Trypanosoma cruzi infection: a continuous invader-host cell cross … · 2003. 7. 24. · sion and cell-extracellular matrix (ECM) in-teraction and is orchestrated by cell adhe-sion

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Braz J Med Biol Res 36(8) 2003

T. cruzi-host cross talk with ECM participationBrazilian Journal of Medical and Biological Research (2003) 36: 1121-1133ISSN 0100-879X

Trypanosoma cruzi infection:a continuous invader-host cellcross talk with participation ofextracellular matrix and adhesionand chemoattractant molecules

1Laboratório de Autoimunidade e Imuno-regulação, and2Laboratório de Imunologia Clínica, Departamento de Imunologia,Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brasil

A.P.M.P. Marino1,A.A. Silva1,

R.T. Pinho2 andJ. Lannes-Vieira1

Abstract

Several lines of evidence have shown that Trypanosoma cruzi inter-acts with host extracellular matrix (ECM) components producingbreakdown products that play an important role in parasite mobiliza-tion and infectivity. Parasite-released antigens also modulate ECMexpression that could participate in cell-cell and/or cell-parasite inter-actions. Increased expression of ECM components has been describedin the cardiac tissue of chronic chagasic patients and diverse targettissues including heart, thymus, central nervous system and skeletalmuscle of experimentally T. cruzi-infected mice. ECM componentsmay adsorb parasite antigens and cytokines that could contribute to theestablishment and perpetuation of inflammation. Furthermore, T.cruzi-infected mammalian cells produce cytokines and chemokinesthat not only participate in the control of parasitism but also contributeto the establishment of chronic inflammatory lesions in several targettissues and most frequently lead to severe myocarditis. T. cruzi-drivencytokines and chemokines may also modulate VCAM-1 and ICAM-1adhesion molecules on endothelial cells of target tissues and play a keyrole in cell recruitment, especially of activated VLA-4+LFA-1+CD8+

T lymphocytes, resulting in a predominance of this cell population inthe inflamed heart, central nervous system and skeletal muscle. TheVLA-4+-invading cells are surrounded by a fine network of fibronec-tin that could contribute to cell anchorage, activation and effectorfunctions. Since persistent “danger signals” triggered by the parasiteand its antigens are required for the establishment of inflammation andECM alterations, therapeutic interventions that control parasitism andselectively modulate cell migration improve ECM abnormalities,paving the way for the development of new therapeutic strategiesimproving the prognosis of T. cruzi-infected individuals.

CorrespondenceJ. Lannes-Vieira

Departamento de Imunologia

Instituto Oswaldo Cruz, Fiocruz

Av. Brasil, 4365

21045-900 Rio de Janeiro, RJ

Brasil

E-mail: [email protected]

Presented at SIMEC 2002

(International Symposiumon Extracellular Matrix),Angra dos Reis, RJ, Brazil,

October 7-10, 2002.

Research supported by CAPES,

CNPq, FAPERJ and PAPES-II-Fiocruz.

Received January 10, 2003

Accepted February 21, 2003

Key words• Trypanosoma cruzi• Extracellular matrix• Cell adhesion• Cell migration• Adhesion molecules• Chemokines

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A.P.M.P. Marino et al.

Introduction

As a common consequence of a combi-natorial interplay between invader and host,inflammatory processes are established lead-ing to invader control but frequently contrib-uting to the establishment of chronic inflam-matory disease. Inflammatory responses totissue infection require cell migration, a wide-spread process that comprises cell-cell adhe-sion and cell-extracellular matrix (ECM) in-teraction and is orchestrated by cell adhe-sion molecules and integrin receptors, ECMcomponents, matrix metalloproteinases andchemoattractant molecules. Migration is alsoa prerequisite for infectious agent spreading.Direct or bridge molecule-mediated interac-tions occur between invaders and host ECMand cells, being a crucial event for success-ful infection. We report here lines of evi-dence that these processes may occur duringTrypanosoma cruzi infection that leads to asevere chronic diffuse and fibrosing myo-carditis with the presence of rare parasites in25-30% of infected patients.

Trypanosoma cruzi-host cellinteraction

The parasite talks

The protozoan T. cruzi, the causativeagent of Chagas’ disease, invades mamma-

lian host cells as trypomastigote or amasti-gote forms to complete its biological cycle.The understanding of the molecular basis ofthe host cell invasion process by T. cruzi hasbeen an almost centenary challenge in para-sitology. Several lines of evidence demon-strate that the ECM glycoprotein fibronectin(FN) promotes adhesion and uptake of try-pomastigotes by macrophages and fibroblasts(1,2) and amastigotes by murine or humanmacrophages (3). Subsequent studies showedthat T. cruzi trypomastigotes specifically rec-ognize and bind to the RGDS amino acidsequence of FN that inhibits the infection ofhost cells (4). Moreover, the expression ofparasite FN receptors characterized as a groupof proteins ranging from 80 to 85 kDa, in-cluding a member of the trans-sialidase su-perfamily, is correlated with the parasite’sability to adhere to and invade host cells(5,6).

It is commonly believed that as a prereq-uisite for host cell invasion, T. cruzi infec-tive forms must cross basement membraneand ECM barriers. Recently, it has beenreported that the binding of T. cruzi to lami-nin (LN), the major constituent of the base-ment membrane, is enhanced by galectin-3(7). Also, a new class of T. cruzi moleculeshas been reported to play a crucial role facili-tating parasite mobilization through the ECM.These T. cruzi ECM-binding proteins, in-cluding the 51 kDa cysteine proteinasecruzipain, present enzymatic activity andmediate type I collagen and FN degradation(8,9). In vivo, cruzipain also binds to the FNnetwork present in the heart tissue of T.cruzi-infected mice (Figure 1), as previouslydemonstrated among the T. cruzi antigensreleased into the cardiac interstitial spaces ofchagasic patients (10). Sites that are crypticor hidden in the native ECM molecules maybecome activated or exposed upon disas-sembly or degradation of the ECM compo-nent structure by proteolytic enzymes thatare released by interstitial parasites or, con-sidering the whole event, secreted by tissue-

Figure 1. Trypanosoma cruzi an-tigen cruzipain (green) is de-tected associated with fibronec-tin fibers (red) in the myocardi-um of chronically (120 days post-infection) infected mice. The ani-mals were infected with 100 try-pomastigotes of the Colombianstrain of T. cruzi. The arrows in-dicate the superposition of thelabeling for fibronectin andcruzipain (yellow). Bar = 25 µm.

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T. cruzi-host cross talk with ECM participation

invading leukocytes. As a biological conse-quence, the ECM breakdown products mayfacilitate invasion of target cells by the para-site. In fact, FN cleavage fragments wereshown to provide a growth factor-like activ-ity for the differentiation of trypomastigotesto amastigotes, a more efficient invasive form(11). In addition, in view of the ongoing T.cruzi-elicited myocarditis, the ECM degra-dation process may modulate ECM interac-tions with cytokines and chemokines in-volved in recruitment and/or activation ofinflammatory cells (12), as discussed below.

The inflamed myocardium is exposed toparasite antigens either released into the car-diac interstitial spaces by extracellular para-sites or from killed organisms. More re-cently, we have shown that proteins mainlyranging from 85 to 170 kDa released by T.cruzi trypomastigotes within membranevesicles adhered to membrane microvilli andwere internalized by noninfected mamma-lian cells, including fibroblast and skeletalmuscle cells. A cysteine proteinase, cruzipain,but not trans-sialidase, was detected amongthe cell-bound antigens. As a consequenceof the adsorption of released antigens thesensitized cells became targets for anti-T.cruzi antibodies and presented increased ex-pression of ECM components (13). Thus,the in vivo released T. cruzi antigens mightbe involved in the establishment of inflam-mation, sensitizing noninfected host cells,triggering an immune response against para-site antigens and modulating ECM expres-sion that could mediate cell-cell or parasite-host cell interactions.

Recent studies have shown that an 80-kDa serine protease located inside a vesicu-lar compartment close to the flagellar pocketwas found to hydrolyze FN and, more impor-tantly, to be a potential target for a potentselective inhibitor that blocks T. cruzi entryinto host cells (8,14). Also, a new member ofthe T. cruzi 80-85-kDa glycoprotein familywas shown to bind to LN (15). Together withparasite-binding sites for host thrombospon-

din, heparin sulfate and FN, this LN-bindingsite has been proposed to be a potentialtarget for inhibition of invasion of host cellsby T. cruzi by RNA aptamers (16,17). Takentogether, these data led to the concept thatspecific interactions between T. cruzi andhost ECM components play a pivotal role inparasite dissemination, mediating basementmembrane and ECM degradation as well asadhesion to and invasion of host cells. More-over, these findings provide evidence thatECM-binding sites present on the T. cruzisurface are new potential therapeutic targetsfor inhibition of T. cruzi dissemination.

The host answers

The heart is the main organ affected dur-ing T. cruzi infection. The pathogenesis oftissue remodeling, particularly that whichcauses the unique apical aneurysm found inchagasic patients, remains unclear. Fibrosisis one of the most important features of thechronic chagasic cardiomyopathy with mul-tifocal scarring frequently observed in theabsence of significant inflammation or para-sitized myocytes (18). More recently, it wasshown that, compared with dilated cardio-myopathy, chronic chagasic myocarditis pre-sents a distinctive pattern of ECM abnor-malities (19). Increased expression of ECMcomponents including FN, LN and types IIIand IV collagen, and remodeling of matrixcomponents associated with the progressionof inflammation have also been detected inthe heart tissue of T. cruzi-infected mice(20). However, focal connective tissue dam-age with multiple areas devoid of ECM com-ponents was observed in inflamed atria ofearly acutely infected mice (21). It is pos-sible that ECM degradation can precede orconcurrently occur with enhanced fibrosis,and account for remodeling of cardiac tis-sue. In fact, alterations of the ECM maycontribute to ventricular dysfunction andgeometric shape changes observed in humanand experimental T. cruzi-induced cardio-

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inflammation composed mainly of mono-nuclear cells is initially detected in the earlyacute phase. It was paralleled by ECM ab-normalities that are observed in areas withinflammatory infiltrates and areas not af-fected by myositis, and related or not to thepresence of parasite pseudocysts and anti-gens. Interestingly, the phenotypic analysisof mononuclear cells isolated enzymatically(23) from skeletal muscle showed that theinflammatory infiltrates were composedmainly of CD8+ T cells (Figure 2). Thisfinding is particularly relevant consideringthe prevalence of this T cell subset in thecardiac tissue of chagasic patients present-ing severe myocarditis (24,25) and in theinflamed heart of C3H/He mice chronicallyinfected with the Colombian strain (23). Inthis experimental model, CD8-mediatedmyocarditis is established during the earlyacute infection (Figure 3) and persists duringthe chronic phase (23). Also, CD8-mediatedmeningoencephalitis restricted to the acutephase is observed in this animal model (26).The findings showing the establishment ofinflammatory infiltrates composed mainlyof CD8+ T cells in several target tissuesduring the early acute phase indicate theimportance of CD8+ T cells in the resistanceto T. cruzi (27). Moreover, these data sup-port the idea that during T. cruzi infectionthe preferential migration of CD8+ T cellstowards target tissues results from the differ-ential activation profile of this cell popula-tion, being coordinated by parasite-drivenchemoattractant molecules expressed in tar-get tissues (23 and Roffê E, Silva AA,Santiago HC, Gazzinelli RT and Lannes-Vieira J, unpublished data).

The intense increase in the expression ofthe cardiac ECM proteins FN, type IV col-lagen and LN exists as an interstitial frame-work surrounding and interconnecting in-fected and noninfected skeletal muscle (Fig-ure 2) or cardiac myocytes (Figures 4 and 5)of Colombian strain-infected C3H/He mice.Laminin expression was significantly in-

Figure 2. A, Conventional histopathological analysis (H&E) of inflammatory infiltrates in theskeletal muscle of Trypanosoma cruzi-infected mice. Amastigote nest containing hundredsof parasites in the absence of inflammatory infiltrates at 35 days post-infection (inset). Bar= 25 µm. B, Flow cytometric analysis of mononuclear cells obtained from skeletal muscleof T. cruzi-infected mice shows the predominance of CD8+ T cells in chronically (120 dayspost-infection) T. cruzi-infected mice. Immunohistochemical analysis showed that com-pared with normal mice (C), chronically T. cruzi-infected mice (D) present increased expres-sion of type IV collagen in the basal lamina of skeletal muscle cells. Bars = 100 µm.

myopathy. These include loss of ECM com-ponents through degradation or mechanicaldestruction producing a thin-walled, dilated,and poorly contracting ventricle, even inthe absence of significant myocyte injury(21).

Abnormal expression of ECM compo-nents seems to be a widespread event duringT. cruzi infection. A progressive and intenseincrease in the intralobular network contain-ing FN and type IV collagen was observed inthe atrophic thymus of T. cruzi-infected mice(22). Further, a remarkable increase in theexpression of FN and the basement mem-brane components LN and type IV collagenwas also detected in other lymphoid (spleenand lymph nodes) and nonlymphoid tissuesincluding skeletal muscle of C3H/He miceinfected with the Colombian strain of T.cruzi (Figure 2). In skeletal muscle diffuse

AB

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3 4

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1000

100

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0 1 10 100 1000CD8

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CD8

CD

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A

B

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17.28 0.86

15.03 62.03

Figure 3. Conventional histo-pathological analysis (H&E) of in-flammatory infiltrates in the car-diac tissue of C3H/He mice dur-ing the acute (A) and chronic (B)phases. Bars = 100 µm. Flowcytometric analysis of mono-nuclear cells obtained from car-diac tissue of acutely (28 dayspost-infection) Trypanosomacruzi-infected C3H/He miceshows the predominance ofCD8+ T cells (C).

creased in the cardiac tissue of T. cruzi-infected mice, particularly in the basal laminaof cardiomyocytes, and absent in areas ofinflammatory infiltrates (Figure 4). In addi-tion, the ß1 integrin LN receptor VLA-6 wasnot immunohistochemically detected on thesurface of infiltrating cells, although rareinterstitial cells were α6+ (Figure 4). Simi-larly, in the inflammation of the central ner-vous system of acutely T. cruzi-infected C3H/He mice, LN expression restricted to thebasement membrane of blood vessels wasenhanced, whereas VLA-6 was absent oninfiltrating perivascular cells (28). Regard-ing the functional role of LN and VLA-6 inthe physiopathogenesis of myocarditis dur-ing T. cruzi infection, it was previously shownthat antibodies recognizing LN and its recep-tor VLA-6 are able to block the migration oflocally injected splenic CD4+ T cells ob-tained from chronically T. cruzi-infected miceto cardiac tissue grafted in the ear of normalmice (29). This finding suggests the partici-pation of LN/VLA-6 interactions in cell mi-gration towards the heart tissue of T. cruzi-infected mice. Our findings do not rule outthis possibility; however, it is possible thatafter transmigration through the endotheliallayer, inflammatory cells may lose or down-modulate the expression of VLA-6 (30).

Interestingly, we have shown that the FNmesh present in the inflamed heart of chroni-cally T. cruzi-infected mice involves VLA-4+

macrophages, and activated VLA-4+LFA-1+

CD4+ and CD8+ T cells (23). Moreover,mononuclear cells expressing VLA-4 andCD44 were detected immunohistochemicallyin the myocardium of chronic chagasic pa-tients (31), suggesting that these inflamma-tory cells are activated and able to interactwith ECM components. When C3H/He micewere inoculated with the Colombian strainduring the early acute phase (14-28 dayspost-infection), most of the infiltrating in-flammatory cells, including both CD4+ andCD8+ T lymphocytes, were VLA-4+, whereasonly a minor population of the mononuclear

cells inside the inflamed cardiac tissue ex-pressed VLA-5 (Figure 6). Furthermore, wehave shown that the predominance of CD8+

T cells in T. cruzi-infected heart observedduring chronic infection (23) is also achievedduring the early acute phase, paralleling theECM alterations.

The role of FN and its receptors in thephysiopathogenesis of the chronic T. cruzi-elicited myocarditis is unclear. The ß1 inte-grin FN receptors on macrophages may con-tribute to T. cruzi dissemination. In fact, ithas been reported that T. cruzi uptake andsubsequent replication inside macrophages

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Figure 4. Confocal analysis of laminin (LN) expression in thecardiac tissue of normal (A) and acutely (B) Trypanosoma cruzi-infected C3H/He mice. LN is not detected surrounding theinflammatory cells (red). Immunohistochemical analysis showedthat the increased expression of LN is restricted to the basallamina of myocytes and basement membrane of blood vessels(C). The ß1 integrin LN receptor VLA-6 is absent on mono-nuclear cells infiltrating the cardiac tissue of T. cruzi-infectedmice (D). Bars = 25 µm.

A B

C D

Figure 5. Panel A, Confocal analysis of fibronectin (FN) expres-sion in the cardiac tissue of normal (a) and acutely Trypanosomacruzi-infected (b) C3H/He mice. Panel B depicts an immunohis-tochemical analysis of the cardiac tissue of T. cruzi-infected miceshowing that a fine FN network surrounds noninfected andinfected (arrow) cardiomyocytes (a). This FN mesh also sur-rounds α4+ mononuclear cells (b) that are CD4+ (c) and mostlyCD8+ (d) lymphocytes. Serial section of 5 µm. Bars = 25 µm.

a b

c d

a b

A

B

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Figure 6. Flow cytometric analysis of mononuclear cells obtained from cardiac tissue ofacutely Trypanosoma cruzi-infected C3H/He mice shows that most of the CD4+ andCD8+ T cells are VLA-4+ (A), whereas a small fraction of CD4+ and CD8+ lymphocytesexpress VLA-5 (B). Immunohistochemical analysis showed that compared with normalmice (C), acutely (28 days post-infection) T. cruzi-infected mice (D) present increasedexpression of the adhesion molecule VCAM-1 on endothelial cells of cardiac tissue. Bars= 50 µm. The perivascular space of VCAM-1+ blood vessels (E) was filled with α4+

mononuclear cells (F). The arrow indicates the presence of an amastigote nest inside acardiomyocyte. Bars = 25 µm.

C D

E F

A

B

0.34 16.83 1.31 66.97

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is inhibited by treatment of mammalian cellswith anti-ß1 integrin antibodies, but not withanti-ß2 integrin. Also, partial inhibition ofparasite uptake by anti-VLA-4 and anti-VLA-5 antibodies suggested that severalmembers of the ß1 integrin family, in addi-tion to the FN receptors VLA-4 and VLA-5,are involved in T. cruzi binding to and entryinto macrophages (32).

A fine network of FN involving VLA-4+

mononuclear inflammatory cells and infectedor noninfected neurons and glial cells (astro-cytes and microglia) was shown to be pres-ent in the central nervous system of Colom-bian strain-infected C3H/He mice duringacute infection; however, this inflammationis self-resolving and chronic alterations arenot present at this site (26,28), whereas aprogressive myocarditis paralleled by ECMabnormalities is observed in these mice (23).The distribution of variant forms of FN inanatomic sites in vivo remains unknown butit has been suggested that VLA-4-dependentco-stimulation of T cells may be restricted tocertain critical anatomic sites in vivo (33). Itis reasonable to propose that interactions ofT cells with the fine FN filamentous networkpresent in the inflamed myocardium viaVLA-4 molecules may provide an anchor forinvading cells and not only function as apathway in the T cell migration processesbut may also influence antigen-specific Tcell recognition, proliferation, survival andeffector activity (34). In addition, FN andother ECM components may also act as che-moattractants for additional invading cellsor provide a substrate for the anchoring andstabilization of chemoattractant molecules(35,36), contributing to the perpetuation ofinflammation in this tissue. Further as dis-cussed above, T. cruzi antigens present pro-teolytic activity resulting in ECM breakdownproducts that are themselves potentially im-munomodulatory and could contribute to themaintenance of chronic inflammation. In-deed, it has been shown that damaged ECMactivates macrophages to secrete IL-12 and

chemokines (37,38), cytokines present inthe inflamed heart of chronic chagasic pa-tients (24,25), and in acute and chronic ex-perimentally T. cruzi-infected mice (23,39,40). These cytokines play a central role bycontrolling tissue parasitism during T. cruziinfection but are also involved in the mainte-nance of chronic myocarditis (41-44).

With respect to putative sources of ECMcomponents in T. cruzi-infected inflamedtarget tissues, the inflammatory mononuclearcells may contribute to FN production asactivated macrophages and T lymphocytesproduce this glycoprotein (45,46). However,the possibility that myocytes and other celltypes present in these tissues could also con-tribute to ECM production cannot be ruledout. In fact, our results show an increasedexpression of FN and LN by infected andnoninfected neurons and glial cells (astro-cytes and microglia) (28) of acutely T. cruzi-infected mice, and FN, type IV collagen andLN in the basal lamina of infected and non-infected cardiac (23) and skeletal musclecells of acutely and chronically infected mice.Also, in vitro studies have shown that sev-eral other cell types when infected with T.cruzi present abnormal quantitative and quali-tative production of ECM components (13,47). In this respect, in addition to a remark-able increase in ECM production by T. cruzi-infected endothelial cells, the heparan sul-fate proteoglycan isolated from this ECMdiffered from the heparan sulfate proteogly-can obtained from the ECM of noninfectedcells by virtue of an eight- to ten-fold in-crease in sulfation besides other biochemi-cal properties. Also, this modified heparansulfate proteoglycan binds more efficientlyto acidic fibroblast growth factor. Moreover,the ECM produced by infected endothelialcells can direct the synthetic patterns of non-infected cells in a manner uniquely observedin infected endothelial cells, suggesting aplausible pathway by which infection of onlya few cells can ultimately result in the coor-dinate responses of neighboring noninfected

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cells (47). Since that gene expression is regu-lated by the surrounding ECM (38), theseresults could partially explain the drasticalterations of ECM expression as well asother products observed during chronic cha-gasic myocardiopathy in the presence of onlyfew parasites.

The modulators of ECM and the mechan-isms leading to increased ECM expressionin the inflamed myocardium during T. cruziinfection are not known. In view of the datadescribed above, the ECM produced by T.cruzi-infected cells could itself modulate theECM synthesized by other cells (38,47). Fur-ther, our findings showing that adsorption ofT. cruzi-released antigens to noninfected cellsinduced an increase in ECM production bysensitized monkey epithelial cells and mu-rine fibroblast and skeletal muscle cells sug-gest that parasite antigens could directly con-tribute to the establishment of ECM abnor-malities and tissue remodeling during acuteand chronic Chagas’ disease (13). It shouldalso be considered that soluble factors suchas cytokines, chemokines and growth fac-tors produced by T. cruzi-infected cells orparasite antigen-sensitized noninfected cellsmay contribute to the overall alterations ofECM in target tissues during T. cruzi infec-tion. In this context, a fibroblast-stimulatingfactor was identified in cell-free conditionedmedia from T. cruzi-infected cultures of fi-broblasts, vascular smooth muscle cells andmyocytes, suggesting that the infection ofmesenchymal cells by T. cruzi could stimu-late the production of modified ECM andthereby initiate the myocardial fibrosis typi-cal of Chagas’ disease (48). More recently itwas shown that T. cruzi-infected macro-phages and cardiomyocytes and antigen-stim-ulated macrophages produce chemokines(40,42,49). Interestingly, a FN mesh sur-rounding IFN-γ-producing inflammatorycells and ECM-associated cytokine was fre-quently detected in the heart tissue of T.cruzi-infected mice (Roffê E and Lannes-Vieira J, unpublished data). Thus, it is pos-

sible that interactions of cytokines andchemokines with FN and other ECM compo-nents or enzymatically modified ECM mayaffect the distribution and pleiotropic activi-ties of these molecules in inflamed tissues,where the cytokines may act as proadhesivemolecules, strengthening cell binding to theECM, thereby promoting cell activation(36,50). Hence, presentation of cytokinesand chemokines by ECM molecules in theinflamed tissue of T. cruzi-infected micecould contribute to cell recruitment, migra-tion and activation leading to the mainte-nance of inflammatory foci in a particularmicroenvironment as well as ECM overpro-duction by mesenchymal or invading inflam-matory cells.

It is also possible that products of theinflammatory cells (such as IFN-γ, TNF-αand chemokines) detected in the heart tissueof chagasic patients (24,25) and heart andcentral nervous system of animals experi-mentally infected with T. cruzi (23,39,40 andRoffê E, Silva AA, Santiago HC, GazzinelliRT and Lannes-Vieira J, unpublished data),demonstrated to modulate ECM expression(38,46,51,52), could contribute to the in-crease in ECM expression in several tissues.Furthermore, systemic hormones such as glu-cocorticoids (53) and cytokines such asIFN-γ (54) and TNF-α (55) detected in el-evated levels in the serum of T. cruzi-in-fected mice may also induce alterations inECM expression in the target tissues. Fur-ther, local and systemically produced pro-inflammatory cytokines may modulate theexpression of adhesion molecules such asICAM-1 and VCAM-1 on the endothelialcells (56,57) and hence promote cellularinfiltration of lymphocytes and other effec-tor cells into the inflamed tissues. In fact,ICAM-1+ and VCAM-1+ blood vessels asso-ciated with CD8+-infiltrating T cells weredetected in the cardiac tissue of chagasicpatients with severe cardiomyopathy (58).More recently, we have shown that duringexperimental chronic chagasic infection there

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is a remarkable increase in the expression ofVCAM-1 on the endothelial cells of heartblood vessels (23). As shown in Figure 6,this up-regulated VCAM-1 expression wasobserved initially during the early acute phaseand paralleled the presence of perivascularVLA-4+-activated mononuclear cells. In ad-dition, ICAM-1 expression that was low onendothelial cells of cardiac tissue of nonin-fected animals was upregulated in T. cruzi-infected mice. Further, LFA-1+ and ICAM-1+

cells were detected on invading inflamma-tory cells (Marino APMP, Azevedo MIP andLannes-Vieira J, unpublished data). Usingmurine models susceptible and resistant tothe development of acute T. cruzi-elicitedmeningoencephalitis, we have demonstratedthat VLA-4/VCAM-1 interactions are cru-cial for the establishment of inflammation inthe central nervous system during chagasicinfection (Roffê E, Silva AA, Marino APMP,dos Santos PVA and Lannes-Vieira J, un-published data). Interestingly, we have re-ported that the distribution of LFA-1+VLA-4+-activated CD4+ and CD8+ T cell populationsin the myocardium mirrors the frequency ofcells expressing the CD62LLowLFA-1High

VLA-4High activation phenotype among CD4+

and CD8+ peripheral blood T cells (23), rein-forcing the importance of adhesion moleculesfor the establishment of inflammation in tar-get tissues during T. cruzi infection. In fact,this was recently confirmed using IFN-γ-and IL-12-immunodeficient mice that pre-sent a decreased myocarditis during the earlyacute infection (15 days post-infection) inparallel to a diminished expression ofVCAM-1 and ICAM-1 on endothelial cellswhen compared with wild-type C57BL/6mice. Moreover, the heart tissue of T. cruzi-infected ICAM-1-deficient mice was devoidof significant inflammation and the animalssuccumbed promptly to infection (Michai-lowsky V, Celes MRN, Marino APMP, RossiM, Gazzinelli RT, Lannes-Vieira J and SilvaJS, unpublished data). The findings discussedabove provide evidence that profound alter-

ations in the expression of ECM, cytokines,and adhesion and chemoattractant moleculesare observed in several target tissues as aconsequence of T. cruzi infection. All ofthese molecules may play a beneficial roleleading to activation, recruitment and migra-tion of cells involved in the control of theparasite but may also contribute to the estab-lishment of adverse consequences such aschronic inflammation and organ dysfunc-tion.

Interfering with the Trypanosomacruzi-host interplay

The pathogenesis of T. cruzi-elicitedmyocarditis remains unknown, but it is prob-ably multifactorial as discussed above. Re-gardless of the mechanisms, ECM alterationsmay result from and/or contribute to thechronic cardiac inflammation. Since the para-site is apparently required for the continuoussupply of “danger signals” that result in dam-age to cardiac tissue (59), the elimination ofthe parasite may cause clearance or partialregression of inflammatory lesions in targettissues. It has been shown that specific che-motherapy leads to reversibility of cardiacfibrosis in mice chronically infected with T.cruzi. Interestingly, the improvement in ECMabnormalities in benznidazole-treated micecorrelated with a drastic reduction in inflam-mation (60). Thus, we may propose that animmunomodulatory treatment that selectivelyblocks or diminishes the entry of deleteriousinflammatory cells into the cardiac tissue ofT. cruzi-infected mice could also be benefi-cial. Our recent findings showing a pre-dominance of CC chemokines, especiallyRANTES (regulated on activation normal Tcell expressed and secreted) and MIP-1α(macrophage inflammatory protein-1α), andcells bearing CC receptors, mainly CCR5, inthe cardiac tissue of T. cruzi-infected miceassociated with preferential expression ofCCR5 on peripheral blood CD8+ T cells (23and Marino APMP, Silva AA, dos Santos

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PVA, Pinto LMO, Gazzinelli RT, TeixeiraMM and Lannes-Vieira J, unpublished data)led us to propose that CC chemokines andtheir receptors are involved in differentialcell migration and pathogenesis of T. cruzi-elicited CD8-mediated myocarditis. Impor-tantly, the treatment of acutely T. cruzi-in-fected mice with methionylated RANTES,an antagonist of CC chemokines, signifi-cantly decreased the inflammatory infiltratesdue to CD4 and CD8 T cells, but decreasedor did not interfere with heart parasitismdepending on the therapeutic scheme used.Furthermore, these effects were accompa-nied by a decrease in ECM expression(Marino APMP, Silva AA, dos Santos PVA,Pinto LMO, Gazzinelli RT, Teixeira MMand Lannes-Vieira J, unpublished data), re-

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inforcing the idea that fibrosis is related tothe presence of T. cruzi and inflammation inthe cardiac tissue during chagasic infection.Thus, we propose that a therapeutic inter-vention ultimately leading to modulation ofECM expression could contribute to improv-ing the prognosis of T. cruzi-infected indi-viduals.

Acknowledgments

The authors are grateful to Patrícia A.Moreno Martinez, Department of Patholo-gy, Oswaldo Cruz Institute, Fiocruz, for Con-focal acquisition and Heloisa Diniz andRodrigo Cunha Mexas, Laboratory of Im-age, Oswaldo Cruz Institute, Fiocruz, forpreparing the figures.

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Trypanosoma cruzi infection: a continuous invader-host cell cross … · 2003. 7. 24. · sion and cell-extracellular matrix (ECM) in-teraction and is orchestrated by cell adhe-sion