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Infection and Immunity, March 2003, p. 1185-1193, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1185-1193.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evidence that Development of Severe Cardiomyopathy in Human Chagas' Disease Is Due to a Th1-Specific Immune Response

J. A. S. Gomes,1,2 L. M. G. Bahia-Oliveira,3 M. O. C. Rocha,4 O. A. Martins-Filho,2 G. Gazzinelli,2 and R. Correa-Oliveira2*

Departamento de Bioquímica e Imunologia,1 Faculdade de Medicina, Curso de Pós-graduação em Medicina Tropical, Universidade Federal de Minas Gerais,4 Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, Minas Gerais,2 Laboratório de Biologia do Reconhecer, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, Rio de Janeiro, Brazil3

Received 11 June 2002/ Returned for modification 27 August 2002/ Accepted 3 December 2002


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ABSTRACT
 
The role of interleukin 10 (IL-10) and gamma interferon (IFN-{gamma}) on the development of pathology in human Chagas' disease was investigated. Two categories of patients, low and high producers of IFN-{gamma}, were identified based on the levels of secretion of this cytokine in the supernatant of peripheral blood mononuclear cell (PBMC) cultures. Eighty-three percent of the patients presenting with cardiac disease (CARD) of different degrees and 59% of the patients with the indeterminate form of disease (IND) were identified as high IFN-{gamma} producers. PBMC from IND patients classified as low IFN-{gamma} producers secreted significantly higher amounts of IL-10 than did those from other groups. Flow cytometry analysis demonstrated that in PBMC from the IND group, the majority of the IL-10-producing cells were monocytes (CD14High+ cells), whereas in the CARD group, the major sources of IFN-{gamma} were T lymphocytes (CD3+ CD4+ cells). These results suggest an association between the production of IFN-{gamma} by CD3+ CD4+ cells and morbidity in Chagas' disease, whereas the production of IL-10 by macrophages/monocytes leads to regulation of the immune response in IND patients. We hypothesize that an exacerbated production of IFN-{gamma} against Trypanosoma cruzi antigens favors the development of a strong Th1 response in CARD patients, which leads to progression of heart disease.


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INTRODUCTION
 
Chagas' disease, caused by the protozoan Trypanosoma cruzi, is an important public health problem on the American continent (34). A wide clinical spectrum is observed in the chronic phase of Chagas' disease. Sixty percent of affected individuals have the indeterminate (IND) form of the disease with morbidity apparently absent, whereas 30% show heart involvement, leading to heart failure and sudden death. Less than 10% of the infected patients display the gastrointestinal form of the disease, which is characterized by pathological dilations of the digestive tract (5).

Although significant controversy still exists regarding the mechanism of pathology, the role of the immune response in mediating tissue destruction has been documented (11, 12, 18, 23, 24, 31) in experimental models of the acute and chronic disease (10, 21, 22, 28). These studies demonstrated that cytokines are important components for building up and controlling an immune response during T. cruzi infection (36).

Higher levels of interleukin 5 (IL-5), IL-10, IL-13, and gamma interferon (IFN-{gamma}) mRNA can be detected on freshly isolated peripheral blood mononuclear cells (PBMC) from chronic chagasic patients, regardless of the clinical form of the disease, than from NI individuals. These results suggest that a fine balance of these cytokines could be the major key in controlling morbidity during chronic disease (6).

Despite the high levels of IFN-{gamma} production by PBMC from cured patients after specific treatment, suggesting a beneficial role of IFN-{gamma} for the effectiveness of chemotherapy (3), the involvement of this cytokine in conjunction with cytotoxic events on the development of pathology has also been reported (18, 30).

In the present study we have investigated the role of IL-10 and IFN-{gamma} and its relationship with the development of pathology in Chagas' disease by evaluating the PBMC response from chronic chagasic individuals, including both IND and cardiac (CARD) patients, with the latter presenting different degrees of heart damage. We also investigated the correlation between the balance of the two cytokines on the evolution of the disease.


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MATERIALS AND METHODS
 
Patient selection. Patient selection was conducted at the Chagas' disease Outpatient Referral Center of the Federal University of Minas Gerais, Belo Horizonte, Brazil, between February 1995 and June 2000. Positive serology for Chagas' disease was determined by two or more tests (indirect immunofluorescence, enzyme-linked immunosorbent assay [ELISA], indirect hemagglutination, or complement fixation). Patients who agreed to participate in this study signed a written informed consent and were subjected to a standard screening protocol that included medical history, physical examination, electrocardiogram (EKG), laboratory and chest X-ray examinations, and echodopplercardiography evolution. The patients were not undergoing chemotherapy treatment, nor had they been previously treated for T. cruzi infection. To define the IND form, esophagograms and barium enemas were also performed. The exclusion criteria included the presence of systemic arterial hypertension, diabetes mellitus, thyroid dysfunction, renal insufficiency, chronic obstructive pulmonary disease, hydroelectrolytic disorders, alcoholism, history suggesting coronary artery obstruction and rheumatic disease, and the impossibility of undergoing the examinations. The study population consisted of 111 patients who completed the screening protocol. All patients were in the chronic phase of the infection. Patient clinical findings are summarized in Table 1.


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  		TABLE 1. Details of the study population

The patients were grouped as follows: IND, patients with the chronic IND form of Chagas ' disease, individuals without significant alteration in the electrocardiography, chest X ray, echodopplercardiogram, esophagogram, and barium enema; and CARD, patients with chronic chagasic cardiopathy, with or without symptomatic cardiomyopathy and different degrees of alterations on the EKG. The CARD patients were subgrouped according to the degree of heart damage (Table 1) into the CARD I, CARD II, and CARD III groups. The CARD I group included those patients in the New York Heart Association (NYHA) functional class I who had no clinical or radiological signs of heart enlargement but with minor electrocardiographic alterations, like low voltage of the QRS complex in the standard leads, left anterior hemiblock, and slight changes in the S-T segment and the T wave. CARD II group were patients in the NYHA functional class I or II with a normal cardiothoracic ratio but presenting remarkable eletrocardiographic alterations, such as right bundle branch block (RBBB), frequent ventricular premature beats, RBBB associated with left anterior hemiblock, abnormal Q waves, diffuse negative symmetric T waves, left bundle branch block, atrial fibrillation, second-degree atrioventricular (AV) block Mobyts type II, and complete AV block (2.3). CARD III group were patients with echocardiographic and/or clinical and radiological signs of heart enlargement, specially a final diastolic diameter of the left ventricle of >55 mm. Healthy individuals, from areas of nonendemicity within the same age range and showing negative serological tests for Chagas' disease were included as a control group (noninfected [NI]).

The study protocol complied with the Helsinki Declaration and was approved by the Ethical Committee of the Hospital das Clinicas of the Universidade Federal de Minas Gerais (UFMG-COEP). Written informed consent was obtained from patients according to the guidelines of the Ethical Committee of the Hospital das Clinicas of the Universidade Federal de Minas Gerais (COEP-ETIC 025/97).

Antigens. Epimastigote (EPI) and trypomastigote (TRYPO) antigens were prepared by using the Y strain of T cruzi (29). EPI or TRYPO were washed three times in cold phosphate-buffered saline (PBS), disrupted by repeated freezing at -70°C and thawing, and homogenized at 4 to 6°C in a Potter-Elvejem centrifuge at 20,000 rpm five times for 60 s, with 30-s intervals on ice. The suspension was subsequently centrifuged at 40,000 x g for 60 min on ice. The clear supernatant was dialyzed for 24 h at 4°C against PBS, filter sterilized on 0.2-µm-pore-size membranes, assayed for protein concentration, aliquoted, and stored at -20°C until use.

Cell preparation and proliferation assay. PBMC were isolated by Ficoll-diatriazoate density gradient centrifugation (LSM; Organon Teknica, Charleston, S.C.) as previously described (7). For antigen stimulation, 106 cells per well were used, and 150,000 cells/well were used for mitogen stimulation. These were the final concentrations per milliliter of culture determined to be optimal at 25 µg of EPI, 20 µg of TRYPO, and 15 ng of staphylococcal enterotoxin B (SEB) (Sigma, St. Louis, Mo.). The culture medium consisted of 90.4% RPMI 1640, 1.6% L-glutamine, 3% antibiotic-antimycotic additions (stock of 10,000 U of penicillin, 10,000 U of streptomycin, and 5 µg of amphotericin B [Fungizone] per ml; GIBCO), and 5% of AB Rh-positive heat-inactivated normal human serum. The cultures were maintained in flat-bottom 24-well tissue culture plates at 37°C, in an atmosphere of 5% CO2.

Cytokine ELISAs. The cytokine concentrations in the supernatants were measured by two-site sandwich ELISAs. To measure IL-10 or IFN-{gamma}, plates were coated overnight with monoclonal antibodies 9D7 (immunoglobulin G1) to IL-10 or A35 (immunoglobulin G1) to IFN-{gamma} and incubated with a standard concentration of recombinant IL-10 or recombinant IFN-{gamma} or with the supernatants. As a detecting antibody, we used anti-IL-10 or anti-IFN-{gamma} monoclonal antibodies bound to nitroiodofenilacetate, and the reaction developed by using horseradish peroxidase-conjugated anti-nitroiodofenilacetate antibodies (all of the antibodies and standard cytokines were a gift from Robert Coffman from the DNAX Institute, Palo Alto, Calif.) and ABTS (2,2-azino-di[3-ethylbenzthizoline] sulfonic acid) (Zymed, San Francisco, Calif.) substrate. The sensitivity of the ELISA was 0.001 ng/ml for IL-10 measurements and 0.19 ng/ml for IFN-{gamma} measurements. The supernatants were collected every day for 6 days of culture to establish the kinetic curve; there was one culture for each day of the kinetic experiments.

Single-cell cytoplasmic cytokine staining. PBMC were cultured in polypropylene tubes (106) for 24 h in 24-well plates (106) for 6 days at 37°C and 5% CO2 in RPMI 1640 supplemented with 1.6% L-glutamine, 3% antibiotic-antimycotic additions (stock of 10,000 U of penicillin, 10,000 U of streptomycin, and 5 µg of amphotericin B per ml; GIBCO) and 5% of AB Rh-positive heat-inactivated normal human serum. PBMC were stimulated with either medium alone, EPI (25 µg/ml), or TRYPO (20 µg/ml). During the last 4 h of culture, brefeldin A (Sigma) (10 µg/ml), which impairs protein secretion by the Golgi complex, was added to the cultures, as described by Jung et al. (13). Cultured cells were washed twice in PBS containing 1% bovine serum albumin and stained with monoclonal antibodies conjugated with fluorescein isothiocyanate for the following cell surface markers (antibody clones are indicated in parentheses): CD3 (UCHT1), CD4 (RPA-T4), CD8 (RPA-T8), CD19 (HIB19), CD16 (3G8), CD14 (M5E2). The cells were then fixed in formaldehyde (4%) and permeabilized in saponin buffer (0.5%) (Sigma) for 15 min. Finally, the cells were incubated with monoclonal antibodies conjugated with phycoerythrin to IL-10 (JES3-9D7) and IFN-{gamma} (B27). Phenotypic analyses were performed by two-color flow cytometry with a Becton Dickinson FACScan flow cytometer. A minimum of 50,000 events were acquired for each assay. This number was required due to the low frequency of positive events being analyzed. Controls of nonstimulated cells versus those stimulated with phorbol myristate acetate (10 ng/ml) and ionomycin (1 µg/ml) were used to standardize the antibodies as positive controls.

Analysis of fluorescence-activated cell sorter data. Lymphocytes were analyzed for their intracellular cytokine expression patterns and frequencies and for surface markers by using the program Cell Quest. The frequency of positive cells was analyzed in three gates for each staining: gate 1 (R1), lymphocyte gate; gate 2 (R2), large lymphocyte blast gate; and gate 3 (R3), the macrophage gate (Fig. 1). Limits for the quadrant markers were always set based on negative populations and isotype controls. This approach allows for the determination of the frequency of populations in subregions of mononuclear cells, taking advantage of the known position of mononuclear cells that is based on their size and granularity profiles. For the analysis of CD4- and CD8-positive lymphocytes, the quadrants were always set for high CD4 and CD8 populations so as not to include CD4 low-positive monocytes and macrophages and CD8 low-positive NK cells, respectively. For analysis of CD14-positive macrophages, we used fluorescence 1 for the CD14 versus granularity. The analyzed population presented intermediary granularity and strong positivity for this cell marker, therefore delineating gate 3 (R3), the macrophage gate. This analytical approach allowed us to obtain a homogeneous and well-defined population, securing its standardized and safe identification. After this region was selected, we analyzed the fluorescence intensity of these cells in R3.



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  		FIG. 1. Representative dot plots of PBMC from patients with Chagas' disease. (A) The forward-versus-side-scatter (FSC x SSC) dot plot demonstrates the placement of the R1 (small lymphocytes) and R2 (blast lymphocytes) gates after 24 h of in vitro stimulation with T. cruzi antigens. (B) The forward-versus-side-scatter dot plot demonstrates the placement of the R1 and R2 gates to select small lymphocytes and blast lymphocytes, respectively, after 6 days of in vitro stimulation with T. cruzi antigens. (C) The dot plot demonstrates the frequency of cytokine-positive cells in the R2 gate. (D) The side-scatter-versus-CD14-positive cells dot plot demonstrates the placement of gate 3 (R3), the macrophage gate. (E) The dot plot demonstrates the frequency of cytokine-positive cells in the R3 gate.

Statistical analysis. The cytokine ELISA data were log transformed before performing statistical analysis. The single-cell cytoplasmic cytokine data were Arcsen [radical]y transformed before performing statistical analysis. The student t test and analyses of variance were used to compare the means between 2 or more groups, respectively. In all tests, a P value of <0.05 was considered significant. The relationships between IL-10 and IFN-{gamma} were examined by the Spearman rank correlation test. The statistical test used to compare proportions of high and low responders between IND and CARD patients was the chi-square test for linear trend. All analyses were performed by using MINITAB, Inc., software. The statistical analysis was performed by using the mean values of each group. The error bar is presented as the standard error of the mean.


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RESULTS
 
IL-10 and IFN-{gamma} production have distinct kinetics. To determine the peak of IL-10 and IFN-{gamma} secretion, we performed a kinetic study, where PBMC from 11 chagasic patients, regardless of the clinical form of disease, were stimulated with EPI or TRYPO and the levels of these cytokines were measured daily for 6 days. The kinetics of IL-10 and IFN-{gamma} production by PBMC from IND and CARD (I, II, and III) patients stimulated with EPI or TRYPO antigens revealed that the peak production of IL-10 in the culture supernatant occurred at 24 h after stimulation, whereas the highest levels of IFN-{gamma} production were observed at day 6 after in vitro stimulation (Fig. 2A and B). Secretion of IL-10 was also observed in the control cultures. The kinetics of IL-10+ and IFN-{gamma}+ cells was also examined by staining PBMC after stimulation with EPI or TRYPO antigens (Fig. 2C and D). The peak of IL-10+ cells occurred at 24 h, and the highest percentage of IFN-{gamma}+ cells occurred on the sixth day (Fig. 2B). No detectable levels of IL-10+ or IFN-{gamma}+ cells were observed in PBMC from NI patients (data not shown). These results revealed that the peak of IL-10+ and IFN-{gamma}+ cells is synchronized with the peak of these cytokines in the supernatants of PBMC cultures.



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  		FIG. 2. PBMC from chagasic patients (n = 11) were cultured without stimulation (Medium) and after in vitro stimulation with antigens from the EPI or TRYPO form of T. cruzi. IL-10 (A) and IFN-{gamma} (B) production were measured in supernatants daily for six consecutive days as described in Materials and Methods. The results are expressed as means ± standard errors (in nanograms/milliliter). The values for each point are the relative means of the 11 individuals independent of the clinical form of the disease. The peak production of IL-10 was observed at 24 h, and the peak production of IFN-{gamma} occurred on the sixth day after stimulation. The kinetics for intracellular staining for IL-10-positive cells (C) and IFN-{gamma}-positive cells (D) were measured daily for six consecutive days as described in Materials and Methods. The results are expressed as percentages of cytokine-positive cells within the blast gate for IL-10 and IFN-{gamma} and the monocyte gate for IL-10. The peak of IL-10+ cells occurred at 24 h, and the peak of IFN-{gamma}+ cells occurred on the sixth day after in vitro stimulation.

Flow cytometric analysis of intracellular IL-10 and IFN-{gamma}. To investigate the cellular sources and frequencies of IL-10- and IFN-{gamma}-producing cells, cytokine profiles after EPI and TRYPO stimulation were determined after 24 h and 6 days of culture, respectively. The analysis was performed by using different gates, as described in Materials and Methods: R1 for lymphocytes, R2 for large lymphocytes (blast cells), and R3 for monocytes macrophages (Fig. 1). The statistical differences in the percentages of IL-10+ and IFN-{gamma}+ cells were observed only within the blast cell gate. Figure 3A and F show the analysis of the total population positive for IL-10 or IFN-{gamma}. It clearly demonstrates that IL-10 is higher in the IND group while IFN-{gamma} is higher in the CARD group of patients. For the identification of the cell population secreting IL-10, CD3, CD19, CD14, and CD16 were used (Fig. 3B, C, D, and E). For the analysis of the IFN-{gamma}-positive population, CD3, CD16, CD4, and CD8 markers were used (Fig. 3G, H, I, and J). It is clear from the data presented that in the IND group, the population secreting IL-10 is the monocytic CD14+ (Fig. 3D) while those secreting IFN-{gamma} are the CD3+CD4+ cells in the CARD group (Fig. 3G and I). Although other cell populations were found to be positive for these cytokines in the different groups, no statistically significant differences were observed.



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  		FIG. 3. Percentage of cytokine expression in different cell populations of chagasic patients after in vitro stimulation with T. cruzi antigens. The percentages of IL-10-positive cells were determined for cells expressing CD3 (T lymphocytes), CD19 (B lymphocytes), CD14 (monocytes/macrophages), and CD16 (NK cells) as described in Materials and Methods. The results given were calculated as the percentages of blast- and monocyte-gated cells. (A) Percentage of IL-10-producing cells in the total lymphocyte population; (B) percentage of CD3+ cells producing IL-10; (C) percentage of CD19+ cells producing IL-10; (D) percentage of CD14+ cells producing IL-10; (E) percentage of CD16+ cells producing IL-10. In IND patients (n = 32), IL-10 is produced predominantly by monocytes (CD14+) when compared with T lymphocytes, B lymphocytes, and NK cells. The percentage of IFN-{gamma}-positive cells was determined for cells expressing CD3 (T lymphocytes), CD16 (NK cells), and T cell subsets CD4 and CD8 as described in Materials and Methods. The results given were calculated as the percentages of blast-gated cells. (F) Percentage of IFN-{gamma}-producing cells in the total lymphocyte population; (G) percentage of CD3+ cells producing IFN-{gamma}; (H) percentage of CD16+ cells producing IFN-{gamma}; (I) percentage of CD4+ cells producing IFN-{gamma}; (J) percentage of CD8+ cells producing IFN-{gamma}. In the CARD I, II, and III groups (n = 9, 16, and 12, respectively), IFN-{gamma} is produced mainly by CD3+ cells when compared with NK cells and CD4+ cells when compared with CD8+ cells. The statistical differences are shown on the figure with P values of <0.05.

IND and CARD patients can be separated into groups of low or high IFN-{gamma} producers. The levels of IFN-{gamma} and IL-10 were measured in PBMC supernatants of 70 CARD (I, II, and III) patients and 41 IND individuals. The levels of both cytokines after stimulation with EPI or TRYPO antigens were not statistically different between the IND and CARD (I, II and III) groups (Fig. 4A and B). However, when patients were classified according to the amounts of IFN-{gamma} produced by T. cruzi antigen-stimulated PBMC as high (>=5 ng/ml) or low (<4.9 ng/ml) producers, this cutoff value corresponded to the mean value of unstimulated control cultures added with 2 values of standard deviation. The CARD group has a significantly higher proportion (P < 0.01) of high IFN-{gamma} producers (83%, 58/70) than the IND group (59%, 24/41) (Fig. 4C). T. cruzi antigen-induced IFN-{gamma} production by PBMC from chagasic patients (IND or CARD) was significantly higher than IFN-{gamma} production by NI individuals (P < 0.05). It is important to mention that the IFN-{gamma}/IL-10 ratio also was significantly higher in the CARD patients(P < 0.05) than that observed in IND individuals.



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  		FIG. 4. Levels of IL-10 and IFN-{gamma} secreted by PBMC from chagasic patients and NI individuals after in vitro stimulation with EPI or TRYPO. The cytokine levels were measured on the first day for IL-10 (A) and on day 6 for IFN-{gamma} (B) independent of the stimuli. The levels of IFN-{gamma} and IL-10 production were not significantly different between IND (n = 41) and CARD (CARD I, II, and III [n = 13, 35, and 22, respectively] were analyzed as a single group) patients after stimulation with EPI or TRYPO. PBMC from NI individuals (n = 13) did not produce IL-10 nor IFN-{gamma} after stimulation with T. cruzi antigens. (C) IND and CARD patients were classified according to the levels of IFN-{gamma} produced after antigenic stimulation as high (>=5.0 ng/ml) and low (<4.9 ng/ml) producers. The percentage of high producers was significantly higher (83%) in the CARD group (CARD I, II, and III [n = 8, 32, and 18, respectively] analyzed as a single group) than in the IND group (59%) (P < 0.01). (D) The levels of IL-10 were plotted for high and low IFN-{gamma} producers. An opposite profile of IFN-{gamma} and IL-10 can be observed. The IND individuals who presented low IFN-{gamma} levels secreted significantly higher levels of IL-10 than did all of the other individuals. The statistical differences are shown on the figure with P values of <0.05.

The comparison of the levels of IL-10 secreted by PBMC from IND patients who are low IFN-{gamma} producers with those from IND patients who are high IFN-{gamma} producers showed that the former secrete significantly (P < 0.05) higher levels of IL-10 (Fig. 4D). However, no statistically significant difference is observed when one compares the levels of IL-10 production by low or high IFN-{gamma} producers belonging to CARD (I, II, and III) groups. When PBMC from CARD (I, II, and III) and IND individuals were stimulated with SEB antigen, no difference between low and high IFN-{gamma} producers was observed (data not shown).

To further confirm the relationship between the CARD and the levels of IL-10 and IFN-{gamma} production described above, we performed a Spearman correlation analysis and observed an inverse correlation between IL-10 and IFN-{gamma} induced by EPI (n = 24, r = -0.628, P < 0.05) and TRYPO (n = 24, r = -0.475, P < 0.05) in the IND high IFN-{gamma} producers.

IFN-{gamma} production in CARD patients can be correlated with the development of heart disease. The relationship between IFN-{gamma} production and the development of heart pathology was examined in CARD I, II, and III patients separately by plotting the degree of heart damage and IFN-{gamma} levels for each group (Fig. 5). The highest level of IFN-{gamma} paralleled the degree of the severity of cardiac involvement. Thus, the levels of IFN-{gamma} secreted by PBMC from CARD II and III patients were significantly higher (P < 0.05) than those of CARD I patients (Fig. 5A). However, the levels of IL-10 produced by all CARD patients were similar in response to the antigenic stimulation and to that of medium alone (Fig. 5B). The levels of IFN-{gamma} and IL-10 of PBMC stimulated with SEB were not statistically different among the groups. While confirming the role of IL-10 and IFN-{gamma} productions described above on the development of cardiac disease, we observed an inverse correlation between IL-10 and IFN-{gamma} induced by EPI and by TRYPO in patients with different degrees of heart function alterations as shown in Table 2.



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  		FIG. 5. PBMC from CARD patients presenting different degrees of heart damage have distinct levels of IFN-{gamma} (A) but not of IL-10 (B). The Cardiac patients were grouped as CARD I (n = 13), II (n = 35), and III (n = 22) as described in Materials and Methods. The levels of IFN-{gamma} in the supernatant of PBMC from CARD II and III patients after antigenic stimulation were significantly higher than those detected for CARD I patients (P < 0.05). No significant difference was observed between the groups after in vitro stimulation of PBMC with SEB. The levels of IL-10 production between the CARD groups stimulated with parasite antigens or SEB are not statistically different. The statistical differences are shown on the figure with P values of <0.05.


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  		TABLE 2. Spearman's rank correlation coefficients between IL-10 and IFN-{gamma} produced by PBMC from chagasic patients


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DISCUSSION
 
The results of the present study revealed that a strong response by CARD patients against T. cruzi antigens with high secretion levels of IFN-{gamma} and low secretion levels of IL-10 is associated with the development of heart damage. The association between secretion of high levels of IFN-{gamma} and the occurrence of cardiac forms of Chagas' disease observed in this study contrasts with previous data, showing a positive correlation between levels of IFN-{gamma} and cure of T. cruzi infection in patients during the acute phase of the infection (3). These apparently contrasting roles of IFN-{gamma} in T. cruzi infections suggest a dual role for this cytokine in Chagas' disease. In this context, it has been proposed that during the acute phase of the infection IFN-{gamma} can act synergistically with the specific treatment to eliminate parasites (16). On the other hand, during the chronic phase of the infection, exacerbation of the immune response with uncontrolled IFN-{gamma} production, particularly in heart tissues, promotes cytolytic destruction of myocardium (2, 24). Recent studies with an experimental murine model also support the dual role for this cytokine during a T. cruzi infection where a dissociation between inflammation and tissue parasitism may depend on the fine balance between Th responses, in which a Th1 response will, on one hand, control parasitism and, on the other hand, enhance heart inflammation throughout the course of the infection (30). These data indirectly reinforce the hypothesis that both the immune response against the parasite and also the documented autoimmune process can together govern the immunopathology of Chagas' disease (32). Thus, the type 1-dependent response to infection, while important in limiting parasite replication during the acute phase (3), may also be involved in the development of severe heart disease. However, an increase in IL-10 secretion during the chronic phase may be associated with protection from the host against excessive pathology induced by type 1 responses.

Due to the importance of these two cytokines in the immune response to the infection, it was important to determine the secretion levels in culture supernatants after in vitro stimulation with parasite antigens. Our data showed that IL-10 and IFN-{gamma} production and expression have distinct kinetics in infected patients, with the peak production and expression of IL-10 in the culture occurring 24 h after stimulation, and the highest levels of IFN-{gamma} production and secretion occurring on day 6 after in vitro stimulation. Similar data were obtained in studies by Kemp et al. (14), where IFN-{gamma} secretion starts on the third day and continues at high levels until the seventh day when both purified protein derivative and Leishmania antigens are used. The differences observed between the ELISA results and single-cell intracytoplasmic cytokine staining may be due to the sensitivity of the ELISA and the fact that the addition of brefeldin A leads to accumulation of the cytokine in the cytoplasm. The latter is therefore more representative of the activity of the cells and allowed the detection of cytokine-producing cells, which generally comprise less than 1% of cells and are thus below the threshold of detection (19), since consumption of the cytokine may occur in the tissue culture.

The determination of cellular sources of IFN-{gamma} and IL-10 in response to T. cruzi antigens was performed by flow cytometry. Analysis of the cell population secreting IFN-{gamma} showed that the majority of IFN-{gamma}+ producers were the CD3+CD4+ cells (Fig. 3), and the CARD group had higher levels of this cytokine than the IND and NI groups. These data suggest that the T. cruzi antigens induce IFN-{gamma} production by CD4+ T cells in the absence of IL-10 and that Th1 CD4+ T cells present in Chagas' disease may also have an important role in disease development. In this context, previous observations demonstrated the role of CD4+ T cells on the development of chronic carditis in an experimental model (26). In a more recent report, these authors showed that an anti-heart tissue CD4+ T cell line obtained from a chronic chagasic mouse had a Th1 cytokine pattern and that it was sufficient to induce an inflammatory heart response (25). This profile is compatible with the pathology of Chagas' disease, since Th1 cells are strong promoters of delayed-type hypersensitivity reaction. Other reports have also shown the role of human CD4+ cytotoxic T cells in infections such as toxoplasmosis (15, 20, 35) and murine malaria (33). More importantly, these cells were IFN-{gamma} secretors. We speculate that the CD4+ T IFN-{gamma}-secreting cells could have a cytotoxic effect themselves or could mediate lesion development by stimulating CD8+ cytotoxic T cells that would react against the heart tissue or both. In agreement with this suggestion, a recent report showed an association between Th1 response and cardiac disease in chagasic patients (1).

The fact that the source of IL-10 differs between CARD and IND patients (Fig. 3), namely, IL-10 is produced mainly by macrophages/monocytes (CD14+high) in the PBMC from the IND group, led us to speculate that macrophages might be important cells involved in the regulation of the immune response in Chagas' disease through the production of IL-10, which can regulate the expression or function of IL-12 and IFN-{gamma} (4, 9). The modulating activity of IL-10-producing macrophages may maintain a balance between parasitism and tissue integrity in IND patients, during which mild inflammatory foci resolve into focal fibrosis, and perhaps a contained Th1 response would keep parasitism under relative control. The progressive destructive process in CARD patients could therefore result from a failure of a pathogenic Th1 response to be down-regulated by IL-10. This failure could, in turn, depend on host genetic characteristics, on age-dependent changes of the immune system, or superposition of infections by unrelated microorganisms and/or by T. cruzi reinfection. In fact, we observed that there is a regulation of IFN-{gamma} production by IL-10 in chagasic patients when we stimulated PBMC with T. cruzi antigens in presence of anti-IL-10 (data not shown).

Patients belonging to the CARD and IND groups could be differentiated according to the levels of IFN-{gamma} production as high and low IFN-{gamma} producers (Fig. 4C). Eighty-three percent of CARD patients were high IFN-{gamma} producers, whereas 59% of IND individuals presented this profile of IFN-{gamma} secretion. This observation suggests that a relationship between IFN-{gamma} production and development of cardiac disease occurs. It is also important to mention that IFN-{gamma} is the cytokine most frequently demonstrated in inflammatory cells from the hearts of patients with Chagas' disease (24). It is also observed that CARD and IND patients may respond differentially to the IFN-{gamma}-inducing stimulus provided by T. cruzi infection (1). Given the T1-type cytokine profile of heart-infiltrating T-cell lines from CARD patients, the ability to mount a vigorous IFN-{gamma} response may play a role in the differential susceptibility to myocardiopathy development.

Although we have found that IFN-{gamma} production may contribute to the development of cardiomyopathy, a large proportion of the IND individuals also produced high levels of IFN-{gamma}, raising the question of the role of this cytokine in this group of patients. One possible explanation is that it is difficult to determine, precisely, the beginning of the T. cruzi infection for each patient; therefore, for IND as well as for CARD patients, the infection and the development of the disease are certainly asynchronous and these patients are at different stages of disease progression. Thus, if IFN-{gamma} production is a key factor for the development of severe cardiomyopathy, it is reasonable to speculate that the IND patients that are high IFN-{gamma} producers are candidates to develop cardiomyopathy sooner than the lower producers. On the other hand, it is probable that IND patients who are able to always keep low levels of IFN-{gamma} and high levels of IL-10 secretion will never develop cardiac disease. The asymptomatic period of the infection constitutes a long chronic stage in which no manifestation of disease is observed. The IND form of T. cruzi infection comprises a long period, usually from 10 to 20 years, between the end of the acute phase and the potential establishment of a late chronic heart disease. Epidemiological studies have shown that some IND individuals will never develop symptoms of T. cruzi infection in their life (5) while 2% of these individuals with become cardiac patients every year. To determine whether the degree of cardiac damage parallels or appears after alterations in the modulation of IFN-{gamma} secretion by IL-10, a longitudinal study with follow up of the IND group is needed. If this is confirmed, mechanisms to block heart disease development will need to be developed, such as the evaluation of the role of treatment during the chronic phase as a method of blocking the development of heart disease.

The suggestion presented here that an exacerbated IFN-{gamma} secretion can be correlated with the development of the severe cardiac form of the T. cruzi infection has been shown to occur in some intracellular bacterial and protozoan infections, such as Listeria monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, and Leishmania major, in which the Th1 response can be protective for the host but can also be detrimental when exacerbated (8, 17, 27).

In summary, we present a set of results evidencing that an exacerbated Th1-like specific immune response against T. cruzi antigens with high levels of IFN-{gamma} and low levels of IL-10 production is established in T. cruzi-infected individuals presenting with cardiac disease. We hypothesize that this type of immune response sustain the cytolytic profile of the cellular infiltrate observed with biopsies and autopsies of chagasic patients presenting with cardiac disease with myocellular destruction.


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ACKNOWLEDGMENTS
 
We thank Fausto G. Araújo (Palo Alto Medical Foundation) for critical reading of the manuscript and suggestions and Jeffrey M. Bethony (Centro de Pesquisas René Rachou-Fundação Oswaldo Cruz) for statistical advice.

Grant support was received from CNPq, FIOCRUZ-PAPES, and FIOCRUZ-CPqRR.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Pesquisas René Rachou/FIOCRUZ, Laboratório de Imunologia Celular e Molecular, Av. Augusto de Lima 1715, 30190-002, Belo Horizonte, MG, Brazil. Phone: 55 31 32953566. Fax: 55 31 32953115. E-mail: correa{at}cpqrr.fiocruz.br. Back

Editor: J. M. Mansfield


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Infection and Immunity, March 2003, p. 1185-1193, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1185-1193.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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