Prevalence of CD8 +αβ T cells in Trypanosoma cruzi -elicited myocarditis is associated with...

Prevalence of CD8+α� T cells in Trypanosomacruzi-elicited myocarditis is associated

with acquisition of CD62LLowLFA-1HighVLA-4High

activation phenotype and expression of IFN-γ-inducible adhesion and chemoattractant molecules

Paula V.A. dos Santosa, Ester Roffêa, Helton C. Santiagob, Renata A. Torresa, Ana Paula M.P. Marinoa,Cláudia N. Paivaa, Andréa A. Silvaa,c, Ricardo T. Gazzinellib,d, Joseli Lannes-Vieiraa*

aLaboratory of Autoimmunity and Immunoregulation, Department of Immunology, IOC-Fiocruz, Av. Brasil 4365, 21045-900, Rio de Janeiro, RJ, BrazilbDepartment of Biochemistry and Immunology, UFMG, Av. Antônio Carlos 6627, 31270-910, Belo Horizonte, MG, Brazil

cDepartment of Pathology, UFF, Av. Marquês do Paraná 303, 24033-900, Niterói, RJ, BrazildLaboratory of Immunopathology, CPqRR-Fiocruz, Av. Augusto de Lima 1715, 30190-002, Belo Horizonte-MG, Brazil

(Received 26 March 2001; accepted 11 June 2001)

ABSTRACT – The determinants of the prevalence of CD8+ T cells in the inflamed myocardium ofTrypanosoma cruzi-infected patients and experimental animals are undefined. Using C3H/He miceinfected with the Colombiana strain of T. cruzi, we found that the distribution of CD4+/CD8– andCD4–/CD8+ T cells in the myocardium mirrors the frequency of cells expressing the CD62LLowLFA-1HighVLA-4High activation phenotype among CD4+/CD8– and CD4–/CD8+ peripheral blood T cells.Consistently, vascular cell adhesion molecule-1-positive endothelial cells and a fine fibronectin networksurrounding VLA-4+ mononuclear cells were found in the inflamed myocardium. Further, interferon γ(IFN-γ) and IFN-γ-induced chemokines (RANTES, MIG and CRG-2/IP-10), as well as JE/MCP-1 andMIP1-α, were found to be the dominant cytokines expressed in situ during acute and chronicmyocarditis elicited by T. cruzi. In contrast, interleukin 4 mRNA was only detected during the chronicphase. Altogether, the results indicate that the distribution of T-cell subsets in the myocardium ofT. cruzi-infected mice reflects the particular profile of adhesion molecules acquired by most peripheralCD8+ T lymphocytes and point to the possibility that multiple IFN-γ-inducible molecules present inthe inflamed tissue contribute to the establishment and maintenance of T. cruzi-induced myocarditis.© 2001 Éditions scientifiques et médicales Elsevier SAS

Trypanosoma cruzi / adhesion molecules / chemokines / inflammation

1. IntroductionThe recruitment and migration of immune cells toward

inflammatory sites in a target tissue is a multi-step processthat depends on the nature and state of activation of theinflammatory cells and is co-ordinated by receptors rec-ognising a mosaic of chemoattractant and pro-inflammatory cytokines, adhesion molecules and extra-cellular matrix components [1–6]. In infectious diseases,

the establishment of inflammatory processes is crucial forelimination of the infectious agent and resolution of theinfection in the target tissues. However, in some instancesinflammation becomes progressive, resulting in chronicdisease. For example, chronic myocarditis is frequentlyassociated with Chagas’ disease, caused by the protozoanTrypanosoma cruzi [7] and one of the leading causes ofdeath in Latin America [8]. In recent years, much effort hasbeen spent attempting to characterise the nature of theinflammatory cells present in the cardiac tissue and themolecular mechanisms involved in the establishment ofthe inflammatory process leading to resolution of parasit

*Correspondence and reprints.E-mail address: [email protected] (J. Lannes-Vieira).

Microbes and Infection, 3, 2001, 971−984© 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

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ism but also contributing to the genesis of T. cruzi-inducedchronic myocarditis [9]. In this respect, the immunohis-tochemical characterisation of the inflammatory cellspresent in the myocardium of animals experimentallyinfected with T. cruzi produced controversial results show-ing the predominance of mononuclear cells and neutro-phils [10], CD4+ T cells [11], or CD8+ T cells [12, 13] inthe inflamed tissue. In chronic cardiac chagasic patientsthe myocarditis is constituted mainly by T cells, predomi-nantly CD8+ T cells [14–17].

Concerning the cytokines that contribute to the estab-lishment of T. cruzi-induced myocarditis, it has beenshown that tumour necrosis factor (TNF)-α and interferon(IFN)-γ play a major role controlling tissue parasitism [18,19]. These two pro-inflammatory cytokines are present inthe inflamed heart of chagasic patients [14, 20] and ofexperimental animals infected with T. cruzi [21, 22], sug-gesting that they could also be involved in the establish-ment of chronic myocarditis. Interestingly, in the myocar-dium of chronic chagasic patients with severe clinicalsigns and heart inflammatory lesions, the presence of IL-4+

cells correlates with the presence of pseudocysts of T. cruziamastigotes [20].

In spite of all the information regarding inflammatorycells as well as cytokines expressed during myocarditiselicited by T. cruzi, the factors determining the composi-tion of the inflammatory infiltrates and contributing to therecruitment, migration, accumulation and positioning ofinflammatory cells into the cardiac tissue during T. cruziinfection are largely unknown. We carried out this study inorder to investigate the mechanisms involved in the recruit-ment and maintenance of CD8+ T cells in the myocardiumof C3H/He mice infected with the T. cruzi Colombianastrain. Emphasis was placed on molecules that could beinvolved in the migration of inflammatory cells and in theperpetuation of the myocarditis, namely adhesion mol-ecules, chemokines, T. cruzi antigens, fibronectin (FN)and its receptors. We found that a high percentage ofcirculating CD8+ T cells as well as most of the T cellspresent in myocardium of T. cruzi-infected animals expressan activation phenotype characterised as L-selectinLow

(CD62LLow), leukocyte function-associated antigen 1High

(LFA-1High) and very late activation antigen 4High (VLA-4High). In addition, our data indicate that various IFN-γ-inducible factors, such as vascular cell adhesion mol-ecules 1 (VCAM-1) and FN, as well as chemokines namelyRANTES (regulated upon activation, normal T-cellexpressed and secreted), MIP-1α (macrophage inflamma-tory protein 1α), MIG (monokine induced by IFN-γ) andCRG-2/IP-10 (cytokine response gene 2/interferonγ-inducible protein 10) are highly expressed in the myo-cardium of infected animals and may contribute to theintense recruitment of activated T cells, and therefore tothe establishment and maintenance of T. cruzi-elicitedmyocarditis.

2. Materials and methods2.1. Animals

Five- to seven-week-old female C3H/He (H-2k) micewere obtained from the animal facilities of the Oswaldo

Cruz Foundation (Rio de Janeiro, Brazil). The animalswere maintained under standard conditions in the animalhouse and all experimental procedures were conductedaccording to institutional guidelines for animal ethics ofthe Oswaldo Cruz Foundation.

2.2. Parasites and experimental infection

Mice were infected intraperitoneally with 100 bloodtrypomastigote forms of the Colombiana type III strain ofT. cruzi [23] isolated from a cardiac chagasic patient [24]and maintained by serial passages from mouse to mouse inthe Laboratory of Autoimmunity and Immunoregulation,IOC-Fiocruz (Rio de Janeiro, Brazil). Parasitaemia wasestimated using 5 µL of blood obtained from the tail veinaccording to Brener’s method [25], and employed as aparameter to establish acute and chronic phases.

2.3. Antibodies

Specific polyclonal antibody recognising T. cruzi anti-gens was a gift from Dr Rosa Teixeira de Pinho (Depart-ment of Immunology, Oswaldo Cruz Institute-Fiocruz,Brazil). Polyclonal antibodies specific for the extracellularmatrix glycoprotein FN, biotinylated monoclonal antibod-ies anti-CD8a-FITC (clone 53-6.7), anti-CD4-PE (GK1.5),anti-T-cell receptor α�-biotin (clone H57-597), purifiedanti-TCRγδ (clone UC7-13D5), anti-CD62L-biotin (cloneMEL-14), anti-CD3-biotin (clone 145-2C11), anti-CD11a-FITC (CD11a/CD18b or LFA-1, clone M17/4), anti-NK(Ly49C antigen, clone 5E6), anti-VLA-4-biotin (CD49d,α-4 chain, clone 9C10), and anti-VCAM-1-biotin (CD106,clone 51-10C9) were purchased from PharMingen (USA).Purified antimacrophage (clone F4/80) antibody, purifiedantineutrophils (clone 7/4), and purified anti-B cell(CD45R, B-220, clone RA3-6B2) were purchased fromCaltag Laboratories (USA). Biotinylated antibodies recog-nising rat or rabbit immunoglobulin, peroxidase-streptavidin complex were purchased from Amersham(UK), TRITC-labelled anti-rabbit immunoglobulin andFITC-labelled anti-rat immunoglobulin were purchasedfrom American Qualex (USA). Appropriate controls wereprepared by replacing primary antibodies with purified ratimmunoglobulin or normal rabbit serum.

2.4. Histopathological studies

Groups of five infected mice were sacrificed underanaesthesia at various time points postinfection. Groups ofthree age-matched control mice were sacrificed at thesame time points. The heart was removed, embedded intissue freezing medium (O.C.T., Tissue Tek, Miles Labora-tories, USA) and stored in liquid nitrogen. Serial 5–7-µm-thick sections were prepared and fixed in cold acetone.These sections were stained with haematoxylin and eosin(H&E) or subjected to indirect immunoperoxidase stainingor immunofluorescence. The H&E-stained sections wereexamined using light microscopy. Tissue parasitism wasscored by counting the total amastigote nests in eachsection (50–80 microscopic fields, 1 × 250 original mag-nification). For the inflammatory infiltrate score, the num-ber of inflammatory foci presenting ten or more cells permicroscopic field was counted in each section and char-acterised as diffuse or focal, depending on how closely the

Original article dos Santos et al.

972 Microbes and Infection2001, 971-0

inflammatory cells were associated, as we previouslydescribed [22].

2.5. Immunohistochemical characterisationof inflammatory infiltrates present in thecardiac tissue of infected mice

The indirect immunoperoxidase technique was devel-oped as previously described [26]. Briefly, serial cryostatsections were mounted on poly-L-lysin-covered glass slidesand fixed for 10 min in cold acetone. Endogenous peroxi-dase and non-specific antibody binding were blocked byincubating the specimens with phosphate-buffered saline(PBS) containing 0.1% sodium azide and normal goatserum diluted 1:50 (Sigma, USA). Thereafter sequentialincubations with primary unlabeled antibodies (anti-T. cruzi antigens, anti-FN or anti-cell markers) or species-matched control immunoglobulins, secondary biotiny-lated antibodies (donkey anti-rabbit immunoglobulin orgoat anti-rat immunoglobulin) and streptavidin-peroxidasecomplex were performed. All incubations were performedfor 1 h with antibodies diluted in PBS containing 1% BSA(Sigma, USA), and were followed by washes in PBS. Theperoxidase reaction was developed with 9-amino-3-ethylcarbazole in the presence of hydrogen peroxide. Thematerial was counter-stained with Mayer’s haematoxylin.Sections of spleen were used as positive controls forlymphocyte staining. Slides were examined using lightmicroscopy. The cells stained for T-cell receptor (TCR)CD4 and CD8 were counted, considering that positivecells were identified by a partial or complete ring of darkcolour outlining the cell membrane. Three sections werecounted for each animal and the data presented as averageand standard deviation of each group of animals.

2.6. Flow cytometry preparations and analysis

This assay was performed as previously described [27].Briefly, animals were sacrificed by blood removal throughcardiac puncture under anaesthesia. Spleen cell suspen-sions were prepared by homogenising the organs in PBScontaining 2% foetal calf serum (FCS) (Hyclone, USA).The suspension obtained was haemolysed in red bloodcell lysis solution (Sigma, USA) and washed in PBS. Sus-pensions of blood mononuclear cells were prepared bypooling 0.3 mL heparinised individual samples (fiveanimals/group) and performing Fycoll Hypaque™(d = 1.077 g/mL) separation. The cells recovered werewashed and resuspended in 1 mL of FCS-PBS. In order toprepare the mononuclear cell suspensions, 10–15 heartswere washed to remove blood clots, minced with scissorsin 1–2-mm fragments and subjected to enzymatic diges-tion using a solution containing 0.015% trypsin (Sigma,T4799, USA) and collagenase A (Boehringer Mannheim,103586, Germany), as previously described [22, 28].

Indirect immunofluorescence labelling proceduresinvolved the sequential incubation of 106 (spleen andblood) or 105 (heart mononuclear cells) viable mono-nuclear cells with primary fluorochromo-conjugated orunlabeled purified antibodies; secondary biotinylated anti-rat (Amersham, UK) and tertiary streptoavidin-PE (Caltag,USA) accompanied by either anti-CD4-FITC (clone GK1.5)or CD8a-FITC (clone 53-6.7), purchased from PharMin-

gen. Optimal concentrations of each antibody were deter-mined in preliminary experiments. All incubations wereperformed for 1 h at 4 °C with antibodies diluted in PBScontaining 1% of normal mouse serum and 2% of FCS,and were followed by washes in a similar medium, tominimise non-specific labelling. Controls of specific label-ling were prepared as described above, replacing the firstincubation by the same mass of normal rat immunoglobu-lin. Indirect immunofluorescence was performed incubat-ing anti-TCR α� chain-biotin, anti-CD62L-biotin or anti-VLA-4-biotin, followed by incubation with streptavidin-Cy-chrome (PharMingen, CA, USA), accompanied by anti-CD11a-FITC, anti-CD4-PE and anti-CD8a-FITC or anti-CD8a-PE. Samples were fixed in cold PBS containing 1%formaldehyde. One-colour-labelled samples were pre-pared to set compensation values.

Flow cytometry studies were performed using a FACS-calibur (FACScalibur, Becton Dickinson, CA, USA). Lym-phocytes were gated using a narrow forward-angle lightscatter parameter to exclude dead cells from analysis. Atleast 10 000 cells were acquired inside this gate. Fluores-cence gates were cut in accordance to labelling controls,respecting curve inflexions. Cytometric analyses were per-formed using the program WinMDI Version 2.5.

2.7. RT-PCR assay for measuring in vivo expressionof cytokine and chemokine mRNA

This assay was performed as previously described [22,29]. Briefly, RNA was isolated from cardiac tissue of miceby acid guanidinium thiocyanate-phenol-chloroformextraction: RNA STAT-60™. Total RNA (0.5 µg) was reversetranscribed by the addition of 10 U RNAsin and 15 ρM ofoligo DT15 (Promega Corp., Madison, WI, USA), AMVReverse Transcriptase (RT) (GIBCO BRL) in 25 µL reactioncontaining 250 mM DNTP, 50 mM Tris-HCl, pH 8.3,75 mM KCl, 3 mM MgCl2, 10 mM DTT. The mixtureswere incubated for 5 min at 95 °C, 5 min on ice and 5 minat 25 °C, at this step 100 U of RT were added to eachsample and the reaction mixture was incubated for 60 minat 37 °C. The temperature was then elevated to 95 °C for5 min and tubes were transferred to ice for 5 min. ThecDNA products were diluted in 0.2 mL of sterile distilledwater and used at 5 µL per reaction for PCR amplifica-tions. The PCR was performed in 25 µL reaction of samplesdiluted in the following buffer: 250 mM of DNTP, 10 mMTris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 10 mM ofeach primer and 0.5 U of Taq polymerase (CENBIOTIC).After initial incubation for 3 min at 95 °C the cycles were:denaturating 1 min at 94 °C; annealing 1 min at 54 °C;and extension 2 min at 72 °C. After the designated cyclenumbers for each primer, the program executed a finalextension of 7 min at 72 °C. The PCR products andmolecular weight marker were electrophoresed in 6%polyacrylamide gel and stained with silver nitrate. Densi-tometry of gels was carried out on a DensitometerCS-9301PC (Shimadzu, Tokyo, Japan). The PCRs werestandardised using HPRT. The primer sequences used tomeasure the messages of IFN-γ, TNF-α and IL-4 were usedin our previous publication [22, 29]. The primer sequencesused are listed below (sense and anti-sense sequence, PCRproduct size and numbers of cycles are indicated. CXC

Adhesion molecules and chemokines in T. cruzi infection Original article


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chemokines: KC: CGC GGA TCC TTG ACC CTG AAGCTC CCT TGG TTC, CGC GGA TCC CGT GCG TGT TGACCA TAC AAT ATG, 521 bp, 35 cycles; CRG-2/IP-10:CGC GGA TCC TGA GCA GAG ATG TCT GAA TC, CGCGGA TCC TCG CAC CTC CAC ATA GCT TAC AG, 399 bp,32 cycles; MIG: GAT CAA ACC TGC CTA GAT CC, GGCTGT GTA GAA CAC AGA GT, 399 bp, 32 cycles; CCchemokines: MIP-1α: CGC GGA TCC CGG AAG ATTCCA CGC CAA TTC, CGC GGA TCC GGT TGA GGAACG TGT CCT GAA G, 448 bp, 32 cycles; MIP-1�: CGCGGA TCC CCC ACT TCC TGC TGT TTC TCT TAC, CGCGGA TCC AGC AGA GAA ACA GCA ATG GTG G,444 bp, 33 cycles; RANTES: CGC GGA TCC CCA CGTCAA GGA GTA TTT CTA CAC C, CGC GGA TCC CTGGTT TCT TGG GTT TGC TGT G, 326 bp, 26 cycles;JE/MCP-1: CCG GAA TTC CAC TCA CCT GCT GCT ACTCAT TCA C, CCG GAA TTC GGA TTC ACA GAG AGGGAA AAA TGG, 505 bp, 30 cycles. HPRT: GTT GGA TACAGG CCA GAC TTT GTT G, GAT TCA ACT TGC GCT CATCTT AGG C, 162 bp, 30 cycles.

2.8. Statistical analysis

Arithmetic or geometric means and standard deviationsof the means were calculated. Student’s t-test was used toanalyse the statistical significance of the observed differ-ences. Differences were considered statistically significantwhen P < 0.05.

3. Results

3.1. Parasitaemia, mortality curve and hearthistopathology of C3H/He mice infectedwith T. cruzi Colombiana

The course of parasitaemia was the criterion used tocharacterise the acute and chronic phases of T. cruzi infec-tion. Circulating parasites were detected 14 days postin-fection (p.i.) of C3H/He mice with 100 parasites of T. cruziColombiana. The peak of parasitaemia occurred 42 daysp.i. and trypomastigotes were rarely found in the blood at63 days p.i., characterising the onset of the chronic phase,as previously described [24, 30]. Around 70% of theinfected mice survived the acute infection and developeda chronic phase (data not shown).

A diffuse inflammatory process was first detected in thecardiac tissue of Colombiana strain-infected C3H/He mice14–21 days p.i. by H&E staining and predominated duringthe initial weeks postinfection. During the early acuteinfection (21–28 days p.i.) amastigote forms were alsofrequently seen inside myocytes, forming large clusters,but no inflammation surrounded the infected cells (figure1A). The peak of cardiac tissue parasitism coincided withthe peak of parasitaemia (42 days p.i.), and at this momentmost of the infected myocytes were surrounded by inflam-matory cells (figure1B). After 63 days p.i. the tissue para-sitism decreased and the parasite pseudocysts were hardlydetected 90 or 120 days p.i.. Although to a lesser degree,focal and diffuse inflammatory infiltrates were detectedduring chronic infection (figure 1C). Normal controls werecompletely devoid of cellular infiltrates (data not shown).

3.2. T. cruzi antigens and inflammatorymononuclear cells present in the cardiac tissueof Colombiana strain-infected C3H/He mice

Figure 2 shows that, using an anti-T. cruzi polyclonalantiserum, the presence of T. cruzi antigens was abundantin acutely (42 days p.i.) infected mice and frequentlyassociated with pseudocysts (figure 2A,B). Conventionalhistopathological examinations showed that during

Figure 1. Histopathological analysis of T. cruzi parasitism andinflammatory infiltrates in the cardiac tissue of C3H/He mice.Panel A shows an amastigote nest containing hundreds of para-sites in the absence of inflammatory infiltrates at 21 days p.i.Panel B shows a nest of amastigotes surrounded by mononuclearinflammatory cells at 42 days p.i. As depicted in C, inflammatoryinfiltrates in the absence of amastigote nest was a commonfinding during chronic infection (120 days p.i.). Arrows indicatethe presence of amastigote nests. H&E. Original magnifica-tion, ×400.



chronic infection the majority of inflammatory lesionsfound in the vicinity of muscle fibres were devoid ofmorphologically identifiable parasites (figure 1C). Despitethese negative results, we were able to detect parasiteantigens in the cardiac tissue of all chronically (120 daysp.i.) infected animals studied, although the positive areaswere scarce, and sometimes several sections of the sameanimal had to be stained in order to find a T. cruzi antigen-positive area (figure 2C,D).

The phenotypic nature of the cells composing theinflammatory infiltrates present in the cardiac tissue ofT. cruzi-infected mice was characterised by means ofimmunohistochemical and flow cytometric assays. Immu-nohistochemically it was demonstrated that during thechronic phase both antigen-free and antigen-containinginflammatory infiltrates were mainly composed of CD8+

cells, although CD4+ cells were also detected in thoseareas (figure 2C–H). In fact, CD8+ lymphocytes outnum-bered CD4+ cells by a factor of two to three in both acutely(42 days p.i.) and chronically (90 and 120 days p.i.)infected mice, as depicted in table I. In addition, a fewmacrophages, characterised as F4/80+ (2–9%) cells, andB220+ B cells (2–3%) were detected in the inflamed myo-cardium of T. cruzi-infected mice during acute and chronicinfections, whereas rare neutrophils were only found dur-ing the chronic phase, and NK cells were never detected(data not shown).

The study of serial sections of cardiac tissue showedcells expressing T-cell receptor � chain in the same areaswhere CD8+ and CD4+ cells were detected, indicatingthat most of the CD8+ and CD4+ cells were α�+ T lympho-cytes (data not shown). These findings were confirmedwhen the mononuclear cells were enzymatically isolatedfrom the inflamed myocardium. In fact, the cytofluorimet-ric analysis showed that all CD8+ and CD4+ cells isolatedfrom the inflamed myocardium during acute and chronicinfection were α�+ T lymphocytes (figure 3). Although aslight increase in the frequency of CD4+ cells was consis-tently observed during chronic infection, the predomi-nance of CD8+ T cells during the acute and chronic phaseswas confirmed and the CD4/CD8 ratio was 0.26–0.40.The few mononuclear cells isolated from normal hearttissue could not be characterised by the panel of antibod-ies used. However, most of the obtained T cells wereCD4+, while a small proportion was CD8+ (figure 3). NoCD4–CD8– (double negative) α� T cells were isolated fromthe myocardium of normal and T. cruzi-infected C3H/Hemice. A small population (1–3%) of CD4+CD8+ (doublepositive) α� T cells was consistently detected among theinflammatory cells isolated from chronically infected mice,while this population was barely measurable (< 0.7%)during acute infection (data not shown).

3.3. Phenotypic analysis of mononuclear cells present inthe peripheral blood and lymphoid tissues of Colombianastrain-infected C3H/He mice

In order to investigate whether the predominance ofCD8+ T cells in the inflamed myocardium was a result ofthe influx of the predominant cell population present inthe peripheral blood and lymphoid tissues (paracardiaclymph nodes and spleen) of T. cruzi-infected mice, we

analysed the cell populations present in these tissues.Immunohistochemical analysis showed that T. cruzi anti-gens were present in both lymphoid and cardiac tissue(figure 4B) and that most of the mononuclear cells presentin the paracardiac lymph nodes were CD4+, whereasCD8+ cells predominated in cardiac tissue (figure 4C,D).

Three independent experiments showed that a signifi-cant (P < 0.001) decrease in the proportion of circulatingCD4+ T cells (from 39.3–41.1% in normal mice to24.0–26.6% in infected mice) and a clear increase(P < 0.001) in the percentage of CD8+ T cells (from15.2–15.3% in normal mice to 32.6–36.5% in infectedmice) were observed during acute infection. Interestingly,minor changes were detected in the percentages of CD4+

T cells (from 46.2–48.5% in normal mice to 37.4–39.8%in infected mice) and CD8+ T cells (from 15.0–18.7% innormal mice to 18.5–29.3% in infected mice) during thechronic phase.

During acute T. cruzi infection, C3H/He mice exhib-ited a severe splenomegaly with splenocyte numbers 2–4-fold higher than those of normal matched controls.Although during chronic infection the T. cruzi-infectedmice still displayed enlarged spleens compared to normalcontrols (1.2–2-fold), the splenomegaly was not as severeas that observed during acute infection (data not shown).The analysis of T-cell populations present in the spleenshowed a slight decrease in the percentage of CD4+ T cellsat day 42 postinfection (from 19.4–21.0% in normal miceto 17.0–17.5% in infected mice) in three independentexperiments. However, a large increase (P < 0.001) in thepercentage of CD8+ T cells (from 7.6–8.3% in normalmice to 16.7–18.0% in infected mice) was detected. Byday 120 postinfection, however, both CD4+ T-cell(23.4–25.6% in normal mice and 22.1–23.5% in infectedmice) and CD8+ T-cell (10.9–12.0% in normal mice and8.9–10.6% in infected mice) compartments were similarin normal and T. cruzi-infected mice.

Taken together, these data indicate that the predomi-nance of CD8+ T cells in the cardiac tissue of T. cruzi-infected mice does not result only from an influx of thepredominant cell population of lymphoid organs into theblood and from there into the cardiac tissue, especiallyduring chronic infection.

3.4. Expression of adhesion molecules by mononuclearcells isolated from myocardium, peripheral bloodand spleen of Colombiana strain-infected C3H/He mice

The molecular mechanisms involved in the recruitmentand accumulation of inflammatory cells into the cardiactissue during Chagas’ infection are unclear. To shed lighton this question, we studied the expression of CD62L,LFA-1 and VLA-4, molecules demonstrated to have afunctional participation in T-cell activation and migration[31, 32], by lymphocytes isolated from myocardium, bloodand spleen of mice chronically infected with T. cruzi. Ourresults show that most of the CD8+ (73–82%) and CD4+

(70–90%) T cells in heart infiltrates did not express CD62Lor expressed low levels (CD62LLow/–), and almost all CD8+

and CD4+ cells expressed high levels of LFA-1 (LFA-1High)(figure 5A,B). Also, as shown in figure 5A,B, the majority ofCD8+ and CD4+ T cells isolated from the myocardium



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during chronic infection expressed high levels of CD49d(VLA-4High), characterising them as activated/memorycells. Taken together, these data indicate a predominanceof activated/memory CD4+ and CD8+ populations in thecardiac tissue of T. cruzi-infected C3H/He mice. Interest-ingly, the predominance of CD8+ T cells in the myocar-dium reflected the prevalence of cells expressingCD62LLow/–, LFA-1High and VLA-4High among peripheralblood CD8+ T lymphocytes of T. cruzi-infected mice,whereas only a small population of the circulating CD4+ Tcells expressed this phenotype (figure 5A,B). Further, apredominance of LFA-1High VLA-4High CD8+ T cells wasalso found in the spleens of mice chronically infected withT. cruzi. For instance, in one representative experiment,65% of the CD8+ T cells were CD8+VLA-4High, whereasamong CD4+ T cells only 35.8% were CD4+VLA-4High

(figure 5A). Altogether, these results strongly suggest that inT. cruzi-infected C3H/He mice the predominance of CD8+

T cells in the cardiac tissue reflects the prevalence of aCD62LLow/– LFA-1High VLA-4High activated/memory T-cellsubset among CD8+ lymphocytes in the peripheral bloodand in the spleen of chronically infected mice.

3.5. Expression of adhesion and chemoattractantmolecules in the myocardium of Colombiana strain-infected C3H/He mice

To study the molecules involved in the transendothelialmigration and positioning of inflammatory cells into thecardiac tissue of T. cruzi-infected mice, we evaluated thein situ expression of VCAM-1 and FN, ligands of VLA-4[32]. The endothelial cells of myocardial blood vessels ofnormal mice did not express VCAM-1 or presented dullexpression (figure 6A), while during both acute and chronicphases the expression of VCAM-1 in the cardiac tissue ofT. cruzi-infected mice was increased (figure 6B). Also, anincreased expression of FN was observed on the endothe-lial cells and myocytes (figure 6C,D) during acute andchronic infection. Moreover, a fine FN network (figure 6D)filled with α4+ mononuclear cells (figure 6F) was detectedin the myocardium during acute and chronic infection.

Further, the analysis of the chemoattractant moleculespresent in the heart tissue of T. cruzi-infected CH3/Hemice demonstrated that RANTES, a C-C chemokine relatedwith the presence of activated T lymphocytes in inflam-matory sites [33], was detected in high levels in the

Figure 2. The presence of T. cruzi antigens was shown andphenotypic analysis of inflammatory infiltrates in frozen sectionsof the cardiac tissue of C3H/He infected mice was performed byimmunohistochemistry using anti-T. cruzi polyclonal serum andmonoclonal antibodies recognising CD4 (clone GK1.5) and CD8(clone 53-6.7) molecules. A shows a tissue section of acutely(42 days p.i.) infected mice presenting several antigen-positiveareas corresponding to amastigote nests surrounded or not byinflammatory infiltrates. Original magnification, ×100. B showsthat diffuse staining of T. cruzi antigens was detected in areaspresenting amastigote forms of the parasite during the acute andchronic (shown here) phases of infection (original magnifica-tion, ×400). The presence of parasite antigens and phenotype ofinflammatory cells were analysed in serial sections (C, E and G; D,F and H) of cardiac tissue of C3H/He mice chronically (120 daysp.i.) infected with T. cruzi. The predominance of CD8+ lympho-cytes was observed in inflamed areas in the absence (C) or presence(D) of T. cruzi antigens; E and F refer to CD8+ lymphocytes, andG and H to CD4+ lymphocytes. Original magnification, ×400.

Table I. Total T cells, CD4+ and CD8+ subpopulations,and CD4/CD8 ratio present per microscopic field ofcardiac tissue sections of C3H/He mice infected with theColombiana strain of T. cruzi.

Dayspostinfection

Total Tcells

CD4+ cells CD8+ cellsCD4+/CD8+

ratio

35 63.5 ± 2.1a 15.5 ± 2.1 48.5 ± 0.7 0.3242 113 ± 45.7 29.7 ± 14.6 83.4 ± 31.6 0.3563 129 ± 9.9 37 ± 4.2 92 ± 5.6 0.4090 108 ± 49.4 27.6 ± 17.9 80.3 ± 32.5 0.34120 160 ± 67 43 ± 9.6 117 ± 58.4 0.37

a Three serial sections of five animals per experimental group wereanalysed at 400× magnification.



cardiac tissue during both acute and chronic phases (fig-ure 7). MIP-1α mRNA expression showed the same patternand paralleled the predominance of CD8+ T cells, whereasa small increase in the expression of MIP-1� mRNA wasdetected during chronic infection, when a small consistentincrease in the proportion of CD4+ and CD4+CD8+ T cellswas observed. The C-C chemokine JE/MCP-1 (monocytechemoattractant protein 1), the IFN-γ-induced non-ELR-C-X-C chemokines CRG-2 and MIG (figure 7), and theELR-C-X-C chemokine KC mRNA (data not shown) werealso detected in the cardiac tissue of T. cruzi-infectedC3H/He mice during both acute and chronic infection.

3.6. Cytokine expression in the cardiac tissueof Colombiana strain-infected C3H/He mice

An increased expression of both TNF-α and IFN-γmRNA was observed in the cardiac tissue of C3H/He miceacutely and chronically infected with T. cruzi. The pre-dominance of these cytokines was clearly observed duringthe acute phase (42 days p.i.). Interestingly, IL-4 mRNAwas not detected during this period, while significantlevels of this cytokine were detected during chronic infec-tion, especially at a later time point, 120 days p.i. (figure8), when parasite antigens were rarely detected and thefocal inflammatory infiltrates persisted.

4. DiscussionIn the present study, we provide evidence that the

predominance of TCRα� CD8+ cells in T. cruzi-antigen-

free and antigen-containing inflammatory infiltratesdetected in the myocardium during chronic infectionreflects a particular profile of the expression of CD62LLow/–,LFA-1High and VLA-4High on peripheral blood TCRα� CD8+

cells. Moreover, we provide evidence to support thatVCAM-1, FN, IFN-γ-induced chemokines and parasiteantigens may contribute to the recruitment, migration andpositioning of inflammatory cells into the heart tissue,leading to the perpetuation of myocarditis in T. cruzi-infected mice.

Using immunohistochemical and flow cytometricassays we showed the predominance of TCRα� CD8+ overCD4+ cells in the cardiac tissue during both acute andchronic infection of C3H/He mice with T. cruzi Colombi-ana. Although others have found the predominance ofmononuclear cells and neutrophils [10] and CD4+ T cells[11] in the cardiac tissue of mice chronically infected withT. cruzi, our results are in agreement with the demonstra-tion that CD8+ cells are the predominant cell populationin the cardiac tissue of chronic chagasic patients [14–17,20], rats acutely infected with T. cruzi [12] and acutelyand chronically infected mice [13, 34]. Interestingly, theCD4/CD8 ratios observed in the heart infiltrates duringchronic infection (0.26–0.40) were similar to thoseobserved in chronic cardiac chagasic patients [14, 15].This result led us to consider that the infection of C3H/Hemice with T. cruzi Colombiana was an appropriate modelto study the differential contribution of inflammatory cellpopulations leading to the establishment of chronic cha-gasic myocarditis.

Figure 3. Flow cytometric analysis of the mononuclear cells obtained from cardiac tissue of normal and T. cruzi-infected C3H/He miceshows that all isolated CD4+ and CD8+ lymphocytes are TCRα�+. The predominance of CD8+ T cells was observed in mice acutely(42 days p.i.) and chronically (120 days p.i.) infected with T. cruzi, while in normal mice the prevalence of CD4+ T cells was observed. Theseresults represent three replica experiments. Each experimental group consisted of 10–15 mice.



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Our results demonstrating that especially during chronicinfection, the predominance of CD8+ T cells in cardiactissue was not a result only of the influx of the predominantcell population in the immune compartments (lymphoidtissues and peripheral blood) led us to investigate themolecules involved in the recruitment and accumulationof inflammatory cells in cardiac tissue, leading to chronicmyocarditis. Initially we investigated the expression ofCD62L, LFA-1 and VLA-4 on cells isolated from hearttissue, peripheral blood and spleen of T. cruzi-infectedmice. The functional participation of these molecules inT-cell activation and migration has been extensively dem-onstrated in other models [31, 32]. Moreover, the effi-ciency of lymphocyte activation correlates with optimallevels of expression of LFA-1 and VLA-4 [35] and low (orabsent) levels of CD62L (L-selectin), and this was used asour criteria for activated lymphocytes [36]. Our resultsshow that a large proportion of peripheral blood CD8+ Tcells present the CD62LLow/–, LFA-1High and VLA-4High

phenotype, while a minor CD4+ population bears thisactivated phenotype. The persistence of high levels ofactivated lymphocytes in the spleen during the chronicphase of murine T. cruzi infection was described manyyears ago [37]. Here we confirm and extend this resultshowing that in the spleen of T. cruzi-infected mice theactivated VLA-4High phenotype was mainly detectedamong the CD8+ T cells. Using the absence of CD28 as anactivation marker, it was shown that a large proportion of

the CD8+ T cells present in the blood of cardiac chagasicpatients were CD28-negative while a minor proportion ofCD4+ T cells had this activation phenotype, although theCD4/CD8 ratio in peripheral blood was similar to that ofnormal individuals [38]. Thus, it is tempting to speculatethat the predominance of CD8+ T cells in the inflamedmyocardium of the T. cruzi-infected host may be a resultof the predominance of CD8+ expressing the activatedphenotype which is acquired in peripheral organs of theimmune system. However, our previous report showingthe presence of encephalitis, with inflammatory infiltratesmainly composed of CD8+ T cells, restricted to the acutephase in these Colombiana strain-infected C3H/He mice[30] provides evidence that the persistence of chronicmyocarditis with the predominance of CD8+ T cells doesnot result only from the activation phenotype expressed byperipheral blood CD8+ T cells. Thus, other factors presentin the cardiac environment could also be involved in themigration, retention and activation of inflammatory cellsleading to perpetuation of myocarditis in these T. cruzi-infected mice. To approach this question we investigatedthe expression of adhesion molecules, FN, cytokines andchemokines in the cardiac tissue of these mice. Weobserved that cardiac endothelial cells of T. cruzi-infectedC3H/He mice express VCAM-1, a VLA-4 ligand, duringacute and chronic infection, whereas the expression ofVCAM-1 by the endothelial cells of the blood vessels ofthe central nervous system of these mice is restricted to the

Figure 4. Phenotypic analysis of the inflammatory infiltrates present in the cardiac tissue and paracardiac lymph node (LN, broken line)of mice chronically (120 days p.i.) infected with T. cruzi. Panel A shows H&E staining. B shows that T. cruzi antigens were detected in theparacardiac LN and cardiac tissue. CD4+ lymphocytes predominated in the LN (C), while predominance of CD8+ lymphocytes wasobserved in the cardiac tissue (D). Arrows indicate the presence of T. cruzi antigens. Original magnification, ×100.



Figure 5. Flow cytometric analysis of the mononuclear cells isolated from the cardiac tissue, peripheral blood and spleen of C3H/He micechronically (90 days p.i.) infected with T. cruzi. A represents one of three independent experiments. The left column shows that thepredominance of CD62LLow/– cells was observed among CD4+ and CD8+ lymphocytes isolated from the cardiac tissue (top panel). Themiddle panel shows that most of the CD8+ T lymphocytes isolated from the peripheral blood were CD62LLow/–, while a minor CD4+

population presented this phenotype. In the spleen (bottom panel) a similar frequency of CD62LLow/– expressing cells was observed amongCD4+ and CD8+ lymphocytes. The middle column shows that most CD4+ and CD8+ lymphocytes isolated from the cardiac tissue wereLFA-1High (top panel). A large proportion of peripheral blood (middle panel) and splenic (bottom panel) CD8+ lymphocytes wereLFA-1High, while a few CD4+ lymphocytes were LFA-1High in these compartments. The right column shows that most CD4+ and CD8+

lymphocytes isolated from the cardiac tissue were VLA-4High (top panel). The middle panel shows that a large proportion of peripheralblood CD8+ cells expressed VLA-4High while only a minor population of CD4+ T cells presented this phenotype. Similar results wereobserved in the cells isolated from the spleen (bottom panel). B: Another independent experiment showed that the majority of the CD4+

and CD8+ T cells isolated from the cardiac tissue expressed the activation CD62LLow/–, VLA-4High and LFA-1High phenotype. While in theperipheral blood most of the CD8+ T cells presented this activated phenotype, only a minor CD4+ population were CD62LLow/–, VLA-4High

and LFA-1High. The results represent the percentage of positive cells for each analysed phenotype. The data represent means of three to fivepools (5–15 animals/pool) and the SD were 8–15% of each obtained value.



979

acute phase (Roffê et al., manuscript in preparation). Fur-ther, it was demonstrated that cardiac endothelial cellsexpress intercellular adhesion molecule-1 (ICAM-1), theLFA-1 ligand, during T. cruzi infection [39]. Altogether,these findings suggest that VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions could contribute to the attachment ofcirculating activated T cells to the activated endothelium,leading to selective migration of these inflammatory cellsinto the T. cruzi-infected myocardium.

Another interesting point concerns the finding that mostof the CD4+ and CD8+ T cells present in the inflamedmyocardium of mice acutely and chronically infectedwith T. cruzi present the CD62LLow/–, LFA-1High and VLA-4High activated phenotype. A previous report using immu-nohistochemistry has shown that LFA-1+ and VLA-4+

mononuclear cells are present in the inflamed myocar-

dium of cardiac chronic chagasic patients [40], suggestingthat at least some of these inflammatory cells are activated.Since all CD8+ and CD4+ T cells present in the inflamedheart tissue of Colombiana strain-infected C3H/He miceduring acute and chronic infection are LFA-1+ and themyocytes express ICAM-1 [39], it is conceivable to con-sider that LFA-1/ICAM-1 interaction could also contributeto the establishment of chronic myocyte injury duringT. cruzi infection, as the ICAM-1+ myocytes could be thetargets of LFA-1+ cytotoxic T cells.

Increased expression of extracellular matrix compo-nents has been previously detected in the myocardium ofT. cruzi-infected mice during acute and chronic phases[41]. Our results confirm this finding and show that a fineFN network involves the inflammatory cells as well as themyocytes. Interestingly, we show that the interstitial FNmesh present in the inflamed heart involves VLA-4+ CD4+

and CD8+ T cells. A similar FN network surroundingVLA-4+ mononuclear inflammatory cells was demon-strated in the central nervous system during acute T. cruziinfection [42]. Concerning the source of this matrix glyco-protein, it is possible that the inflammatory mononuclearcells may contribute to the FN production, as activatedmacrophages and T lymphocytes produce this glycopro-tein [43, 44]. However, the possibility that myocytes andother cell types present in the inflamed myocardium canalso contribute to FN production cannot be ruled out. Thefine FN filamentous network present in the inflamed myo-cardium may function as a pathway in T-cell migrationprocess via VLA-4 interactions [1, 32] but may also influ-ence T-cell activation. In fact, it was demonstrated thatantibodies against VLA-4 increase the anti-CD3-inducedT-cell adherence to FN and synergistically induce T-cellproliferation [1]. Thus, it is reasonable to propose thatinteractions of CD4+ and CD8+ VLA-4+ T cells present inthe inflamed heart with the FN mesh could contribute tothe perpetuation of the inflammation in this tissue, influ-encing antigen-specific T-cell recognition, activation, pro-liferation, survival and effector activity. This hypothesis ispresently under investigation.

The FN modulators and the mechanisms leading toincreased FN expression in the inflamed myocardiumduring T. cruzi infection are not known. However, onecould consider that products of inflammatory cells (suchas IFN-γ and TNF-α) detected in inflamed heart tissue anddemonstrated to modulate FN expression [44–46] maycontribute to the observed increase in FN expression. Inaddition, systemic hormones such as glycocorticoids [47],and cytokines such as IFN-γ [48] and TNF-α [49], detectedin elevated levels in the serum of T. cruzi-infected mice,may also induce the alterations in FN expression detectedin the heart tissue, as they modulate FN production [27,44, 46]. The local and systemically produced pro-inflammatory cytokines may also modulate the expressionof adhesion molecules such as ICAM-1 and VCAM-1 onthe endothelial cells [50, 51] and hence promote cellularinfiltration of lymphocytes and other effector cells into theinflamed tissue.

In the present study, we show that TNF-α and IFN-γmRNA are present in the heart tissue during acute T. cruziinfection persisting during the chronic phase. Moreover,

Figure 6. Immunohistochemical analysis showed that VCAM-1+

endothelial cells were detected in myocardial blood vessels ofT. cruzi-infected mice. A, normal mice; B, chronically (120 daysp.i.) infected mice (×400). Immunohistochemistry detection ofan FN network involving VLA-4+ inflammatory mononuclearcells present in the cardiac tissue of mice acutely (42 days p.i.)infected with T. cruzi. C and D, The expression of FN in thecardiac tissue of normal (C) and T. cruzi-infected mice (D). E andF, The expression of VLA-4 in normal (E) and T. cruzi-infected (F)myocardium. Original magnification, ×100.



IFN-γ-secreting cells were detected in antigen-free andantigen-containing areas during the acute and chronicphase (our unpublished results). Although these cytokinesare proposed to control parasitism [20, 52], they are alsorelated to the establishment of chronic inflammatory pro-cesses [19]. Thus, it appears that the cytokines that areproduced in the heart tissue during the initial immuneresponse can also influence the regulation of the subse-quent immune reaction; our results show that the expres-sion of IFN-γ-induced chemokines, namely RANTES, MIGand CRG-2, are also increased in inflamed heart tissueduring acute infection, persisting during the chronic phase.RANTES, a C-C chemokine produced by activated T cellsand macrophages, has been identified as a T-cell-activatingfactor and has the ability to induce T-lymphocyte recruit-ment and proliferation [34, 53], whereas MIG and CRG-2,C-X-C chemokines are involved in the selective recruit-ment of Th1 cells [6]. Furthermore, the expression ofmRNA coding the C-C chemokines MIP-1α and JE/MCP-1is also enhanced in heart tissue of T. cruzi-infected C3H/Hemice. A recent study has shown that during Listeria mono-cytogenes infection, CD8+ T cells are the main source ofMIP-1α and that this chemokine has the ability to prefer-entially recruit the CD8+ T-cell subpopulation [54]. Inaddition, in vitro experiments showed that MIP-1α prefer-entially induces chemotaxis of CD8+ T lymphocytes,whereas MIP-1� is related to the preferential recruitmentof CD4+ T cells [55]. Also, it was demonstrated thatJE/MCP-1 and KC direct the migration of macrophages andneutrophils [6]. All these findings may provide an expla-nation to our results showing a predominance of activatedCD8+ T cells but also the presence of CD4+ lymphocytes,macrophages and neutrophils in the inflamed myocar-dium during chronic infection of C3H/He mice withT. cruzi Colombiana. However, further experiments char-

acterising the expression of chemokine receptors onperipheral blood and cardiac inflammatory T cells areneeded to confirm this possibility.

In addition to being essential in leukocyte recruitment,chemokines also appear to affect several other immuno-logical phenomena [6, 56]. In this respect, a recent studyhas shown that in vivo and in vitro T. cruzi-infected peri-toneal macrophages produce RANTES, MIP-1α andJE/MCP-1 and that these chemokines induce T. cruziuptake leading to enhanced nitric oxide production andcontrol of parasite replication [57]. Thus, it is possible thatthe chemokines found in the inflamed heart during T. cruziinfection could also play a role in the control of parasitismor in the survival of parasites in this tissue, contributing tothe perpetuation of myocarditis, either directly or viamodulation of other effector mechanisms.

Finally, we also detected IL-4 mRNA in the inflamedheart during chronic infection associated with the persis-tence of T. cruzi antigens. Although others were not ableto detect IL-4 in the heart tissue of T. cruzi-infected mice[58], recently we showed the presence of mRNA in hearttissue of C57BL/6 acutely and chronically infected withT. cruzi Colombiana [22], suggesting that different strainsof this parasite trigger differential cytokine profiles thatcould determine the fate of the myocarditis. In fact, recentstudy showed an association between high levels of IL-4mRNA expression and susceptibility to myocarditis elic-ited by the Brazil strain of T. cruzi [59]. Although themechanism by which increased IL-4 production is inducedinside the inflamed heart during chronic T. cruzi infectionis not clear, previous studies have demonstrated that mul-tiple or repeated stimulation of T lymphocytes via theirTCR or with anti-CD3 results in induction and increasedproduction of IL-4 [60]. It has also been demonstrated thatJE/MCP-1 costimulation of mitogen-activated splenic T

Figure 7. Kinetics of C-C and C-X-C chemokine mRNA expression in the cardiac tissue of T. cruzi-infected mice determined by RT-PCR.Total RNA was extracted from cardiac tissue of normal and T. cruzi-infected mice at different times postinfection, reverse transcribed andused as template for PCR reaction employing primers specific for HPRT, C-C (MIP-1α, MIP-1�, JE/MCP-1, RANTES) and C-X-C(CRG-2, MIG) chemokines. A: Silver-stained gel for the chemokines analysed in cardiac tissue. B: Semi-quantitative analysis of chemokineexpression. The data represent one out of three experiments. Each experimental group consisted of 10–15 mice.



981

cells increases IL-4 production, whereas MIP-1α signifi-cantly decreases IL-4 production in these cell populations[61]. It is possible that parasite antigens contribute to theexpression of IL-4 in the myocardium during chronicinfection. This possibility is in agreement with the findingthat, in chronic chagasic patients with severe myocardio-pathy, a high frequency of IL-4+ cells paralleled the pres-ence of pseudocysts of T. cruzi amastigotes [21]. Thesefindings are consistent with the hypothesis that IL-4 isdirectly involved in the increase in parasitism and decreasein immune resistance to T. cruzi infection [20, 52]. Thus,the expression of IL-4 during the chronic phase couldresult from continuous (parasite or self) antigen stimula-tion as well as from the chemokine milleu.

Considering a general model for Chagas’ pathogenesis,our results suggest that the predominance of CD8+ T cellsin the myocardium of T. cruzi-infected individuals reflects

the profile of adhesion molecules and chemokine recep-tors displayed by the circulating CD8+ T cells. Moreover,our data point to the possibility that adhesion moleculesexpressed by the cardiac endothelial cells, and cytokines,chemokines, FN and parasite antigens present in inflamedcardiac tissue contribute to the establishment and perpetu-ation of T. cruzi-induced myocarditis. Further studies mayprovide additional insights into the mechanisms by whichthese molecules contribute to the establishment and regu-lation of the immune response in the inflamed heart andmay unravel novel therapeutic strategies aimed at limitingthe inflammatory damage to cardiomyocytes besides thespecific antiparasite drugs. However, it remains to bedemonstrated by functional experiments whether the acti-vated CD8+ and CD4+ cells found within the inflamedheart are mounting a specific anti-T. cruzi and/or anautoimmune response, or represent polyclonally activatedcells. Experiments aiming to characterise the specificity ofthe T cells isolated from the heart tissue of T. cruzi-infectedC3H/He mice are presently under way.

Acknowledgments

This work was partially supported by grants from PAPES-2-Fiocruz , FAPEMIG, WHO, CNPq, FAPERJ and TDR(970728) and fellowships from CNPq (JLV and RTG),CAPES (PVAS and CNP) and FAPERJ (ER). The authors aregrateful to Heloisa Diniz and Romney Lima for preparingthe figures. We are deeply indebted to Dr Lain CarlosPontes de Carvalho (CPqGM/Fiocruz, Brazil) and Dr MauroTeixeira (UFMG, Brazil) for critically reading this manu-script.

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