, but the marked change incytokine profile and the timing of cytokineproduction in primigravidae may be involved(12).

Most studies of maternal malaria havebeen carried out in P. falciparum-infectedwomen. However, pregnant women are sus-ceptible to all four human malaria parasites,including P. vivax, the most prevalent para-site in Brazil and elsewhere outside sub-Saharan Africa. A study in Thailand revealedthat primigravidae had a significantly higherrisk of P. vivax infection than multigravidae.Moreover, P. vivax infection has also beenshown to be significantly associated withmaternal anemia and risk of LBW, althoughthese outcomes were more marked in multi-gravidae (13). The deposition of malariapigment in the placenta has been observed inP. vivax-infected women (14), and variantantigens encoded by a specific P. vivax mul-tigene family have been identified (15). How-ever, hard data on P. vivax cytoadhesion tothe placental syncytiotrophoblast or endo-thelial cells remain scarce.

In Brazil, where malaria transmission isunstable, P. falciparum and P. vivax infec-tions account for 15.1 and 84.4% of casesamong non-pregnant women (16). However,the corresponding proportions for pregnantwomen are 29.7% for falciparum and 67.7%for vivax malaria (16). This corresponds to asignificant, 2.5 times increase in the fre-quency of P. falciparum infection for the195 cases of malaria in pregnant womenanalyzed (16). The precise reason for thisshift in prevalence is unclear and furtherstudies with a larger number of patients arerequired.

Ligands and receptors involved inPAM

The DBL-γ3 domain of PfEMP-1, en-coded by the var1CSA gene, was initially

thought to be the ligand responsible for para-site cytoadhesion in PAM (17). However,several recent studies have suggested thatthe protein encoded by the var2CSA genemay be the principal ligand involved in pla-cental cytoadhesion. Unlike var1CSA, var2CSA

is up-regulated in placental parasites afterselection for adhesion to the CSA receptor invitro (18-20); indeed it is the only geneshown to be transcriptionally up-regulatedfollowing such selection. var2CSA knockoutparasites are unable to recover their initialCSA binding, even after repeated exposureto CSA (21,22). The PfEMP-1 encoded bythe var2CSA gene has been shown to have astructure different from that of other vargenes, in that it lacks the CIDR, DBL-γ andN-terminal DBL-α domains (Figure 2B).The PfEMP-1 encoded by the 3D7 var2CSA

gene (3D7gDNA-PFL0030cvar) has sixDBL motifs: DBL4-ε, DBL5-ε and DBL6-ε,and a further three motifs that do not fit intothe current classification: DBL1-X, DBL2-X and DBL3-X. The DBL2-X and DBL6-εdomains are able to bind CSA (Figure 2B)(18).

A recent mass spectrometry-based prote-omics study identified novel parasite anti-gens, which might be expressed on the IEsurface, exclusively in CSA-binding or pla-cental parasites, but did not evaluate thebinding of these antigens to host receptors(23).

There is evidence that CSA is not theonly placental receptor and that a subpopu-lation of parasites collected from infectedplacentas may also bind to HA and to non-immune IgG via their F(ab’) moieties. How-ever, recent cytoadhesion assays using pla-cental parasites collected from 60 pregnantTanzanian women with malaria showed thatalmost all placental parasites capable of bind-ing to at least one host receptor were alsoable to adhere to immobilized CSA, and thatonly three of 46 of these parasites adhered toimmobilized HA. In binding inhibition as-says using soluble CSA as a competitor,

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adhesion to placental sections was signifi-cantly abolished in all placental parasitestested. In contrast, soluble HA, non-immuneIgG and protein A failed to inhibit parasitebinding to placental cryosections. These find-ings strongly suggest that CSA is the majorplacental receptor, and support the develop-ment of vaccines targeting parasite ligandsto CSA (24).

The CSA or chondroitin 4-sulfate (C4S)receptor is a glycosaminoglycan present inthe extracellular matrix, and was first identi-fied as a receptor for parasite binding toSaimiri monkey endothelial brain cells andto Chinese hamster ovary cells (25,26). Gysinet al. (27) showed that thrombomodulin witha CSA chain was the dominant proteoglycaninvolved in the sequestration of CSA-bind-ing parasites in the placenta and that a CSAchain at least 9 kDa in size was required tosustain the adhesion of CSA-binding para-sites (28). Achur et al. (29) subsequentlypurified and identified several types of chon-droitin sulfate proteoglycan (CSPG) fromthe human placenta, showing that these natu-ral CSPGs present in the intervillous spacecontained unusually low levels of sulfateand served as receptors for PAM parasiteadhesion. The same group went on to definethe structures required for parasite cyto-adhesion as C4S dodecasaccharides, andshowed that these placental CSPGs have aunique distribution of sulfate groups through-out the second and third semesters of preg-nancy (30-32). It was recently shown thatthe ability of antiadhesive molecules to in-hibit C4S-specific binding also depends onthe sulfation partner of these CSPGs (33).

Does the antibody-mediatedimmune response play a role inPAM?

Despite pregnancy-related immunosup-pression, pregnant women with malaria de-velop antibodies that inhibit the binding ofIE to CSA, and these antibodies are associ-

ated with protection against placental infec-tion. Primigravidae have a much higher sus-ceptibility to maternal malaria than multi-gravidae, because the antibodies acquiredafter multiple pregnancies are associated witha reduction in the number of IE in the pla-centa (34). In addition, higher levels of theseCSA adhesion-blocking antibodies are cor-related with less pronounced maternal ane-mia and with higher birth weight for babiesborn at term (35,36).

For var2CSA parasites, a recent studyshowed that rabbit antibodies raised againsttwo VAR2CSA recombinant proteins, cor-responding to the DBL1-X and DBL5-ε do-mains, recognize only the surface proteinsof PAM parasites (20). Plasma samples fromGhanaian individuals recognized these re-combinant proteins in a sex- and parity-dependent manner in ELISA tests; this wasparticularly true for the recombinant proteinbased on the DBL5-ε domain (20). Highplasma levels of anti-VAR2CSA antibodiesin women were also found to be correlatedwith a lower risk of LBW (20). Finally,monoclonal antibodies (mAbs) that recog-nize VAR2CSA DBL domains inhibit, tovarious extents, the adhesion of a placentalisolate to placental cryosections under flowconditions. Moreover, sera from mice im-munized with native VAR2CSA domaincomplexes with specific mAbs strongly in-hibit PAM parasite cytoadhesion to CSA onthe surface of endothelial cells (37).

These observations suggest that one prob-able mechanism controlling placental para-sitemia and attenuating clinical outcome maybe based on adhesion-blocking antibodiesagainst CSA-binding domains. However, asprimigravidae and multigravidae present sig-nificant levels of adhesion-blocking anti-bodies at term, the timing of acquisition ofthese antibodies may be important in im-mune protection. O’Neil-Dunne et al. (38)studied 198 pregnant Cameroonian womenand showed that multigravidae began mount-ing an antibody-based immune response af-

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ter just 12 weeks of gestation, whereas primi-gravidae took eight weeks longer to developsuch adhesion-blocking antibodies. How-ever, as cytophilic antibodies have been col-lected from infected pregnant women (39), itshould be borne in mind that other antibody-dependent mechanisms, such as phagocyto-sis and complement activation, may alsohelp to protect against PAM, in addition toblocking adhesion. Consistent with the ex-istence of additional antibody mechanisms,Megnekou et al. (40) showed that IgG1 andIgG3 were the most prevalent subclasses ofPAM antibodies in Cameroonian women,and that larger amounts of these antibodieswere found in pregnant multiparous women.

Is the cell-mediated immuneresponse involved in PAMpathogenesis or protection?

The precise mechanism by which PAMparasites evade the immune system and thepossible involvement of a cell-mediated im-mune response in protection remains unre-solved. However, it has been shown thatmassive sequestration of the parasite in theplacenta leads to a switch in the cell-medi-ated immune response, typically from TH2to TH1, resulting in the clinical manifesta-tion of PAM, characterized by an increase inthe level of pro-inflammatory cytokine pro-duction (10). Several studies have shownthat high levels of IFN-γ and TNF-α, mainlysecreted by placental macrophages, are as-sociated with poor clinical outcome in pa-tients with PAM and with the concentrationof hemozoin in the placenta (41). Placentalinfection increases the levels of α- and ß-chemokines, which, in turn, increase im-mune cell recruitment to the placenta (10,42).

In contrast, IFN-γ levels have been re-ported to be higher following in vitro stimu-lation of blood mononuclear cells from mul-tigravidae than following the stimulation ofsuch cells from women in their first or sec-ond pregnancy. The cells collected from

women in their second pregnancy secretedhigh levels of IL-4 and IL-10 (43). Multi-gravidae have been shown to present higherlevels of lymphocyte proliferation and natu-ral killer cell cytotoxic activity in responseto CSA-binding parasites than primigravidaewomen (10). These observations suggest thatIFN-γ is involved in immunity to PAM.Further evidence for the protective role ofIFN-γ is provided by the higher susceptibil-ity to PAM of women with both malaria andHIV infection (10). Indeed, it has been re-cently shown that neonates born to motherswith active placental infection have lowerlevels of PAM-parasite antigen-specific IFN-γ T cells and higher levels of IL-10 CD4 Tcells than do pregnant infected women treatedfor malaria (44). However, TGF-ß, an anti-inflammatory cytokine, is produced in largeramounts in multigravidae than in primi-gravidae, suggesting a possible role in con-trolling the manifestation of PAM clinicalsymptoms.

A recent study in monkeys showed thatinfection with P. coatneyi did not result inhigher levels of CD4 and CD8 T lympho-cytes than observed in infected non-preg-nant monkeys. Indeed, the pregnant infectedmonkeys had lower levels of monocytes andmacrophages in peripheral blood than didthe non-pregnant infected monkeys (45).Conversely, high levels of mononuclear cellaccumulation have been associated with poorPAM outcomes (46). It should be noted that,in these studies, cells were counted in pe-ripheral blood, so we cannot exclude thepossibility that this modulation may alter thelevels of these cells in placental compart-ments. In PAM, T cells collected from pe-ripheral blood proliferate more efficientlythan those collected from the intervillousblood, whereas intervillous and peripheralmonocytes are equally able to present anti-gens (47).

These observations suggest a dual effecton cytokine production in PAM and that afine balance in the timing and levels of pro-

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and anti-inflammatory cytokines dictateswhether an individual will manage to con-trol PAM or whether the clinical symptomsassociated with the disease will develop.This dual effect may depend on regulation ofthe macrophage migration inhibitory factor,a specific cytokine that counter-regulatesthe immunosuppressive effects of pregnancy.Placental infection was recently shown toincrease macrophage migration inhibitoryfactor production in the presence of cytotro-phoblast-adherent IE (48).

Is an anti-PAM vaccine feasible?

The first evidence that it might be pos-sible to develop an anti-PAM vaccine wasprovided by the study of Fried and Duffy(9,35) and Staalsoe et al. (34,36) showingthat multiparous women were less suscep-tible to PAM than women in their first preg-nancy, that infected women developed highlevels of adhesion-blocking antibodiesagainst PAM parasites after several preg-nancies (9,34) and that these antibodies wereassociated with attenuation of the clinicaloutcome of PAM. Antibodies against var2CSA

parasites have also been shown to cross-react with genetically different P. falcipa-rum strains (49). Cross-reactivity betweenthe DBL-γ3CSA domain and var2CSA-encodedantigens has been observed (50). Further-more, mAbs raised against var2CSA parasitesurface antigens have also proved to be pan-reactive with CSA-binding parasites fromdifferent geographical origins (37). More-over, molecular analysis of the var1CSA DBL-γ3 minimal binding domain revealed 37%sequence identity to the var2CSA DBL3-Xdomain (51). These somewhat surprising pan-reactivity results are probably related to con-formational similarities.

In light of the antibody-mediated im-mune response in PAM, an efficient vaccinewould probably elicit large amounts of ad-hesion-blocking antibodies, mainly againstconserved binding motifs. However, as the

cell-mediated immune response also seemsto be involved in immune protection againstPAM, immunization regimes and adjuvantsshould aim to induce high levels of IFN-γ-secreting T cells. Immunization regimensbased on naked DNA for priming and re-combinant viral vectors or proteins for boost-ing have been shown to elicit high levels ofCD4- and CD8-producing T cells able toinduce immune protection against severalviral and protozoan diseases (52). Finally,anti-PAM vaccines may contain other para-site antigens since sera collected from Cam-eroonian women showed a significant corre-lation between low or null levels of anti-bodies against the carboxyl-terminal 19-kDasegment of the P. falciparum merozoite sur-face protein-1 and the risk of PAM (53).

Is it possible to model PAM?

Reliable in vitro adhesion models arerequired for the evaluation of potential vac-cine candidates and the antibody-mediatedimmune response directed against them. Mostof the existing in vitro models of IE seques-tration were developed for studying IE adhe-sion in parasites thought to be involved incerebral malaria. Many knob+ laboratory-adapted P. falciparum strains adhere in vitroto various cell types, including human um-bilical vein endothelial cells, C32 amelanoticmelanoma cells, human dermal microvascu-lar endothelial cells, human brain capillaryendothelial cells, human monocytes andplatelets, and transfected COS cells (54).However, in 1995, Gay et al. (55) describedthe use of Saimiri microvascular brain endo-thelial cell clones differing in terms of theexpression of several combined surface mol-ecules such as CD36, ICAM-1, E-selectin,and CSA, permitting for the first time theselection by cytoadhesion of distinct mono-morphic adhesion phenotypes.

Each model attempts to simulate the in-teraction between IE and the cerebral endo-thelial cells, but none can be considered

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ideal. The Saimiri cell model has some ad-vantages in that it allows in vitro cytoadhesionstudies, the results of which can be con-firmed or rejected using the homologousSaimiri/P. falciparum monkey model (56).This model is also considered to be morerelevant than non-primate cell models due tothe phylogenetic proximity to humans. Theuse of organ-specific endothelial cells ap-pears to be particularly useful for structure-function studies in which the native confor-mation of a receptor is critical. A placentalBeWo-derived cell line has been success-fully used to select monomorphic CSA-bind-ing parasites (57). Since placental CSPGshave an unusual sulfation pattern, BeWocells provide an alternative to cells of non-placental origin in CSA-binding studies.

In all the parasite-binding assays de-scribed above, cytoadhesion was investi-gated under static conditions, in which sus-pensions of IE were allowed to settle on aconfluent monolayer of cultured cells. Thismethod is technically simple, making it pos-sible to carry out a large number of assayssimultaneously. However, static assays donot model the dynamic blood flow condi-tions encountered by IE in vivo.

In 1995, Cooke and Coppel (54) devel-oped an in vitro assay for visualizing andquantifying the adhesion of IE to endothelialcells or to immobilized adhesion receptorsunder flow conditions. The flow assemblyused consisted of a parallel-plate flow cham-ber or a glass microcapillary tube (microslide)on which a monolayer of endothelial cells,such as human umbilical vein endothelialcells, C32 melanoma cells, or Saimiri micro-vascular brain endothelial cell clones (58),can be cultured, or a plastic slide coated withpurified proteins, such as CD36, ICAM-1 orthrombospondin, by adsorption. Adhesionunder dynamic flow conditions can be quan-tified by counting adherent IE directly underthe microscope.

Nevertheless, with the exception of thisflow adhesion model using endothelial cells

and immobilized proteins, very few modelshave been developed for studying placentalmalaria. One example is the Saimiri (squir-rel monkey) microvascular endothelial cellline Sc17, which expresses a thrombomodu-lin bearing only a CSA chain and a CD44-csa isoform. The presence of the chondroitinsulfate of thrombomodulin, or a CD44isoform, on endothelial cells mimics, to someextent, the presence of CSA on the surface ofthe syncytiotrophoblast, thereby providing aclear advantage over previous cell models orcommercial CSA preparations from variousnon-placental sources (59). Conversely, onemajor disadvantage of the use of cell lines asmodels for CSA binding is the presence ofunidentified or unknown adhesion receptorsin addition to CSA on the surface of endo-thelial cells. Another disadvantage of theseassays is that CSA preparations from vari-ous sources, used by different laboratories,can generate conflicting results. Thus, theconformational modification of CSA by add-ing dipalmitoyl-diphosphatidylethanolaminemay bias results, especially when this sys-tem is used to select CSA-binding IE bypanning. The addition of charged groupsseems to be problematic for the specificselection of CSA-binding IE from labora-tory strains and field isolates (28). Further-more, cytoadhesion inhibition assays withSaimiri brain endothelial or Chinese ham-ster ovary cells cannot distinguish betweensubpopulations of CSA-binding parasites infield samples.

However, most of these problems can besolved by using normal and at-term humanplacental cryosections (60), as IE bind al-most exclusively to syncytiotrophoblast andin the intervillous space containing proteo-glycans with low levels of sulfation (61).The use of human placental tissue makes itmuch easier to count IE under flow thanunder static conditions, in which parasitesalso bind to CSA and to other receptorswithin the villi. A comparison of inhibitionassays carried out under flow and static con-

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ditions revealed significant differences inthe presence of soluble CSA, or inhibitormAbs in serum samples from primi- andmultigravidae (61). Static parasite adhesionassays are subject to considerable inter-ex-perimental variation, due primarily to differ-ences in washing procedures.

The use of placental cryosections made itpossible, for the first time, to measure theshear-stress resistance of CSA-binding IE.Distinct subpopulations within the CSA-binding phenotype were identified by in-creasing the flow rate gradually from 0.2 to3.2 Pascal (Pa). For example, at 0.6 Pa,which exceeds the normal shear stress in theplacenta (0.05 Pa), 70% of IE remained ad-herent for the laboratory strain FCR3CSA,and 25% of IE resisted a shear stress >3.2 Pa(60).

These results strongly suggest that theinitial FCR3CSA strain was composed of amixture of strong (≥3.2 Pa) and weak (≤0.8Pa) CSA-binding parasites, confirming theexistence of distinct adhesion subpopula-tions among the CSA phenotypes of variousstrains (58; Nogueira PA, Costa FT and GysinJ, unpublished data), and supporting the hy-pothesis that only some subpopulations ofCSA-binding IE have the potential for se-questration in the microvasculature (58). Thishypothesis is based on the notion that IE inthe placenta are not normally exposed toshear stresses exceeding 0.05 Pa, whereasshear stresses in the postcapillary venulesvary from 0.1 to 1 Pa (58).

Conclusions

There is now considerable evidence thatPAM is a particularly severe form of ma-laria, and that primigravidae and their off-spring have a higher risk of developing PAMthan do multigravidae and their children.These observations, and others, provide an

impetus for the development of an anti-PAMvaccine. However, several issues concern-ing the expression of antigens by PAM para-sites and the precise immunological mechan-isms involved in protection remain unre-solved in the context of PAM. First, does theunique set of hormones and cytokines in-duced during gestation play a role in antigenor placental host receptor expression? Sec-ond, why is CSA the major PAM receptor,given that this glycosaminoglycan is foundin various organs other than the placenta?Third, how many antigenically differentPAM parasites exist, and what are their rela-tive prevalences in infected pregnant women?Fourth, what is the evolutionary importanceof this pan-reactivity and the presence ofmultiple CSA-binding domains? Can thesedomains be ordered into a hierarchy? Fi-nally, as some of the poor placental out-comes observed in falciparum malaria arealso observed in vivax malaria, is IEcytoadhesion in the placenta an exclusivefeature of P. falciparum parasites? Or, likethe inhabitants of “Plato’s Cave”, are wemerely watching the “theater of shadows” ofreal PAM parasite interactions and mechan-isms reflected on the cave wall?

Acknowledgments

We would like to thank Dr. Artur Scherf(Institut Pasteur, Paris, France) and Dr.Daniela D. Carvalho (Universidade Estadualde Campinas, Campinas, SP, Brazil) for criti-cally reading the manuscript and RafaelChaves (Universidade Estadual de Campi-nas, Campinas, SP, Brazil) for editorial as-sistance. We would like to apologize to thoseauthors whose published articles have notbeen cited in this review due to space con-straints and the huge body of literature avail-able.

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Cytoadhesion of Plasmodium falciparum-infected erythrocytes … · 2006. 12. 13. · Globally, malaria is the most widespread human parasitic disease, affecting 300 to 500 million