Kinetics of cytokine gene expression inexperimental chagasic cardiomyopathy: tissueparasitism and endogenous IFN-γ as importantdeterminants of chemokine mRNA expression

during infection with Trypanosoma cruziAndré 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*

aDepartment of Biochemistry and Immunology, ICB, UFMG, Av. Antônio Carlos 6627, 31270–910 Belo Horizonte, MG, BrazilbLaboratory of Immunopathology, CPqRR - FIOCRUZ, Av. Augusto de Lima 1715, 30190–002 Belo Horizonte, MG, Brazil

cDepartment of Parasitology, Microbiology and Immunology, School of Medicine from Ribeirão Preto, USP, Av. Bandeirantes 3900, 14049–900 RibeirãoPreto, SP, Brazil

dDepartment of Immunology, Instituto Oswaldo Cruz - FIOCRUZ, Av. Brasil 4365, 21045–900 Rio de Janeiro, RJ, BrazileDepartment of Parasitology, ICB, USP, Av. Prof. Lineu Prestes 1374, 05508–900 São Paulo, SP Brazil

fLaboratory 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, andcytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombianastrain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 daysand was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that theinflammatory infiltrate was dominated by the presence of α�Τ CD3+ CD4+ CD8–, α�Τ CD3+

CD4–CD8+ lymphocytes and macrophages. The mRNA expression of the monokines IL-1� and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-α mRNAwas expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokineexpression was dominated by the presence of IFN-γ mRNA, whereas at 60 days or later time points thebalance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. Thechemokine mRNAs encoding JE, MIP-1α, MIP-1�, KC, and MIP-2 were all mainly expressed at 15and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10mRNAs 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 decreasedtissue parasitism, may be largely responsible for the control of inflammation and immunopathologyobserved in the cardiac tissue of animals infected with T. cruzi. © 2000 Éditions scientifiques etmédicales Elsevier SAS

Trypanosoma cruzi / chemokines / macrophages / inflammation

1. IntroductionThe intracellular protozoan parasite Trypanosoma cruzi

is the etiological agent of Chagas' disease [1, 2]. In humans,

the acute infection with T. cruzi lasts for 2–4 months and ischaracterized by the presence of parasites in the blood-stream and different host tissues. In addition, various non-specific symptoms and myocarditis are common featuresduring the early stage of Chagas' disease [3]. After devel-opment of immunity, both parasitemia and tissue parasit-* Correspondence and reprints

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ism are controlled and myocarditis is resolved. However, acardiopathy will reemerge in 20–40% of chronic chagasicpatients 10 or 20 years after the initial infection by a yetundefined mechanism [3, 4]. Nevertheless, follow-up stud-ies on chagasic patients suggest that the intensity of clini-cal symptoms as well as pathophysiological alterationsthat occur during the acute phase of the disease correlatepositively with the severity of cardiac disease observedduring the chronic phase of Chagas' disease [5, 6].

The mechanisms by which parasite replication is con-trolled during the acute phase of infection are not com-pletely understood. Nevertheless, different studies usingexperimental models of Chagas' disease indicate the cru-cial role of a few cytokines (e.g., IFN-γ, TNF-α, and IL-12)in resistance to T. cruzi [7–11]. After replication in tissuesfrom the vertebrate host, amastigotes and trypomastigotesstart expressing molecules that initiate the synthesis ofseveral pro-inflammatory cytokines, among them IL-12and TNF-α, by cells of the macrophage lineage [12]. TheIL-12 produced initiates the synthesis of IFN-γ by differentcell populations from the lymphocyte lineage, such as NKcells, CD4+ CD8– α� and CD4–CD8+ α� T cells. IFN-γcombined with TNF-α will play a major role in resistanceby activating macrophages to produce high levels of reac-tive nitrogen intermediates (RNI) [8, 13, 14], which willeffectively control parasite replication both in vitro and invivo [15–17].

The strong activation of the cellular compartment of theimmune system by T. cruzi may also result in some sideeffects [18]. In fact, the parasite-elicited inflammation andimmune responses appear to be largely responsible for thetissue damage and neuronal destruction in the cardiactissue observed in the acute stage of disease. Thus, in micelacking CD4+ CD8– α� and/or CD4–CD8+ α� T cells, acutemyocarditis is largely abolished despite higher tissue para-sitism [19–21]. However, the dynamics of the parasite-induced inflammation in the cardiac tissue is poorly under-stood. The present study was undertaken to determine thekinetics of tissue parasitism, inflammatory cell infiltratesand expression of different members of the CC and CXCchemokine subfamilies, as well as other pro-inflammatorycytokines, in heart tissue of C57BL/6 mice infected withthe Colombiana strain, a myotropic strain of T. cruzi. Ourresults show that C57BL/6 mice inoculated with 50trypomastigotes/each developed subpatent parasitemiaand an intense cardiac inflammation, which peakedaround 30-60 days postinfection (p.i.) and was correlatedwith tissue parasitism. The inflammatory infiltrate wascharacterized mainly by the presence of CD4+ CD8– α�,CD4–CD8+ α� T cells and macrophages. Among variouschemokines we found that JE, MIP-1α, MIP-1�, MIP-2 andKC mRNAs were expressed in the heart mainly at 15 and30 days p.i., whereas RANTES, MIG and IP-10 mRNAswere enhanced during the early and late stages of infec-tion. Cytokine expression in cardiac tissue was character-ized by an early type 1 response, with elevated message ofIL-12(p40) and IFN-γ and late type 2 response with domi-nant expression of IL-4 and IL-10. Our results also showthat the downregulation of IFN-γ and most chemokinemRNA was associated with control of parasite replicationin cardiac tissue. Taken together, these findings further

indicate the importance of parasite components as stimu-lants of inflammation during experimental chagasic myo-carditis.

2. Material and methods2.1. Animals

Five- to six-week-old female C57BL/6 or outbred Swissmice were obtained from the Oswaldo Cruz Foundation –FIOCRUZ (Rio de Janeiro, Brazil) and maintained understandard conditions in the animal house of the Centro dePesquisas René Rachou – FIOCRUZ (Belo Horizonte, Bra-zil).

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 macroph-age experiments. For the culture-derived trypomastigotes,the LLCMK2 cell line was infected with trypomastigotesand maintained in DMEM supplemented with 5% FCS at33 °C in 5% CO2. After 4–5 days, the trypomastigoteswere collected daily, centrifuged at 40 × g at 4 °C for 10min for separation of debris, and centrifuged again at 700× g at 4 °C for 10 min. The resulting pellet containing liveparasites was used for purification of tGPI-mucins or inmacrophage experiments employing live parasites. ThetGPI-mucins were isolated from T. cruzi trypomastigotesas previously described [12, 23], using sequential organicextraction followed by hydrophobic-interaction chroma-tography on an octyl-Sepharose column (Pharmacia Bio-tech, Uppsala, Sweden) and elution with a propan-1-olgradient (5–60%).

The Colombiana strain was used in all in vivo experi-ments as well as in some in vitro experiments with inflam-matory peritoneal macrophages. T. cruzi Colombiana wasisolated by Frederici et al. [24] and maintained by serialpassages from mouse to mouse in the Laboratory of Cha-gas' Disease - CPqRR-FIOCRUZ (Belo Horizonte, Brazil).C57BL/6 mice were infected intraperitoneally with 50blood-derived trypomastigotes of T. cruzi Colombiana.The levels of parasitemia were evaluated using 5 µL ofblood obtained from the tail vein of infected mice aspreviously described [25]. Animals were sacrificed ondays 0, 15, 30, 60 and 120 p.i. and cardiac tissue wasdivided and stored under specific conditions for differentassays.

2.3. Histologic evaluation

Groups of four animals were sacrificed on different daysafter T. cruzi infection. The myocardium was fixed inneutral 10% formalin, embedded in paraffin, sectioned,stained with hematoxylin & eosin (HE) and examined bylight microscopy. Tissue parasitism was scored by count-ing the total number of amastigote nests in 12 microscopefields (1 × 100 magnification) per histopathological sec-tion. Four sections were counted for each animal and theindividual data was determined as the mean result of thefour sections. The data presented for each group are themean and standard deviation of four animals.

An inflammatory infiltrate was considered to be presentwhen we detected 50 leukocytes or more in each inflam-

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matory focus. In addition, the inflammatory foci weresubdivided into focal and diffuse infiltrates, depending onhow closely the inflammatory cells were associated. Mostcells from the focal inflammatory infiltrate were in directcontact with each other, forming a continuous site ofinflammation. In contrast, the diffuse inflammatory infil-trate was defined as a high density of mononuclear cellsscattered throughout the cardiac tissue and, sometimes,also composed of one or more small inflammatory focicontaining less than 50 inflammatory cells. For the inflam-matory infiltrate score, the total number of focal or diffuseinflammatory foci was counted in 12 microscope fields (1× 100 magnification) per cardiac section. Four sectionswere counted for each animal and individual data wasdetermined as the mean result of the four sections. Thedata presented for each group are the mean and standarddeviation of four animals.

2.4. Characterization of the inflammatory infiltratepresent in cardiac tissue from infected mice

Fragments of cardiac tissue were frozen in liquid N2

and stored until use. The frozen tissue was then embeddedin tissue-tek (OCT, Miles, USA), sectioned with a cryostatand fixed in cold acetone. Rat anti-mouse CD4, anti-mouse CD8, anti-mouse Mac-1 or anti-mouse CD3 anti-bodies (Pharmingen, San Diego, CA) were incubated withmouse cardiac tissue previously incubated with normalgoat serum. Thereafter the sections were incubated withbiotinylated secondary anti-rat antibodies, followed byincubation with a peroxidase-streptavidine conjugate. Thereaction was developed with 3-amino 9-ethyl-carbazolein sodium acetate solution in the presence of H2O2. Slideswere examined by light microscopy. For the confocalmicroscopic studies, the same methodology as describedabove was used, except that rat anti-mouse CD8 (Pharm-ingen) and anti-mouse CD4 (Pharmingen) antibodies weredirectly labeled with FITC and PE, respectively. Hearts ofthree animals were analyzed for each time p.i. For eachanimal, 20 inflammatory infiltrates were analyzed by con-focal microscopy or conventional immunocytochemistry.

2.5. FACS analysis

In order to isolate the mononuclear cells from themyocardium, hearts from 10–20 animals, at each time p.i.,were washed to remove the blood clots, pooled, mincedwith scissors in 1–2 mm fragments and subjected to enzy-matic digestion using a solution of 0.015% trypsin (Sigma,St Louis, USA) and 0.01% collagenase A (BoehringerMannheim Biochemicals, Mannheim, Germany). Theinflammatory cells were then purified in a FicollHypaqueTM gradient (d = 1.077 g/mL). Flow cytometryanalyses of inflammatory cells recovered from heart tissuefrom C57BL/6 mice infected with T. cruzi were performedas previously described [26, 27]. Briefly, single cell sus-pensions were then stained with FITC- or PE-labeled anti-bodies against CD3, CD4, CD8, TCR-α� or MAC-1 mol-ecules. Viable cells (3 × 105) were analyzed with a FACS440 (Becton and Dickson, San Jose, USA) as determinedby narrow forward-angle light scatter and exclusion ofpropidium iodide.

2.6. Macrophage culture

C57BL/6 mice were inoculated intraperitoneally with 2mL of 3% thioglycollate, and four days later, the elicitedperitoneal exudate cells were harvested in cold serum-freeDMEM [12, 13]. The medium used in the macrophagecultures (MacMed) consisted of DMEM (Gibco, GrandIsland, NY) supplemented with 40 µg/mL gentamicin and5% heat-inactivated FCS. Macrophages were resuspendedin MacMed at 2 × 106/mL, and 0.5 mL aliquots weredispensed into the wells of a 24-well plate. Cells wereallowed to adhere for 3h at 37 °C in the presence of 5%CO2, and then washed once with serum-free DMEM, and1.0 mL of MacMed was added to each well. The plateswere incubated overnight at 37 °C in the presence of 5%CO2 and in the absence or presence of 50 units/mL ofrecombinant murine IFN-γ (Genentech, San Francisco,CA, USA). The macrophages were washed and then cul-tured 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 macroph-ages were lysed and total RNA extracted according to theprotocol described below.

2.7. Quantification of murine IFN-γ

Suspensions of splenocytes from uninfected andinfected mice were washed in Hank's balanced salt solu-tion (HBSS) and treated with lysing buffer (nine parts of0.16 M NH4Cl and one part of 0.17 M Tris-HCl, pH 7.5) for2 min. The erythrocyte-free cells were then washed threetimes in HBSS and adjusted to 3 × 106 cells of RPMI-1640(Flow Laboratories, Inc., McLean, VA) supplemented with10% fetal calf serum (Hyclone, Logan, UT),2-mercaptoethanol (5 × 10–5 M), L-glutamine (2 mM), andantibiotics (all purchased from Sigma). The cell suspen-sion was distributed (1 mL/well) in 24-well tissue cultureplates (Corning, Corning, NY) and cultured for 48 h at 37°C in a humidified 5% CO2 atmosphere, in the presence orabsence of T. cruzi lysate (TcAg) (10 µg/mL), or Con-A(2 µg/mL).

The levels of IFN-γ in splenocyte culture supernatantsand sera were assayed in a two-site ELISA using a ratanti-IFN-γ mAb R46A2 (ATCC, Rockville, USA) and apolyclonal rabbit serum specific for the cytokine, as pre-viously described [12]. IFN-γ levels were calculated byreference to a standard curve constructed with recombi-nant cytokine (Genzyme, Cambridge, MA, USA). Sensitiv-ity of this method was 100 pg/mL.

2.8. Detection of parasite specific DNA in the cardiactissue of infected mice

Cardiac fragments were used as DNA source for thedetection of a T. cruzi specific gene. DNA was extractedwith phenol:chloroform:isoamylic alcohol, precipitated inisopropanol and washed in 70% ethanol. The DNA prepa-ration was resuspended in water and the concentrationadjusted to 10 and 100 ng/µL and used as template for PCRusing specific primers for T. cruzi guanine hypoxanthinephosphoribosyltransferase (HGPRT - 412 bp) geneHGPRT1 (forward): 5'- CTACAAGGGAAAGGGTCTGC-3'and HGPRT2 (reverse): 5'-ACCGTAGCCAATCACA-AAGG-3' designed from the complete nucleotide

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sequence of the HGPRT gene [28]. Each amplificationreaction was performed in a final volume of 10 µL, con-taining 0.5 U of Taq DNA polymerase (Cenbiot, RS, Brazil)/200 µM of each dNTP/15 mM MgCl2/50 mM KCl/10 mMTris-HCl pH 8.5/5 pmol of each primer and 10 or 100 ng ofDNA extracted from infected animals. After heat denatur-ation for 5 min at 95 °C, samples were submitted to 30cycles at three temperatures (95 °C/1 min, 55 °C/1 min and72 °C/1 min). In the final cycle an extension step at 72 °Cwas performed for 5 min. After amplification, 3 µL of eachreaction was eletrophoresed in 6% acrylamide gel andsilver stained [29].

2.9. RT-PCR assay for measuring in vivo or in vitroexpression of cytokine mRNA

RNA was isolated from cardiac tissue of mice by acidguanidinium thiocyanate-phenol-chloroform extraction:RNA STAT-60TM [30]. One microgram of total RNA wasreverse transcribed by the addition of 2.5 U RNAsin(Promega Corporation, Madison, WI), 2.5 mM deoxy-nucleotides (dNTPS) (Boehringer Mannheim), 0.1 M dithio-threitol - DTT (Gibco BRL Life Technologies, Inc., GrandIsland, NY), 1X Moloney murine leukemia virus RNAaseH

–reverse transcriptase buffer (Life Technologies), 25 ng

random hexamer oligonucleotides (Boehringer Man-nheim), and 200 U Moloney murine leukemia virus RNAseH– reverse transcriptase (Life Technologies) in a total vol-ume of 20 µL. The reaction proceeded for 1 h at 37 °C andwas terminated by boiling for 5 min after the addition of175 µL H2O. Five microliters of cDNA was used for ampli-fication in a 30 µL PCR reaction containing 2.5 mM dNTPs(Pharmacia), a 0.2-mM concentration of the 3' and 5'exter-nal primers, 1.5 mM MgCl2; 1X GeneAmp PCR buffer and0.5 U Taq DNA polymerase (Promega). PCR conditionswere 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, 7min (final cycle). The primers used are shown below. PCRproducts and molecular weight marker were run on 6%polyacrylamide gel and stained with silver nitrate [26].Plasmids containing chemokine/cytokine-encodingsequences were used to establish the PCR conditions.

PCR products on silver-stained gels were quantifiedwith a densitometer (Shimadzu Corporation, Tokyo, Japan)using a CS-9301 PC program. The densitometry value foreach cytokine/chemokine was corrected for the mousehypoxanthine phosphoribosyltransferase (HPRT) value forthe same sample and divided by the average value for thatcytokine/chemokine obtained from uninfected controls. Alow level of constitutive expression of cytokines/chemokines was observed in samples from cardiac tissueof noninfected controls or unstimulated macrophages.The results are reported as fold increase over uninfectedcontrol or unstimulated macrophages.

The primer (sense and antisense) sequence from 5'to 3',followed by number of cycles and expected product sizeof PCR presented within parentheses is indicated below.CXC chemokines: MIP-2, CGC-GGA-TCC-CCT-GGT-TCA-GAA-AAT-CAT-CC, CGC-GGA-TCC-TCC-CCA-GTC-TCT-TTC-ACT-GT (33,468 bp); KC, CGC-GGA-TCC-TTG-ACC-CTG-AAG-CTC-CCT-TGG-TTC, CGC-GGA-TCC-CGT-GCG-TGT-TGA-CCA-TAC-AAT-ATG (35,521 bp);

IP-10, CGC-GGA-TCC-TGA-GCA-GAG-ATG-TCT-GAA-TC, CGC-GGA-TCC-TCG-CAC-CTC-CAC-ATA-GCT-TAC-AG (33,399 bp); MIG, GAT-CAA-ACC-TGC-CTA-GAT-CC, GGC-TGT-GTA-GAA-CAC-AGA-GT (35,399bp); SDF-1α, CTC-CAA-ACT-GTG-CCC-TTC-AG, AAA-GCT-CCA-TTG-TGC-ACG-GG (35,348 bp); SDF-1�, CCG-GAA-TTC-CTC-CAA-ACT-GTG-CCC-TTC-AG, CCG-GAA-TTC-GCC-TGT-CAC-CAA-TGA-CGT-TG (35,368bp), LIX, GCC-GGA-ATT-CGG-GAT-CTT-GTC-CAC-AAT-GAC, AAC-TGC-AGC-AGG-GAC-AAT-GGT-TTC-CCT-T(35, 547 bp). CC chemokines: MIP-1α, CGC-GGA-TCC-CGG-AAG-ATT-CCA-CGC-CAA-TTC, CGC-GGA-TCC-GGT-TGA-GGA-ACG-TGT-CCT-GAA-G (35,448 bp);MIP-1�, CGC-GGA-TCC-CCC-ACT-TCC-TGC-TGT-TTC-TCT-TAC, CGC-GGA-TCC-AGC-AGA-GAA-ACA-GCA-ATG-GTG-G (33,444 bp); JE, CCG-GAA-TTC-CAC-TCA-CCT-GCT-GCT-ACT-CAT-TCA-C, CCG-GAA-TTC-GGA-TTC-ACA-GAG-AGG-GAA-AAA-TGG (30,505 bp);RANTES, CGC-GGA-TCC-CCA-CGT-CAA-GGA-GTA-TTT-CTA-CAC-C, CGC-GGA-TCC-CTG-GTT-TCT-TGG-GTT-TGC-TGT-G (26,326 bp); TCA-3, TGT-TAC-AGA-AAG-ATG-GGC-TCC-TCC, TCC-AAG-AAA-CAG-AGG-CAG-CG (33,324 bp); eotaxin, CAC-GAA-GCT-TTA-GGT-AAG-CAG-TAA-CTT-CCA-TCT-GTC-TC, GCG-GCT-AGC-TGA-CTA-AAT-CAA-GCA-GTT-CTT-AGG-CTC-TG(35,380 bp).

The sequence of primers used to measure the messagesof IL-1�, IL-12(p40), TNF-α, IL-2, IL-4, IL-5, IL-10 andIFN-γ was the same as those used in our previous studies[30, 31].

2.10. Statistical analysis

Arithmetic or geometric means (parasitemia and densi-tometric levels) and standard deviations of the means werecalculated. The Student's t-test was used to analyze thestatistical significance of the differences observed inRT-PCR analysis. Differences were considered statisticallysignificant when P < 0.05.

3. Results3.1. Parasitemia, cardiac tissue parasitism and mortalitycurves of C57BL/6 mice infected with T. cruzi Colombiana

C57BL/6 mice were inoculated with 50 parasites; mostanimals survived the parasitemia peak (figure 1A) at 30days of infection, and about 25% of the animals survivedup to 120 days p.i. (figure 1B). Figure 2A (top panel) showsnine amastigote nests in a field of cardiac tissue from amouse at 30 days p.i. Considering that the size of amastig-ote nests present in cardiac tissue of animals infected for15, 30 and 60 days was not statistically different, we usedthe number of amastigote nests as an indicator of tissueparasitism. Our results indicate that the peak of tissueparasitism (figure 2B) coincides with the peak of para-sitemia (figure 1A) occurring at 30 days p.i. At this time ofinfection we observed an average 30 amastigote nests in12 histopathology fields (HE, 1 × 100) of cardiac tissue.After 30 days of infection, tissue parasitism decreased toundetectable levels until about 120 days p.i. Despite thenegative results of the histological examinations, by using

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a more sensitive method we were able to detect T. cruzigenomic DNA in the cardiac tissue of all animals even at120 days p.i. (figure 2C).

3.2. Analysis of T. cruzi-induced histopathology in cardiactissue

Using conventional HE staining we also investigatedthe effects of parasitic infection on cardiac tissue inflam-mation at different times after infection of C57BL/6 micewith T. cruzi Colombiana (figure 3A and 3B). The inflam-matory process around cardiac fibers began about 15 daysafter the initial infection. With the increase of amastigotenests at 30 days p.i., we observed a prominent inflamma-tory infiltrate along the cardiac fibers. As shown infigure 2B, the diffuse inflammatory infiltrate peaked at 30days of infection. After this period, the intensity of the focalinflammatory infiltrate increased, peaking at 60 days p.i.and coinciding with elimination of T. cruzi parasites. Lowlevels of a diffuse inflammatory process were observed inthe cardiac tissue of all animals even at 120 days p.i. withT. cruzi Colombiana (figure 3B).

3.3. Characterization of the cellular infiltrate in cardiactissue from mice infected with T. cruzi Colombiana

The characterization of leukocyte phenotypes in thecellular infiltrate from the cardiac tissue of C57BL/6 miceinfected with T. cruzi Colombiana was performed usingconventional immunocytochemistry, confocal micros-copy, and FACS analysis and shown in figure 4 and table I.Our results indicate a dominance of CD4+ T cells overCD8

+T lymphocytes during the initial phase of the infec-

tion, i.e., at 15 days p.i. (figure 4A – top panels). As shownin figure 4A and table I, a significant number of CD8+ Tlymphocytes was present in the cardiac tissue of miceinfected for 30 days. The number of CD8+ lymphocytesbecame dominant at 42 and persisted until 90 days afterinfection with T. cruzi Colombiana.

We also observed a gradual and significant increase inmononuclear cells expressing Mac-1 (CD11b) receptors

Figure 1. Natural course of T. cruzi infection in C57BL/6 mice. A. Parasitemia and B. mortality curve of C57BL/6 mice that wereintraperitoneally infected with 50 blood trypomastigote forms of T. cruzi Colombiana.

Figure 2. Analysis of T. cruzi parasitism in cardiac tissue ofC57BL/6 mice infected with T. cruzi. A. Nine amastigote nestscontaining variable numbers of parasites are shown in one field ofcardiac tissue from an animal at 30 days p.i. (HE, 1 × 200). Thearrows indicate amastigote nests. B. Number of amastigote nestsin 12 histopathological fields (magnification, × 56) obtained fromcardiac sections of mice at different times p.i. Each value pre-sented in figure 1B is the average and standard deviation of fouranimals. Similar results were obtained in two other experiments.C. Percentage of positive animals in a PCR using primers specificfor T. cruzi HGPRT. DNA from five distinct animals was used foreach time point. Similar results were obtained in a second experi-ment.

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(figure 4B – bottom panels). These cells were most likelymacrophages, since polymorphonuclear cells were rarelyseen at 30 days or later p.i. The relative number of mac-rophages increased from 40 and 20% of the inflammatoryinfiltrate at 42 and 90 days, respectively, to 50% of allinflammatory cells at 120 days p.i. as determined by FACSanalysis. A small percentage of polymorphonuclear cells(i.e., neutrophils) was observed in cardiac tissue during theearly stages of infection, i.e., at 15 and 30 days p.i., but notat later time points (data not shown).

3.4. Kinetics of monokine mRNA expression by cardiactissue from C57BL/6 mice infected with T. cruziColombiana

The results shown in figure 5 illustrate the expression ofthe monokines IL-1, TNF-α and IL-12(p40) at differenttimes p.i. with T. cruzi Colombiana. A small but measur-able increase in IL-1� was observed at 15 days p.i. At thelevel of mRNA expression, TNF-α was the dominantmonokine expressed in the heart of infected animals.TNF-α mRNA increased at 15 days p.i. and peaked at 30days p.i. In addition, we observed an increased expressionof TNF-α mRNA at late time points, i.e., 60 and 120 days

p.i. IL-12(p40) mRNA increased only at 15 days p.i. andthen declined thereafter, since at no other time point didwe observe increased expression of the IL-12(p40) gene inthe cardiac tissue of infected animals.

3.5. Kinetics of cytokine mRNA expression by cardiactissue from C57BL/6 mice infected with T. cruziColombiana

Regarding the mRNA expression of cytokine in cardiactissue from infected animals, we also observed an increasein both IFN-γ and IL-4 levels at days 15 and 30 p.i. Thisincrease was also accompanied by an increase in IL-10mRNA expression. No expression of IL-2 (figure 6) or IL-5(data not shown) mRNAs was observed at any time ofinfection. IFN-γ was the dominating lymphokine mRNA at15 days p.i., whereas at day 30 days after infection weobserved 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 persis-tence and dominance of type 2 cytokine mRNAs (i.e., IL-4and IL-10), whereas IFN-γ mRNA was regulated almost tothe basal levels (figure 6).

We also analyzed the levels of IFN-γ in the supernatantsof spleen cells and sera from animals at different times ofinfection with T. cruzi Colombiana. As shown in table II,maximal levels of IFN-γ were measured in supernatants ofsplenocytes 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 insupernatants of splenocytes and sera from animals at 60and 120 days p.i.

3.6. Macrophages as a source of chemokines in cardiactissue from mice infected with T. cruzi Colombiana

Our group [9, 10, 12, 13] and others [32, 33] have nowrepeatedly shown that T. cruzi trypomastigotes are potentstimulators of cytokine synthesis by macrophages. Becausemacrophages are an important source of various chemok-ines and were found to be present in large amounts incardiac tissue from animals infected with T. cruzi Colom-biana, we decided to investigate the ability of T. cruzitrypomastigotes to induce expression of chemokinemRNAs by inflammatory peritoneal macrophages. Theresults presented in figure 7 show that either live tissueculture trypomastigotes (tTryp) or the glycosylphosphati-dylinositol linked mucin-like glycoproteins isolated fromT. cruzi trypomastigotes (tGPI-mucins) were able to induceexpression of RANTES, MIP-1α, MIP-1�, KC and IP-10mRNAs by inflammatory macrophages. Expression of someof these chemokines was upregulated (RANTES and IP-10)or downregulated (KC) by IFN-γ. In vitro MIG was onlyinduced by IFN-γ (data not shown). Although live trypo-mastigotes enhanced expression of MIG induced by IFN-γ(data not shown), parasite products alone were unable totrigger expression of MIG mRNA.

3.7. Kinetics of CC chemokine mRNA expression bycardiac tissue from C57BL/6 mice infected with T. cruziColombiana

RANTES, JE and MIP-1� were found to be the main CCchemokine mRNAs expressed in cardiac tissue during theacute phase of experimental Chagas' disease. We also

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

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Figure 4. Characterization of infiltrating cells in cardiac tissue obtained from mice infected with T. cruzi. A. Frozen sections from cardiactissue of animals obtained at different times p.i. with T. cruzi were stained with FITC-labeled anti-CD8 and PE-labeled anti-CD4 mAbsand analyzed with a confocal microscope. Top panels show the staining in control and infected mice, at 15 and 30 days p.i. Red and greenstaining indicate the presence of CD4+ CD8– and CD4–CD8+ T lymphocytes, respectively. B. Frozen sections from cardiac tissue of animalsobtained at different times p.i. with T. cruzi were stained with anti-MAC-1 biotinylated mAb, vizualized by using a streptavidin-peroxidaseconjugate and analyzed under the light microscope (magnification, × 385). Four panels show the staining in tissue from an uninfectedcontrol (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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observed a small increase in the expression of MIP-1α at15 days p.i. Expression of JE and MIP-1� mRNAs were atbasal levels at 60 and 120 days p.i., whereas RANTESmRNA was still elevated at 120 days p.i. (figure 8). Wewere unable to detect expression of TCA-3 or EotaxinmRNA at any time during experimental infection with T.cruzi (data not shown).

3.8. Kinetics of CXC chemokine mRNA expression bycardiac tissue from C57BL/6 mice infected with of T. cruziColombiana

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

4. DiscussionIn the present study we used a previously described

model [24] to investigate the kinetics of tissue parasitism,inflammation and cytokine synthesis during infection withT. cruzi Colombiana. The advantage of this model for theimmunopathology of Chagas' disease is related to theintense cardiomyopathy associated with the high rate ofanimal survival, which enables the study of induction andresolution of cardiomyopathy during experimental infec-tion with T. cruzi. For this purpose, we infected animalswith 50 trypomastigotes from T. cruzi Colombiana andanalyzed the kinetics of tissue parasitism, inflammatoryinfiltrate, as well as lymphokine, monokine and chemok-ine expression in the cardiac tissue of infected animals.Our results show that control of tissue parasitism is accom-panied by decreased inflammation and expression of dif-ferent chemokines and IFN-γ mRNAs. In contrast, theexpression of type 2 cytokines (i.e., IL-4 and IL-10) incardiac tissue was maintained at later times of infectionwith T. cruzi, when tissue parasitism was controlled.

In our initial studies we decided to follow the kinetics oftissue parasitism and inflammation in cardiac tissue ofanimals infected with T. cruzi Colombiana. Our resultsshow that tissue parasitism initiates at about 15 days p.i.,peaking at 30 days and already declining at 60 days p.i. A

close correlation was also observed in terms of tissueparasitism and intensity of inflammation in the cardiactissue. The diffuse and focal inflammation peaked at 30and 60 days p.i., respectively, and declined thereafter.However, low levels of cardiac tissue inflammation wereobserved even at 120 days p.i., when amastigote nestswere not detectable by conventional histopathology. Nev-ertheless, the vast majority of animals were positive whenwe searched for T. cruzi genomic DNA using PCR. Thelatter results are consistent with the hypothesis that,although at low levels, tissue parasitism is an essentialcomponent of inflammation during the chronic phase ofChagas'disease [34–36].

The phenotypic analysis revealed that at different timesof infection the inflammatory reaction was dominated bymononuclear cells expressing the MAC.1 marker (i.e.,macrophages) and CD3+ α�TCR+ lymphocytes expressingeither CD4 and CD8 markers. Consistent with previousstudies using human cardiac sections [37, 38] or themouse model [20, 21] with other T. cruzi strains, ourkinetic studies showed a dominance (i.e., over 60% oftotal T lymphocytes) of CD8+ T lymphocytes after 30 daysof infection. Although there was not much variation interms of relative amount of different inflammatory cells,the absolute number of inflammatory cells was muchlower from 60 to 120 days p.i.

We also analyzed the expression of the monokines,TNF-α, IL-12(p40) and IL-1�. Our results show that TNF-αmRNA was already enhanced at 15 days p.i. and expressedat high levels in cardiac tissue from infected animals.Although at lower levels, TNF-α mRNA was detectableabove control values, even at 120 days after infection,when the inflammatory infiltrate was controlled. Thesefindings are consistent with those obtained for humansections which demonstrate expression of high levels ofTNF-α protein during chronic Chagas' disease [38]. Incontrast, expression of IL-12(p40) and IL-1� mRNAs wasincreased in cardiac tissue at day 15 p.i., and decreased tobasal levels at 30 days p.i. or at later time points.

Of the 13 chemokine mRNA [39, 40] tested in thecardiac tissue from animals infected with T. cruzi at differ-ent times p.i., there was a dominance of chemokinesinduced by IFN-γ, namely MIG [41], RANTES [42] andIP-10 [43]. The expression of MIG, RANTES and IP-10mRNAs persisted even at 120 days p.i., despite low levelsof expression of IFN-γ mRNA in heart tissue at 60 and 120days p.i. A possible explanation for this observation could

Table I. Distribution of CD3+CD4+CD8– and CD3+CD4–CD8+ α�+ T lymphocytes in cardiac tissue of animalsinfected with Trypanosoma cruzi Colombiana*.

Days postinfection CD4+CD8– CD4–CD8+ CD3+/TCRα�+ CD4–CD8–CD3–/TCRα�–

0 0.6 % 0.4 % 1.2 % 98.8 %42 8.5 % 28.9 % 37.5 % 62.4 %90 17.3 % 51.1 % 73.6 % 12.4 %120 7.4 % 8.4 % 15.0 % 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 intwo different experiments.

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be that infection with T. cruzi elicits high enough levels ofIFN-γ in the circulation that are sufficient to stimulate cellsin heart tissue to express MIG, RANTES and IP-10 mRNAs[44]. Alternatively, parasites present in cardiac tissue, asindicated by T. cruzi specific PCR, would be sufficient tokeep stimulating the expression of mRNAs specific for

these three chemokines. Consistent with this hypothesiswe showed the ability of T. cruzi parasites or their products(i.e., tGPI-mucins) to induce inflammatory macrophagesto express IP-10 and RANTES. The dominant expression ofMIG, RANTES and IP-10 is also consistent with the pres-ence of a large proportion of T lymphocytes in the inflam-

Figure 5. Kinetics of monokine expression in the cardiac tissue from C57BL/6 animals infected with 50 blood trypomastigote forms ofT. cruzi as determined by RT-PCR. Total RNA was extracted from cardiac tissue, obtained at different times after T. cruzi infection, reversedtranscribed and used as template for PCR employing primers specific for HPRT, TNF-α, IL-1� and IL-12(p40). Top panel A. shows asilver-stained gel containing PCR products for some of the monokine analyzed. Each lane of the gel corresponds to the result of RT-PCRobtained from a single animal. Bottom panel B. shows the semi-quantitative analysis of monokine mRNA expression in the cardiac tissueof infected mice. Individual values were pooled from two different experiments and used to obtain the arithmetic means and standarddeviations 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 indicatethat 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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Figure 6. Kinetics of type 1 and type 2 cytokine expression in the cardiac tissue from C57BL/6 animals infected with 50 bloodtrypomastigote forms of T. cruzi as determined by RT-PCR. Total RNA was extracted from cardiac tissue of animals, obtained at differenttimes 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-5and IL-10. Top panel A. shows a silver-stained gel containing PCR products obtained by RT-PCR. Each lane of the gel corresponds to theresult of RT-PCR obtained from a single animal. ND (i.e., not done) on silver-stained gels indicates that cytokine specific RT-PCR was notperformed on that sample. Bottom panel B. shows the semi-quantitative analysis of type 1 and type 2 cytokine mRNA expression in cardiactissue of infected mice. Individual values were pooled from two different experiments and used to obtain the arithmetic means and standarddeviations 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 indicatethat 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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matory infiltrates found in cardiac tissue of animals infectedwith T. cruzi, since these chemokines are chemoattracta-nts for lymphocytes [45–47].

In addition to inducing expression of RANTES andIP-10, T. cruzi trypomastigotes were also able to trigger thesynthesis of JE, MIP-1α, MIP-1� and KC by inflammatorymacrophages, and their expression was also augmented inthe cardiac tissue of infected animals. In contrast, we wereunable to detect any message in the cardiac tissue ofinfected animals for the following chemokines: SDF1α,SDF1�, TCA3, eotaxin and LIX. In general, the kinetics forexpression of chemokine mRNA (i.e., KC, MIP-1α, MIP-1�, JE and RANTES) was similar to the pattern of intensityof parasitism in the cardiac tissue of animals infected withT. cruzi. Therefore despite the controversy about the majortarget(s) (i.e., self-antigens or parasite antigens) for inflam-matory lymphocytes [48, 49] in the experimental model ofmyocarditis induced by T. cruzi, the presence of parasitesin cardiac tissue above a certain limit may be essential foreliciting expression of chemokines and recruitment ofinflammatory cells leading to chagasic myocarditis.

Interestingly, we found that the pattern of chemokinemRNA expressed by macrophages exposed to tTryp ortGPI-mucins was very similar to that expressed in thecardiac tissue of infected animals. KC, IP-10, MIP-1α,MIP-1�, RANTES and JE were all induced, whereas SDF-1α, SD-F1�, TCA3, LIX and eotaxin were all negative inmacrophages exposed to T. cruzi or its products or cardiactissue from infected animals. This similarity in the panel ofchemokine mRNA expression suggests that macrophagesmay indeed be an important source of chemokines incardiac tissue of animals infected with T. cruzi.

In terms of type 1 and type 2 cytokines, we found thatIFN-γ, IL-4 and IL-10 but not IL-2 or IL-5 mRNAs wereupregulated 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-2were not detectable at any time of infection. There was aconcomitant fall in the levels of IFN-γ mRNA, but not ofIL-4 or IL-10 mRNAs, with the resolution of inflammationin the heart of chagasic animals.

Modulation of IFN-γ and upregulation of IL-4 and IL-10proteins during the chronic phase of Chagas'disease havealso been found in cardiac tissue of animals infected withthe Brazil strain of T. cruzi [52], thus suggesting that thismay not be a specific phenomenon for the infection withthe Colombiana strain. The dominance of type 2 over type

Table II. Levels of IFN-γ synthesis induced during infection with Trypanosoma cruzi Colombiana1.

Splenocyte culture supernatants (ng/mL)

Days p.i. (n) Medium TcAg ConA Sera (ng/mL)

0 5 < 0.5 1.2 ± 0.2 12.3 ± 0.8 < 0.515 5 3.2 ± 1.3** 22.1 ± 5.1** 20.3 ± 2.1** 4.7 ± 1.5**30 5 2.1 ± 0.9* 14.5 ± 2.7** 17.7 ± 2.3* 3.2 ± 1.5**60 5 1.6 ± 0.5* 8.7 ± 1.7** 14.9 ± 3.1 2.5 ± 0.7*120 5 1.3 ± 0.2* 6.1 ± 2.1** 11.5 ± 1.5 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 significantwhen compared to uninfected animals. Similar results were obtained in a second experiment.

Figure 7. Identification of macrophages exposed to T. cruziparasites or parasite glycoconjugates as a major cellular source ofchemokine mRNAs. Thioglycollate-elicited macrophages fromC57BL/6 were cultured in medium alone (control), with tissueculture trypomastigote forms (tTryp) of T. cruzi Colombiana ortGPI-mucins extracted from tissue culture trypomastigotes. TotalRNA was extracted from macrophages 6 h after macrophagestimulation and levels of cytokine transcripts were measured byRT-PCR. Top panel A. shows silver-stained gel containing PCRproducts obtained by RT-PCR. Bottom panel B. shows thesemi-quantitative analysis of chemokine mRNA expression inmacrophages exposed to either live trypomastigotes or tGPI-mucins. Similar results were obtained in a second experiment.

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1 cytokines at 60 and 120 days p.i. may be occurring onlyin cardiac tissue, since our ex-vivo experiments with sple-nocytes demonstrated that both IFN-γ and IL-4 were pro-duced at high levels at 15 days p.i. and at lower levelsthereafter, without showing a dominance of IL-4 duringthe late stages of infection (data not shown). Because IL-4and IL-10 possess a downregulatory activity on the devel-opment and effector functions of cell-mediated immunity,it is tempting to speculate that the decline in IFN-γ andpersistence of IL-4 and IL-10 expression may be an impor-tant event responsible for controlling the strong cell-mediated immunity and immunopathology elicited by T.cruzi infection. This hypothesis remains to be experimen-tally demonstrated. Nevertheless, different studies have

demonstrated the ability of IL-4 and IL-10 to regulateinflammatory processes involved in the tissue damage thatoccurs during different parasitic infections [53, 54] andautoimmune diseases [55, 56].

Powell and colleagues found an association betweenhigh levels of IL-4 mRNA expression and susceptibility tomyocarditis elicited by the Brazil strain of T. cruzi [57].Thus, it is possible that the regulatory effect of IL-4 andIL-10 on cell-mediated immunity will enhance suscepti-bility to T. cruzi. Additional studies using IL-4 knockoutmice will be necessary to further analyze the role of IL-4 inthe immunopathology of experimental Chagas' disease.Nevertheless, the local action of T lymphocytes, IFN-γ andmacrophage may no longer be essential once a T. cruzi

Figure 8. Kinetics of CC and CXC chemokine expression in the cardiac tissue from C57BL/6 animals infected with 50 bloodtrypomastigote 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-1�,TCA3, and eotaxin. A. Top panel shows silver-stained gel containing PCR products for some of the CC chemokines analyzed. Each lane ofthe gel corresponds to the result of RT-PCR obtained from a single animal. ND (i.e., not done) on silver-stained gels indicates that cytokinespecific-RT-PCR was not performed on that sample. B. Bottom panel shows the semi-quantitative analysis of CC chemokine expression inthe cardiac tissue of infected mice. Individual values were pooled from two different experiments and used to obtain the arithmetic meansand 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 twoasterisks indicate that differences are statistically significant at P < 0.05 and P < 0.01 levels, respectively, when comparing expression of acytokine/chemokine at different times p.i. with those of uninfected mice.

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specific humoral response develops. In fact, different stud-ies suggest that B lymphocytes and antibodies have animportant role in resistance to chronic infection with T.cruzi [58, 59]. Finally, it is also possible that the reactiva-tion of cardiopathy may emerge from a breakdown ofimmunological control of T. cruzi and/or from immuno-regulatory activity on cell-mediated immunity in cardiactissue during chronic Chagas' disease. Consistent with thishypothesis are the studies showing a more intense cardi-opathy and lethality in mice lacking a functional IL-10gene [54], and an increased IFN-γ response to parasiteantigens in patients with the cardiac form of Chagas'disease [60]. We believe that infection of mice with theColombiana strain will be a useful model to analyze therole of cytokines on induction and regulation of cardiacpathology elicited by T. cruzi.

Acknowledgments

We thank Denise C. Cara and Mauro M. Teixeira forhelp with the analysis of histopathology data and forcritically reading this manuscript. This work was partiallysupported 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 researchfellows from CNPq.

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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 astemplate for PCR reaction employing primers specific for HPRT, MIG, IP-10, MIP-2, KC, and SDF1-α. A. Top panel shows silver-stainedgels 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 thesemi-quantitative analysis of CXC chemokines expression in the cardiac tissue of infected mice. Individual values were pooled from twodifferent experiments and used to obtain the arithmetic means and standard deviations shown. The total number of animals varied from 6to 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 andP < 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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