Kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous IFN-γ as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi

Microbes and Infection, 2, 2000, 851−866 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457900003889/FLA

Kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous IFN-γ as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi André Talvania,b, Cristiana S. Ribeiroa, Júlio C.S. Alibertic, Vladimir Michailowskyb, Paula V.A. Santosd, Silvane M.F. Murtab, Alvaro J. Romanhab, Igor C. Almeidae, Joshua Farberf, Joseli Lannes-Vieirad, João S. Silvac, Ricardo T. Gazzinellia,b* a

Department of Biochemistry and Immunology, ICB, UFMG, Av. Antônio Carlos 6627, 31270–910 Belo Horizonte, MG, Brazil b Laboratory of Immunopathology, CPqRR - FIOCRUZ, Av. Augusto de Lima 1715, 30190–002 Belo Horizonte, MG, Brazil c Department of Parasitology, Microbiology and Immunology, School of Medicine from Ribeirão Preto, USP, Av. Bandeirantes 3900, 14049–900 Ribeirão Preto, SP, Brazil d Department of Immunology, Instituto Oswaldo Cruz - FIOCRUZ, Av. Brasil 4365, 21045–900 Rio de Janeiro, RJ, Brazil e Department of Parasitology, ICB, USP, Av. Prof. Lineu Prestes 1374, 05508–900 São Paulo, SP Brazil f Laboratory of Clinical Investigation, NIAID, NIH, 9000 Rockville Pike, 20892 Bethesda, MD, USA (Received 3 January 2000; accepted 5 April 2000)

ABSTRACT – We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of αβΤ CD3+ CD4+ CD8–, αβΤ CD3+ CD4–CD8+ lymphocytes and macrophages. The mRNA expression of the monokines IL-1β and IL12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-α mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-γ mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1α, MIP-1β, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-γ and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi. © 2000 Éditions scientifiques et médicales Elsevier SAS Trypanosoma cruzi / chemokines / macrophages / inflammation

1. Introduction The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease [1, 2]. In humans, * Correspondence and reprints Microbes and Infection 2000, 851-866

the acute infection with T. cruzi lasts for 2–4 months and is characterized by the presence of parasites in the bloodstream and different host tissues. In addition, various nonspecific symptoms and myocarditis are common features during the early stage of Chagas' disease [3]. After development of immunity, both parasitemia and tissue parasit851

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ism are controlled and myocarditis is resolved. However, a cardiopathy will reemerge in 20–40% of chronic chagasic patients 10 or 20 years after the initial infection by a yet undefined mechanism [3, 4]. Nevertheless, follow-up studies on chagasic patients suggest that the intensity of clinical symptoms as well as pathophysiological alterations that occur during the acute phase of the disease correlate positively with the severity of cardiac disease observed during the chronic phase of Chagas' disease [5, 6]. The mechanisms by which parasite replication is controlled during the acute phase of infection are not completely understood. Nevertheless, different studies using experimental models of Chagas' disease indicate the crucial role of a few cytokines (e.g., IFN-γ, TNF-α, and IL-12) in resistance to T. cruzi [7–11]. After replication in tissues from the vertebrate host, amastigotes and trypomastigotes start expressing molecules that initiate the synthesis of several pro-inflammatory cytokines, among them IL-12 and TNF-α, by cells of the macrophage lineage [12]. The IL-12 produced initiates the synthesis of IFN-γ by different cell populations from the lymphocyte lineage, such as NK cells, CD4+ CD8– αβ and CD4–CD8+ αβ T cells. IFN-γ combined with TNF-α will play a major role in resistance by activating macrophages to produce high levels of reactive nitrogen intermediates (RNI) [8, 13, 14], which will effectively control parasite replication both in vitro and in vivo [15–17]. The strong activation of the cellular compartment of the immune system by T. cruzi may also result in some side effects [18]. In fact, the parasite-elicited inflammation and immune responses appear to be largely responsible for the tissue damage and neuronal destruction in the cardiac tissue observed in the acute stage of disease. Thus, in mice lacking CD4+ CD8– αβ and/or CD4–CD8+ αβ T cells, acute myocarditis is largely abolished despite higher tissue parasitism [19–21]. However, the dynamics of the parasiteinduced inflammation in the cardiac tissue is poorly understood. The present study was undertaken to determine the kinetics of tissue parasitism, inflammatory cell infiltrates and expression of different members of the CC and CXC chemokine subfamilies, as well as other pro-inflammatory cytokines, in heart tissue of C57BL/6 mice infected with the Colombiana strain, a myotropic strain of T. cruzi. Our results show that C57BL/6 mice inoculated with 50 trypomastigotes/each developed subpatent parasitemia and an intense cardiac inflammation, which peaked around 30-60 days postinfection (p.i.) and was correlated with tissue parasitism. The inflammatory infiltrate was characterized mainly by the presence of CD4+ CD8– αβ, CD4–CD8+ αβ T cells and macrophages. Among various chemokines we found that JE, MIP-1α, MIP-1β, MIP-2 and KC mRNAs were expressed in the heart mainly at 15 and 30 days p.i., whereas RANTES, MIG and IP-10 mRNAs were enhanced during the early and late stages of infection. Cytokine expression in cardiac tissue was characterized by an early type 1 response, with elevated message of IL-12(p40) and IFN-γ and late type 2 response with dominant expression of IL-4 and IL-10. Our results also show that the downregulation of IFN-γ and most chemokine mRNA was associated with control of parasite replication in cardiac tissue. Taken together, these findings further 852

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indicate the importance of parasite components as stimulants of inflammation during experimental chagasic myocarditis.

2. Material and methods 2.1. Animals

Five- to six-week-old female C57BL/6 or outbred Swiss mice were obtained from the Oswaldo Cruz Foundation – FIOCRUZ (Rio de Janeiro, Brazil) and maintained under standard conditions in the animal house of the Centro de Pesquisas René Rachou – FIOCRUZ (Belo Horizonte, Brazil). 2.2. Parasites and experimental infection

Tissue culture trypomastigotes of the Y strain of T. cruzi [22] were used as a source of tGPI-mucins or in macrophage experiments. For the culture-derived trypomastigotes, the LLCMK2 cell line was infected with trypomastigotes and maintained in DMEM supplemented with 5% FCS at 33 °C in 5% CO2. After 4–5 days, the trypomastigotes were collected daily, centrifuged at 40 × g at 4 °C for 10 min for separation of debris, and centrifuged again at 700 × g at 4 °C for 10 min. The resulting pellet containing live parasites was used for purification of tGPI-mucins or in macrophage experiments employing live parasites. The tGPI-mucins were isolated from T. cruzi trypomastigotes as previously described [12, 23], using sequential organic extraction followed by hydrophobic-interaction chromatography on an octyl-Sepharose column (Pharmacia Biotech, Uppsala, Sweden) and elution with a propan-1-ol gradient (5–60%). The Colombiana strain was used in all in vivo experiments as well as in some in vitro experiments with inflammatory peritoneal macrophages. T. cruzi Colombiana was isolated by Frederici et al. [24] and maintained by serial passages from mouse to mouse in the Laboratory of Chagas' Disease - CPqRR-FIOCRUZ (Belo Horizonte, Brazil). C57BL/6 mice were infected intraperitoneally with 50 blood-derived trypomastigotes of T. cruzi Colombiana. The levels of parasitemia were evaluated using 5 µL of blood obtained from the tail vein of infected mice as previously described [25]. Animals were sacrificed on days 0, 15, 30, 60 and 120 p.i. and cardiac tissue was divided and stored under specific conditions for different assays. 2.3. Histologic evaluation

Groups of four animals were sacrificed on different days after T. cruzi infection. The myocardium was fixed in neutral 10% formalin, embedded in paraffin, sectioned, stained with hematoxylin & eosin (HE) and examined by light microscopy. Tissue parasitism was scored by counting the total number of amastigote nests in 12 microscope fields (1 × 100 magnification) per histopathological section. Four sections were counted for each animal and the individual data was determined as the mean result of the four sections. The data presented for each group are the mean and standard deviation of four animals. An inflammatory infiltrate was considered to be present when we detected 50 leukocytes or more in each inflamMicrobes and Infection 2000, 851-866

In vivo chemokine mRNA expression induced by T. cruzi

matory focus. In addition, the inflammatory foci were subdivided into focal and diffuse infiltrates, depending on how closely the inflammatory cells were associated. Most cells from the focal inflammatory infiltrate were in direct contact with each other, forming a continuous site of inflammation. In contrast, the diffuse inflammatory infiltrate was defined as a high density of mononuclear cells scattered throughout the cardiac tissue and, sometimes, also composed of one or more small inflammatory foci containing less than 50 inflammatory cells. For the inflammatory infiltrate score, the total number of focal or diffuse inflammatory foci was counted in 12 microscope fields (1 × 100 magnification) per cardiac section. Four sections were counted for each animal and individual data was determined as the mean result of the four sections. The data presented for each group are the mean and standard deviation of four animals. 2.4. Characterization of the inflammatory infiltrate present in cardiac tissue from infected mice

Fragments of cardiac tissue were frozen in liquid N2 and stored until use. The frozen tissue was then embedded in tissue-tek (OCT, Miles, USA), sectioned with a cryostat and fixed in cold acetone. Rat anti-mouse CD4, antimouse CD8, anti-mouse Mac-1 or anti-mouse CD3 antibodies (Pharmingen, San Diego, CA) were incubated with mouse cardiac tissue previously incubated with normal goat serum. Thereafter the sections were incubated with biotinylated secondary anti-rat antibodies, followed by incubation with a peroxidase-streptavidine conjugate. The reaction was developed with 3-amino 9-ethyl-carbazole in sodium acetate solution in the presence of H2O2. Slides were examined by light microscopy. For the confocal microscopic studies, the same methodology as described above was used, except that rat anti-mouse CD8 (Pharmingen) and anti-mouse CD4 (Pharmingen) antibodies were directly labeled with FITC and PE, respectively. Hearts of three animals were analyzed for each time p.i. For each animal, 20 inflammatory infiltrates were analyzed by confocal microscopy or conventional immunocytochemistry. 2.5. FACS analysis

In order to isolate the mononuclear cells from the myocardium, hearts from 10–20 animals, at each time p.i., were washed to remove the blood clots, pooled, minced with scissors in 1–2 mm fragments and subjected to enzymatic digestion using a solution of 0.015% trypsin (Sigma, St Louis, USA) and 0.01% collagenase A (Boehringer Mannheim Biochemicals, Mannheim, Germany). The inflammatory cells were then purified in a Ficoll HypaqueTM gradient (d = 1.077 g/mL). Flow cytometry analyses of inflammatory cells recovered from heart tissue from C57BL/6 mice infected with T. cruzi were performed as previously described [26, 27]. Briefly, single cell suspensions were then stained with FITC- or PE-labeled antibodies against CD3, CD4, CD8, TCR-αβ or MAC-1 molecules. Viable cells (3 × 105) were analyzed with a FACS 440 (Becton and Dickson, San Jose, USA) as determined by narrow forward-angle light scatter and exclusion of propidium iodide. Microbes and Infection 2000, 851-866

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2.6. Macrophage culture

C57BL/6 mice were inoculated intraperitoneally with 2 mL of 3% thioglycollate, and four days later, the elicited peritoneal exudate cells were harvested in cold serum-free DMEM [12, 13]. The medium used in the macrophage cultures (MacMed) consisted of DMEM (Gibco, Grand Island, NY) supplemented with 40 µg/mL gentamicin and 5% heat-inactivated FCS. Macrophages were resuspended in MacMed at 2 × 106/mL, and 0.5 mL aliquots were dispensed into the wells of a 24-well plate. Cells were allowed to adhere for 3h at 37 °C in the presence of 5% CO2, and then washed once with serum-free DMEM, and 1.0 mL of MacMed was added to each well. The plates were incubated overnight at 37 °C in the presence of 5% CO2 and in the absence or presence of 50 units/mL of recombinant murine IFN-γ (Genentech, San Francisco, CA, USA). The macrophages were washed and then cultured in the absence or presence of either tGPI-mucins (100 ng/mL) or live tTryp (2:1 parasite:macrophage ratio) in a final volume of 1.0 mL/well. Six hours later macrophages were lysed and total RNA extracted according to the protocol described below. 2.7. Quantification of murine IFN-γ

Suspensions of splenocytes from uninfected and infected mice were washed in Hank's balanced salt solution (HBSS) and treated with lysing buffer (nine parts of 0.16 M NH4Cl and one part of 0.17 M Tris-HCl, pH 7.5) for 2 min. The erythrocyte-free cells were then washed three times in HBSS and adjusted to 3 × 106 cells of RPMI-1640 (Flow Laboratories, Inc., McLean, VA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 2-mercaptoethanol (5 × 10–5 M), L-glutamine (2 mM), and antibiotics (all purchased from Sigma). The cell suspension was distributed (1 mL/well) in 24-well tissue culture plates (Corning, Corning, NY) and cultured for 48 h at 37 °C in a humidified 5% CO2 atmosphere, in the presence or absence of T. cruzi lysate (TcAg) (10 µg/mL), or Con-A (2 µg/mL). The levels of IFN-γ in splenocyte culture supernatants and sera were assayed in a two-site ELISA using a rat anti-IFN-γ mAb R46A2 (ATCC, Rockville, USA) and a polyclonal rabbit serum specific for the cytokine, as previously described [12]. IFN-γ levels were calculated by reference to a standard curve constructed with recombinant cytokine (Genzyme, Cambridge, MA, USA). Sensitivity of this method was 100 pg/mL. 2.8. Detection of parasite specific DNA in the cardiac tissue of infected mice

Cardiac fragments were used as DNA source for the detection of a T. cruzi specific gene. DNA was extracted with phenol:chloroform:isoamylic alcohol, precipitated in isopropanol and washed in 70% ethanol. The DNA preparation was resuspended in water and the concentration adjusted to 10 and 100 ng/µL and used as template for PCR using specific primers for T. cruzi guanine hypoxanthine phosphoribosyltransferase (HGPRT - 412 bp) gene HGPRT1 (forward): 5'- CTACAAGGGAAAGGGTCTGC-3' and HGPRT2 (reverse): 5'-ACCGTAGCCAATCACAAAGG-3' designed from the complete nucleotide 853

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sequence of the HGPRT gene [28]. Each amplification reaction was performed in a final volume of 10 µL, containing 0.5 U of Taq DNA polymerase (Cenbiot, RS, Brazil)/ 200 µM of each dNTP/15 mM MgCl2/50 mM KCl/10 mM Tris-HCl pH 8.5/5 pmol of each primer and 10 or 100 ng of DNA extracted from infected animals. After heat denaturation for 5 min at 95 °C, samples were submitted to 30 cycles at three temperatures (95 °C/1 min, 55 °C/1 min and 72 °C/1 min). In the final cycle an extension step at 72 °C was performed for 5 min. After amplification, 3 µL of each reaction was eletrophoresed in 6% acrylamide gel and silver stained [29]. 2.9. RT-PCR assay for measuring in vivo or in vitro expression of cytokine mRNA

RNA was isolated from cardiac tissue of mice by acid guanidinium thiocyanate-phenol-chloroform extraction: RNA STAT-60TM [30]. One microgram of total RNA was reverse transcribed by the addition of 2.5 U RNAsin (Promega Corporation, Madison, WI), 2.5 mM deoxynucleotides (dNTPS) (Boehringer Mannheim), 0.1 M dithiothreitol - DTT (Gibco BRL Life Technologies, Inc., Grand Island, NY), 1X Moloney murine leukemia virus RNAase – H reverse transcriptase buffer (Life Technologies), 25 ng random hexamer oligonucleotides (Boehringer Mannheim), and 200 U Moloney murine leukemia virus RNAse H– reverse transcriptase (Life Technologies) in a total volume of 20 µL. The reaction proceeded for 1 h at 37 °C and was terminated by boiling for 5 min after the addition of 175 µL H2O. Five microliters of cDNA was used for amplification in a 30 µL PCR reaction containing 2.5 mM dNTPs (Pharmacia), a 0.2-mM concentration of the 3' and 5'external primers, 1.5 mM MgCl2; 1X GeneAmp PCR buffer and 0.5 U Taq DNA polymerase (Promega). PCR conditions were as follows: 95 °C, 3 min, 94 °C, 1 min, 54 °C, 1 min (first cycle), 72 °C, 2 min, 54 °C, 1 min (n cycles), 72 °C, 7 min (final cycle). The primers used are shown below. PCR products and molecular weight marker were run on 6% polyacrylamide gel and stained with silver nitrate [26]. Plasmids containing chemokine/cytokine-encoding sequences were used to establish the PCR conditions. PCR products on silver-stained gels were quantified with a densitometer (Shimadzu Corporation, Tokyo, Japan) using a CS-9301 PC program. The densitometry value for each cytokine/chemokine was corrected for the mouse hypoxanthine phosphoribosyltransferase (HPRT) value for the same sample and divided by the average value for that cytokine/chemokine obtained from uninfected controls. A low level of constitutive expression of cytokines/ chemokines was observed in samples from cardiac tissue of noninfected controls or unstimulated macrophages. The results are reported as fold increase over uninfected control or unstimulated macrophages. The primer (sense and antisense) sequence from 5'to 3', followed by number of cycles and expected product size of PCR presented within parentheses is indicated below. CXC chemokines: MIP-2, CGC-GGA-TCC-CCT-GGTTCA-GAA-AAT-CAT-CC, CGC-GGA-TCC-TCC-CCA-GTCTCT-TTC-ACT-GT (33,468 bp); KC, CGC-GGA-TCC-TTGACC-CTG-AAG-CTC-CCT-TGG-TTC, CGC-GGA-TCCCGT-GCG-TGT-TGA-CCA-TAC-AAT-ATG (35,521 bp); 854

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IP-10, CGC-GGA-TCC-TGA-GCA-GAG-ATG-TCT-GAATC, CGC-GGA-TCC-TCG-CAC-CTC-CAC-ATA-GCTTAC-AG (33,399 bp); MIG, GAT-CAA-ACC-TGC-CTAGAT-CC, GGC-TGT-GTA-GAA-CAC-AGA-GT (35,399 bp); SDF-1α, CTC-CAA-ACT-GTG-CCC-TTC-AG, AAAGCT-CCA-TTG-TGC-ACG-GG (35,348 bp); SDF-1β, CCGGAA-TTC-CTC-CAA-ACT-GTG-CCC-TTC-AG, CCGGAA-TTC-GCC-TGT-CAC-CAA-TGA-CGT-TG (35,368 bp), LIX, GCC-GGA-ATT-CGG-GAT-CTT-GTC-CAC-AATGAC, AAC-TGC-AGC-AGG-GAC-AAT-GGT-TTC-CCT-T (35, 547 bp). CC chemokines: MIP-1α, CGC-GGATCC-CGG-AAG-ATT-CCA-CGC-CAA-TTC, CGC-GGATCC-GGT-TGA-GGA-ACG-TGT-CCT-GAA-G (35,448 bp); MIP-1β, CGC-GGA-TCC-CCC-ACT-TCC-TGC-TGT-TTCTCT-TAC, CGC-GGA-TCC-AGC-AGA-GAA-ACA-GCAATG-GTG-G (33,444 bp); JE, CCG-GAA-TTC-CAC-TCACCT-GCT-GCT-ACT-CAT-TCA-C, CCG-GAA-TTC-GGATTC-ACA-GAG-AGG-GAA-AAA-TGG (30,505 bp); RANTES, CGC-GGA-TCC-CCA-CGT-CAA-GGA-GTATTT-CTA-CAC-C, CGC-GGA-TCC-CTG-GTT-TCT-TGGGTT-TGC-TGT-G (26,326 bp); TCA-3, TGT-TAC-AGAAAG-ATG-GGC-TCC-TCC, TCC-AAG-AAA-CAG-AGGCAG-CG (33,324 bp); eotaxin, CAC-GAA-GCT-TTAGGT-AAG-CAG-TAA-CTT-CCA-TCT-GTC-TC, GCG-GCTAGC-TGA-CTA-AAT-CAA-GCA-GTT-CTT-AGG-CTC-TG (35,380 bp). The sequence of primers used to measure the messages of IL-1β, IL-12(p40), TNF-α, IL-2, IL-4, IL-5, IL-10 and IFN-γ was the same as those used in our previous studies [30, 31]. 2.10. Statistical analysis

Arithmetic or geometric means (parasitemia and densitometric levels) and standard deviations of the means were calculated. The Student's t-test was used to analyze the statistical significance of the differences observed in RT-PCR analysis. Differences were considered statistically significant when P < 0.05.

3. Results 3.1. Parasitemia, cardiac tissue parasitism and mortality curves of C57BL/6 mice infected with T. cruzi Colombiana

C57BL/6 mice were inoculated with 50 parasites; most animals survived the parasitemia peak (figure 1A) at 30 days of infection, and about 25% of the animals survived up to 120 days p.i. (figure 1B). Figure 2A (top panel) shows nine amastigote nests in a field of cardiac tissue from a mouse at 30 days p.i. Considering that the size of amastigote nests present in cardiac tissue of animals infected for 15, 30 and 60 days was not statistically different, we used the number of amastigote nests as an indicator of tissue parasitism. Our results indicate that the peak of tissue parasitism (figure 2B) coincides with the peak of parasitemia (figure 1A) occurring at 30 days p.i. At this time of infection we observed an average 30 amastigote nests in 12 histopathology fields (HE, 1 × 100) of cardiac tissue. After 30 days of infection, tissue parasitism decreased to undetectable levels until about 120 days p.i. Despite the negative results of the histological examinations, by using Microbes and Infection 2000, 851-866

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Figure 1. Natural course of T. cruzi infection in C57BL/6 mice. A. Parasitemia and B. mortality curve of C57BL/6 mice that were intraperitoneally infected with 50 blood trypomastigote forms of T. cruzi Colombiana. a more sensitive method we were able to detect T. cruzi genomic DNA in the cardiac tissue of all animals even at 120 days p.i. (figure 2C). 3.2. Analysis of T. cruzi-induced histopathology in cardiac tissue

Using conventional HE staining we also investigated the effects of parasitic infection on cardiac tissue inflammation at different times after infection of C57BL/6 mice with T. cruzi Colombiana (figure 3A and 3B). The inflammatory process around cardiac fibers began about 15 days after the initial infection. With the increase of amastigote nests at 30 days p.i., we observed a prominent inflammatory infiltrate along the cardiac fibers. As shown in figure 2B, the diffuse inflammatory infiltrate peaked at 30 days of infection. After this period, the intensity of the focal inflammatory infiltrate increased, peaking at 60 days p.i. and coinciding with elimination of T. cruzi parasites. Low levels of a diffuse inflammatory process were observed in the cardiac tissue of all animals even at 120 days p.i. with T. cruzi Colombiana (figure 3B). 3.3. Characterization of the cellular infiltrate in cardiac tissue from mice infected with T. cruzi Colombiana

The characterization of leukocyte phenotypes in the cellular infiltrate from the cardiac tissue of C57BL/6 mice infected with T. cruzi Colombiana was performed using conventional immunocytochemistry, confocal microscopy, and FACS analysis and shown in figure 4 and table I. Our +results indicate a dominance of CD4+ T cells over CD8 T lymphocytes during the initial phase of the infection, i.e., at 15 days p.i. (figure 4A – top panels). As shown in figure 4A and table I, a significant number of CD8+ T lymphocytes was present in the cardiac tissue of mice infected for 30 days. The number of CD8+ lymphocytes became dominant at 42 and persisted until 90 days after infection with T. cruzi Colombiana. We also observed a gradual and significant increase in mononuclear cells expressing Mac-1 (CD11b) receptors Microbes and Infection 2000, 851-866

Figure 2. Analysis of T. cruzi parasitism in cardiac tissue of C57BL/6 mice infected with T. cruzi. A. Nine amastigote nests containing variable numbers of parasites are shown in one field of cardiac tissue from an animal at 30 days p.i. (HE, 1 × 200). The arrows indicate amastigote nests. B. Number of amastigote nests in 12 histopathological fields (magnification, × 56) obtained from cardiac sections of mice at different times p.i. Each value presented in figure 1B is the average and standard deviation of four animals. Similar results were obtained in two other experiments. C. Percentage of positive animals in a PCR using primers specific for T. cruzi HGPRT. DNA from five distinct animals was used for each time point. Similar results were obtained in a second experiment. 855

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p.i. IL-12(p40) mRNA increased only at 15 days p.i. and then declined thereafter, since at no other time point did we observe increased expression of the IL-12(p40) gene in the cardiac tissue of infected animals. 3.5. Kinetics of cytokine mRNA expression by cardiac tissue from C57BL/6 mice infected with T. cruzi Colombiana

Figure 3. A Diffuse and focal inflammatory infiltrates in cardiac tissue from a mouse at 60 days p.i. (HE, 1 × 200). The arrows indicate the focal infiltrates. The star is in the middle of a diffuse inflammatory infiltrate. Bottom panel shows the number of diffuse (white bars) and focal (black bars) inflammatory infiltrates present in 12 histopathological fields (magnification, × 57) obtained from cardiac sections of mice at different times p.i. Each value is the average and standard deviation of four animals. Similar results were obtained in two other experiments.

(figure 4B – bottom panels). These cells were most likely macrophages, since polymorphonuclear cells were rarely seen at 30 days or later p.i. The relative number of macrophages increased from 40 and 20% of the inflammatory infiltrate at 42 and 90 days, respectively, to 50% of all inflammatory cells at 120 days p.i. as determined by FACS analysis. A small percentage of polymorphonuclear cells (i.e., neutrophils) was observed in cardiac tissue during the early stages of infection, i.e., at 15 and 30 days p.i., but not at later time points (data not shown). 3.4. Kinetics of monokine mRNA expression by cardiac tissue from C57BL/6 mice infected with T. cruzi Colombiana

The results shown in figure 5 illustrate the expression of the monokines IL-1, TNF-α and IL-12(p40) at different times p.i. with T. cruzi Colombiana. A small but measurable increase in IL-1β was observed at 15 days p.i. At the level of mRNA expression, TNF-α was the dominant monokine expressed in the heart of infected animals. TNF-α mRNA increased at 15 days p.i. and peaked at 30 days p.i. In addition, we observed an increased expression of TNF-α mRNA at late time points, i.e., 60 and 120 days 856

Regarding the mRNA expression of cytokine in cardiac tissue from infected animals, we also observed an increase in both IFN-γ and IL-4 levels at days 15 and 30 p.i. This increase was also accompanied by an increase in IL-10 mRNA expression. No expression of IL-2 (figure 6) or IL-5 (data not shown) mRNAs was observed at any time of infection. IFN-γ was the dominating lymphokine mRNA at 15 days p.i., whereas at day 30 days after infection we observed high levels of IFN-γ, IL-4 and IL-10 mRNAs. Interestingly, at later time points, 60 and 120 days p.i., when inflammation was decreased, we observed a persistence and dominance of type 2 cytokine mRNAs (i.e., IL-4 and IL-10), whereas IFN-γ mRNA was regulated almost to the basal levels (figure 6). We also analyzed the levels of IFN-γ in the supernatants of spleen cells and sera from animals at different times of infection with T. cruzi Colombiana. As shown in table II, maximal levels of IFN-γ were measured in supernatants of splenocytes and sera from animals at 15 and 30 days p.i. Nevertheless, when compared with uninfected controls, higher levels of IFN-γ were still produced and detected in supernatants of splenocytes and sera from animals at 60 and 120 days p.i. 3.6. Macrophages as a source of chemokines in cardiac tissue from mice infected with T. cruzi Colombiana

Our group [9, 10, 12, 13] and others [32, 33] have now repeatedly shown that T. cruzi trypomastigotes are potent stimulators of cytokine synthesis by macrophages. Because macrophages are an important source of various chemokines and were found to be present in large amounts in cardiac tissue from animals infected with T. cruzi Colombiana, we decided to investigate the ability of T. cruzi trypomastigotes to induce expression of chemokine mRNAs by inflammatory peritoneal macrophages. The results presented in figure 7 show that either live tissue culture trypomastigotes (tTryp) or the glycosylphosphatidylinositol linked mucin-like glycoproteins isolated from T. cruzi trypomastigotes (tGPI-mucins) were able to induce expression of RANTES, MIP-1α, MIP-1β, KC and IP-10 mRNAs by inflammatory macrophages. Expression of some of these chemokines was upregulated (RANTES and IP-10) or downregulated (KC) by IFN-γ. In vitro MIG was only induced by IFN-γ (data not shown). Although live trypomastigotes enhanced expression of MIG induced by IFN-γ (data not shown), parasite products alone were unable to trigger expression of MIG mRNA. 3.7. Kinetics of CC chemokine mRNA expression by cardiac tissue from C57BL/6 mice infected with T. cruzi Colombiana

RANTES, JE and MIP-1β were found to be the main CC chemokine mRNAs expressed in cardiac tissue during the acute phase of experimental Chagas' disease. We also Microbes and Infection 2000, 851-866

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Figure 4. Characterization of infiltrating cells in cardiac tissue obtained from mice infected with T. cruzi. A. Frozen sections from cardiac tissue of animals obtained at different times p.i. with T. cruzi were stained with FITC-labeled anti-CD8 and PE-labeled anti-CD4 mAbs and analyzed with a confocal microscope. Top panels show the staining in control and infected mice, at 15 and 30 days p.i. Red and green staining indicate the presence of CD4+ CD8– and CD4–CD8+ T lymphocytes, respectively. B. Frozen sections from cardiac tissue of animals obtained at different times p.i. with T. cruzi were stained with anti-MAC-1 biotinylated mAb, vizualized by using a streptavidin-peroxidase conjugate and analyzed under the light microscope (magnification, × 385). Four panels show the staining in tissue from an uninfected control (0) and mice infected for 15, 30 and 60 days p.i. The brown staining indicates the presence of MAC1+ cells.

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Table I. Distribution of CD3+CD4+CD8– and CD3+CD4–CD8+ αβ+ T lymphocytes in cardiac tissue of animals infected with Trypanosoma cruzi Colombiana*. Days postinfection 0 42 90 120

CD4+CD8–

CD4–CD8+

CD3+/TCRαβ+

CD4–CD8–CD3–/TCRαβ–

0.6 % 8.5 % 17.3 % 7.4 %

0.4 % 28.9 % 51.1 % 8.4 %

1.2 % 37.5 % 73.6 % 15.0 %

98.8 % 62.4 % 12.4 % 84.2 %

* Inflammatory cells were obtained from 10–20 hearts pooled from animals at different times of infection with T. cruzi. Similar results were obtained in two different experiments.

observed a small increase in the expression of MIP-1α at 15 days p.i. Expression of JE and MIP-1β mRNAs were at basal levels at 60 and 120 days p.i., whereas RANTES mRNA was still elevated at 120 days p.i. (figure 8). We were unable to detect expression of TCA-3 or Eotaxin mRNA at any time during experimental infection with T. cruzi (data not shown). 3.8. Kinetics of CXC chemokine mRNA expression by cardiac tissue from C57BL/6 mice infected with of T. cruzi Colombiana

The non-ELR-CXC chemokine IP-10 and MIG mRNAs, which are induced by IFN-γ, were expressed at high levels at 15 and 30 days as well as at later time points of infection. Expression of ELR-CXC chemokine KC and MIP-2 mRNAs peaked at 15 and 30 days p.i. However, the expression was mostly absent at 60 and 120 days p.i. (figure 9). No expression of chemokine LIX, SDF-1α and SDF-1β mRNA was noted in any of our samples (figure 9 and data not shown).

4. Discussion In the present study we used a previously described model [24] to investigate the kinetics of tissue parasitism, inflammation and cytokine synthesis during infection with T. cruzi Colombiana. The advantage of this model for the immunopathology of Chagas' disease is related to the intense cardiomyopathy associated with the high rate of animal survival, which enables the study of induction and resolution of cardiomyopathy during experimental infection with T. cruzi. For this purpose, we infected animals with 50 trypomastigotes from T. cruzi Colombiana and analyzed the kinetics of tissue parasitism, inflammatory infiltrate, as well as lymphokine, monokine and chemokine expression in the cardiac tissue of infected animals. Our results show that control of tissue parasitism is accompanied by decreased inflammation and expression of different chemokines and IFN-γ mRNAs. In contrast, the expression of type 2 cytokines (i.e., IL-4 and IL-10) in cardiac tissue was maintained at later times of infection with T. cruzi, when tissue parasitism was controlled. In our initial studies we decided to follow the kinetics of tissue parasitism and inflammation in cardiac tissue of animals infected with T. cruzi Colombiana. Our results show that tissue parasitism initiates at about 15 days p.i., peaking at 30 days and already declining at 60 days p.i. A 858

close correlation was also observed in terms of tissue parasitism and intensity of inflammation in the cardiac tissue. The diffuse and focal inflammation peaked at 30 and 60 days p.i., respectively, and declined thereafter. However, low levels of cardiac tissue inflammation were observed even at 120 days p.i., when amastigote nests were not detectable by conventional histopathology. Nevertheless, the vast majority of animals were positive when we searched for T. cruzi genomic DNA using PCR. The latter results are consistent with the hypothesis that, although at low levels, tissue parasitism is an essential component of inflammation during the chronic phase of Chagas'disease [34–36]. The phenotypic analysis revealed that at different times of infection the inflammatory reaction was dominated by mononuclear cells expressing the MAC.1 marker (i.e., macrophages) and CD3+ αβTCR+ lymphocytes expressing either CD4 and CD8 markers. Consistent with previous studies using human cardiac sections [37, 38] or the mouse model [20, 21] with other T. cruzi strains, our kinetic studies showed a dominance (i.e., over 60% of total T lymphocytes) of CD8+ T lymphocytes after 30 days of infection. Although there was not much variation in terms of relative amount of different inflammatory cells, the absolute number of inflammatory cells was much lower from 60 to 120 days p.i. We also analyzed the expression of the monokines, TNF-α, IL-12(p40) and IL-1β. Our results show that TNF-α mRNA was already enhanced at 15 days p.i. and expressed at high levels in cardiac tissue from infected animals. Although at lower levels, TNF-α mRNA was detectable above control values, even at 120 days after infection, when the inflammatory infiltrate was controlled. These findings are consistent with those obtained for human sections which demonstrate expression of high levels of TNF-α protein during chronic Chagas' disease [38]. In contrast, expression of IL-12(p40) and IL-1β mRNAs was increased in cardiac tissue at day 15 p.i., and decreased to basal levels at 30 days p.i. or at later time points. Of the 13 chemokine mRNA [39, 40] tested in the cardiac tissue from animals infected with T. cruzi at different times p.i., there was a dominance of chemokines induced by IFN-γ, namely MIG [41], RANTES [42] and IP-10 [43]. The expression of MIG, RANTES and IP-10 mRNAs persisted even at 120 days p.i., despite low levels of expression of IFN-γ mRNA in heart tissue at 60 and 120 days p.i. A possible explanation for this observation could Microbes and Infection 2000, 851-866

In vivo chemokine mRNA expression induced by T. cruzi

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Figure 5. Kinetics of monokine expression in the cardiac tissue from C57BL/6 animals infected with 50 blood trypomastigote forms of T. cruzi as determined by RT-PCR. Total RNA was extracted from cardiac tissue, obtained at different times after T. cruzi infection, reversed transcribed and used as template for PCR employing primers specific for HPRT, TNF-α, IL-1β and IL-12(p40). Top panel A. shows a silver-stained gel containing PCR products for some of the monokine analyzed. Each lane of the gel corresponds to the result of RT-PCR obtained from a single animal. Bottom panel B. shows the semi-quantitative analysis of monokine mRNA expression in the cardiac tissue of infected mice. Individual values were pooled from two different experiments and used to obtain the arithmetic means and standard deviations shown. The total number of animals varied from 6 to 8 animals at 0, 15, 30, 60 and 120 days p.i. One and two asterisks indicate that differences are statistically significant at P < 0.05 and P < 0.01 levels, respectively, when comparing expression of a cytokine/ chemokine at different times p.i. with those of uninfected mice.

be that infection with T. cruzi elicits high enough levels of IFN-γ in the circulation that are sufficient to stimulate cells in heart tissue to express MIG, RANTES and IP-10 mRNAs [44]. Alternatively, parasites present in cardiac tissue, as indicated by T. cruzi specific PCR, would be sufficient to keep stimulating the expression of mRNAs specific for Microbes and Infection 2000, 851-866

these three chemokines. Consistent with this hypothesis we showed the ability of T. cruzi parasites or their products (i.e., tGPI-mucins) to induce inflammatory macrophages to express IP-10 and RANTES. The dominant expression of MIG, RANTES and IP-10 is also consistent with the presence of a large proportion of T lymphocytes in the inflam859

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Figure 6. Kinetics of type 1 and type 2 cytokine expression in the cardiac tissue from C57BL/6 animals infected with 50 blood trypomastigote forms of T. cruzi as determined by RT-PCR. Total RNA was extracted from cardiac tissue of animals, obtained at different times p.i. with T. cruzi, reversed transcribed and used as template for PCR employing primers specific for HPRT, IL-2, IFN-γ, IL-4, IL-5 and IL-10. Top panel A. shows a silver-stained gel containing PCR products obtained by RT-PCR. Each lane of the gel corresponds to the result of RT-PCR obtained from a single animal. ND (i.e., not done) on silver-stained gels indicates that cytokine specific RT-PCR was not performed on that sample. Bottom panel B. shows the semi-quantitative analysis of type 1 and type 2 cytokine mRNA expression in cardiac tissue of infected mice. Individual values were pooled from two different experiments and used to obtain the arithmetic means and standard deviations shown. The total number of animals varied from 6 to 8 animals at 0, 15, 30, 60 and 120 days p.i. One and two asterisks indicate that differences are statistically significant at P < 0.05 and P < 0.01 levels, respectively, when comparing expression of a cytokine/ chemokine at different times p.i. with those of uninfected mice.

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Table II. Levels of IFN-γ synthesis induced during infection with Trypanosoma cruzi Colombiana1. Splenocyte culture supernatants (ng/mL) Days p.i. 0 15 30 60 120

(n)

Medium

TcAg

ConA

Sera (ng/mL)

5 5 5 5 5

< 0.5 3.2 ± 1.3** 2.1 ± 0.9* 1.6 ± 0.5* 1.3 ± 0.2*

1.2 ± 0.2 22.1 ± 5.1** 14.5 ± 2.7** 8.7 ± 1.7** 6.1 ± 2.1**

12.3 ± 0.8 20.3 ± 2.1** 17.7 ± 2.3* 14.9 ± 3.1 11.5 ± 1.5

< 0.5 4.7 ± 1.5** 3.2 ± 1.5** 2.5 ± 0.7* 1.8 ± 0.3*

1 One (P < 0.05) and two (P < 0.01) asterisks indicate that differences in results obtained from experimentally infected animals are statistically significant when compared to uninfected animals. Similar results were obtained in a second experiment.

matory infiltrates found in cardiac tissue of animals infected with T. cruzi, since these chemokines are chemoattractants for lymphocytes [45–47]. In addition to inducing expression of RANTES and IP-10, T. cruzi trypomastigotes were also able to trigger the synthesis of JE, MIP-1α, MIP-1β and KC by inflammatory macrophages, and their expression was also augmented in the cardiac tissue of infected animals. In contrast, we were unable to detect any message in the cardiac tissue of infected animals for the following chemokines: SDF1α, SDF1β, TCA3, eotaxin and LIX. In general, the kinetics for expression of chemokine mRNA (i.e., KC, MIP-1α, MIP1β, JE and RANTES) was similar to the pattern of intensity of parasitism in the cardiac tissue of animals infected with T. cruzi. Therefore despite the controversy about the major target(s) (i.e., self-antigens or parasite antigens) for inflammatory lymphocytes [48, 49] in the experimental model of myocarditis induced by T. cruzi, the presence of parasites in cardiac tissue above a certain limit may be essential for eliciting expression of chemokines and recruitment of inflammatory cells leading to chagasic myocarditis. Interestingly, we found that the pattern of chemokine mRNA expressed by macrophages exposed to tTryp or tGPI-mucins was very similar to that expressed in the cardiac tissue of infected animals. KC, IP-10, MIP-1α, MIP-1β, RANTES and JE were all induced, whereas SDF1α, SD-F1β, TCA3, LIX and eotaxin were all negative in macrophages exposed to T. cruzi or its products or cardiac tissue from infected animals. This similarity in the panel of chemokine mRNA expression suggests that macrophages may indeed be an important source of chemokines in cardiac tissue of animals infected with T. cruzi. In terms of type 1 and type 2 cytokines, we found that IFN-γ, IL-4 and IL-10 but not IL-2 or IL-5 mRNAs were upregulated in the cardiac tissue of infected animals. Although we found high levels of IFN-γ mRNA expression, consistent with previous studies [50, 51] messages for IL-2 were not detectable at any time of infection. There was a concomitant fall in the levels of IFN-γ mRNA, but not of IL-4 or IL-10 mRNAs, with the resolution of inflammation in the heart of chagasic animals. Modulation of IFN-γ and upregulation of IL-4 and IL-10 proteins during the chronic phase of Chagas'disease have also been found in cardiac tissue of animals infected with the Brazil strain of T. cruzi [52], thus suggesting that this may not be a specific phenomenon for the infection with the Colombiana strain. The dominance of type 2 over type Microbes and Infection 2000, 851-866

Figure 7. Identification of macrophages exposed to T. cruzi parasites or parasite glycoconjugates as a major cellular source of chemokine mRNAs. Thioglycollate-elicited macrophages from C57BL/6 were cultured in medium alone (control), with tissue culture trypomastigote forms (tTryp) of T. cruzi Colombiana or tGPI-mucins extracted from tissue culture trypomastigotes. Total RNA was extracted from macrophages 6 h after macrophage stimulation and levels of cytokine transcripts were measured by RT-PCR. Top panel A. shows silver-stained gel containing PCR products obtained by RT-PCR. Bottom panel B. shows the semi-quantitative analysis of chemokine mRNA expression in macrophages exposed to either live trypomastigotes or tGPImucins. Similar results were obtained in a second experiment. 861

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Figure 8. Kinetics of CC and CXC chemokine expression in the cardiac tissue from C57BL/6 animals infected with 50 blood trypomastigote forms of T. cruzi as determined by RT-PCR. Total RNA was extracted from cardiac tissue, obtained at different times p.i. with T. cruzi, reversed transcribed and used as template for PCR employing primers specific for HPRT, RANTES, JE, MIP-1α, MIP-1β, TCA3, and eotaxin. A. Top panel shows silver-stained gel containing PCR products for some of the CC chemokines analyzed. Each lane of the gel corresponds to the result of RT-PCR obtained from a single animal. ND (i.e., not done) on silver-stained gels indicates that cytokine specific-RT-PCR was not performed on that sample. B. Bottom panel shows the semi-quantitative analysis of CC chemokine expression in the cardiac tissue of infected mice. Individual values were pooled from two different experiments and used to obtain the arithmetic means and standard deviations shown. The total number of animals varied from 6 to 8 animals at 0, 15, 30, 60 and 120 days p.i. One and two asterisks indicate that differences are statistically significant at P < 0.05 and P < 0.01 levels, respectively, when comparing expression of a cytokine/chemokine at different times p.i. with those of uninfected mice.

1 cytokines at 60 and 120 days p.i. may be occurring only in cardiac tissue, since our ex-vivo experiments with splenocytes demonstrated that both IFN-γ and IL-4 were produced at high levels at 15 days p.i. and at lower levels thereafter, without showing a dominance of IL-4 during the late stages of infection (data not shown). Because IL-4 and IL-10 possess a downregulatory activity on the development and effector functions of cell-mediated immunity, it is tempting to speculate that the decline in IFN-γ and persistence of IL-4 and IL-10 expression may be an important event responsible for controlling the strong cellmediated immunity and immunopathology elicited by T. cruzi infection. This hypothesis remains to be experimentally demonstrated. Nevertheless, different studies have 862

demonstrated the ability of IL-4 and IL-10 to regulate inflammatory processes involved in the tissue damage that occurs during different parasitic infections [53, 54] and autoimmune diseases [55, 56]. Powell and colleagues found an association between high levels of IL-4 mRNA expression and susceptibility to myocarditis elicited by the Brazil strain of T. cruzi [57]. Thus, it is possible that the regulatory effect of IL-4 and IL-10 on cell-mediated immunity will enhance susceptibility to T. cruzi. Additional studies using IL-4 knockout mice will be necessary to further analyze the role of IL-4 in the immunopathology of experimental Chagas' disease. Nevertheless, the local action of T lymphocytes, IFN-γ and macrophage may no longer be essential once a T. cruzi Microbes and Infection 2000, 851-866

In vivo chemokine mRNA expression induced by T. cruzi

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Figure 9. Total RNA was extracted from cardiac tissue, obtained at different times p.i. with T. cruzi, reversed transcribed and used as template for PCR reaction employing primers specific for HPRT, MIG, IP-10, MIP-2, KC, and SDF1-α. A. Top panel shows silver-stained gels for some of the CXC chemokine analyzed. Each lane of the gel corresponds to the result of RT-PCR obtained from a single animal. ND (i.e., not done) in silver-stained gel indicates that cytokine specific-RT-PCR was not performed on that sample. B. Bottom panel shows the semi-quantitative analysis of CXC chemokines expression in the cardiac tissue of infected mice. Individual values were pooled from two different experiments and used to obtain the arithmetic means and standard deviations shown. The total number of animals varied from 6 to 8 animals at 0, 15, 30, 60 and 120 days p.i. One and two asterisks indicate that differences are statistically significant at P < 0.05 and P < 0.01 levels, respectively, when comparing expression of a cytokine/chemokine at different times p.i. with those of uninfected mice.

specific humoral response develops. In fact, different studies suggest that B lymphocytes and antibodies have an important role in resistance to chronic infection with T. cruzi [58, 59]. Finally, it is also possible that the reactivation of cardiopathy may emerge from a breakdown of immunological control of T. cruzi and/or from immunoregulatory activity on cell-mediated immunity in cardiac tissue during chronic Chagas' disease. Consistent with this hypothesis are the studies showing a more intense cardiopathy and lethality in mice lacking a functional IL-10 gene [54], and an increased IFN-γ response to parasite antigens in patients with the cardiac form of Chagas' disease [60]. We believe that infection of mice with the Colombiana strain will be a useful model to analyze the role of cytokines on induction and regulation of cardiac pathology elicited by T. cruzi. Microbes and Infection 2000, 851-866

Acknowledgments We thank Denise C. Cara and Mauro M. Teixeira for help with the analysis of histopathology data and for critically reading this manuscript. This work was partially supported by PAPES-FIOCRUZ (#2), FAPEMIG (CBS 1208/ 95), FAPESP, CNPq (522.056/95-4) and WHO/TDR (970506 and 970728). RTG, AJR, JLV and JSS are research fellows from CNPq.

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Microbes and Infection 2000, 851-866

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