Type 1 chemokine receptor expression in Chagas' disease correlates with morbidity in cardiac patients

Abstract

Chemokines and chemokine receptors (CKRs) control the migration of leukocytes during the inflammatory process and are important immunological markers of type 1 (CCR5 and CXCR3) and type 2 (CCR3 and CCR4) responses. The coexpression of CKRs (CCR2, CCR3, CCR5, CXCR3, and CXCR4) and intracellular cytokines (interleukin-10 [IL-10], IL-4, tumor necrosis factor alpha [TNF-alpha], and gamma interferon [IFN-gamma]) on T CD4+ and CD8+ peripheral cells from individuals with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas' disease after in vitro stimulation with Trypanosoma cruzi antigens, were evaluated in this study. The percentage of T CD4+ and CD8+ cells coexpressing CCR5 and IFN-gamma, CXCR3 and IFN-gamma, and CXCR3 and TNF-alpha were higher in CARD than in IND individuals; on the other hand, the percentage of T CD4+ or CD8+ cells coexpressing CCR3 and IL-10 or coexpressing CCR3 and IL-4 were lower in CARD individuals than in IND individuals. In addition, a significant positive correlation between the expression of CCR5 or CXCR3 and IFN-gamma was observed in CARD individuals contrasting with a significant positive correlation between the expression of CCR3 and IL-4 and of CCR3 and IL-10 in IND patients. These results reinforce the hypothesis that a T. cruzi-exacerbated specific type 1 immune response developed by CARD chagasic patients is associated with the development of heart pathology.

FIG. 1.

Representative dot plots of PBMC from patients with Chagas disease. (A) Forward (FSC) and side (SSC) scatter dot plots demonstrate R1 (small lymphocytes) and R2 (lymphocyte blast gate) gates after 6 days of in vitro culture with T. cruzi antigens. (B) Dot plot representation of the frequencies of chemokine receptor-expressing cells (e.g., CXCR4) in R2 after 6 days of culture with T. cruzi antigens. (C) Dot plot side scatter and CD4⁺ cells after 6 days of culture with T. cruzi antigens showing low granularity and strong CD4 positivity is delineated in gate 3 (R3). (D) Dot plot showing the frequency of cells expressing CXCR3 and/or IFN-γ identified using directly phycoerythrin-conjugated CXCR3 antibodies (y axis) or fluorescein isothiocyanate-conjugated anti-IFN-γ antibodies (x axis) after 6 days of in vitro culture with T. cruzi antigens in R3.

FIG. 2.

Chemokine receptor (CCR2, CCR5, CXCR3, CXCR4, and CCR3) (A) and intracellular cytokine (IL-10, IL-4, TNF-α, and IFN-γ) (B) expression by PBMC from chagasic patients was evaluated after in vitro stimulation with T. cruzi antigens. The results given were calculated as the percentage of blast-gated cells. NI, noninfected individual (n = 10); IND, indeterminate patients (n = 15); CARD, cardiac patients (n= 15). The asterisks indicate statistical differences (P < 0.05) between groups.

FIG. 3.

The percentage of CD4⁺ or CD8⁺ T cells coexpressing chemokine receptors and cytokines was determined by multiparametric fluorescence-activated cell sorting analysis as described in Materials and Methods. The results given were calculated as the percentage of R3 gated cells. (A) Percentage of CD4⁺ cells coexpressing CCR3-IL-4 or CCR3-IL-10; (B) percentage of CD8⁺ cells coexpressing CCR3-IL-4 or CCR3-IL-10; (C) percentage of CD4⁺ cells coexpressing CCR5-IFN-γ or CCR5-TNF-α; (D) percentage of CD8⁺ cells coexpressing CCR5-IFN-γ or CCR5-TNF-α; (E) percentage of CD4⁺ cells coexpressing CXCR3-IFN-γ or CXCR3-TNF-α; (F) percentage of CD8⁺ cells coexpressing CXCR3-IFN-γ or CXCR3-TNF-α. NI, noninfected individuals (n = 10); IND, indeterminate patients (n = 15); CARD, cardiac patients (n= 15). Asterisks indicate statistical differences (P < 0.05) between groups.