Monocytes from patients with indeterminate and cardiac forms of Chagas' disease display distinct phenotypic and functional characteristics associated with morbidity

Abstract

Many studies have demonstrated that monocyte-derived macrophages display critical activities in immunity to parasites. The ability of these cells to process and present antigens, produce cytokines, and provide costimulatory signals demonstrates their pivotal role in initiating immune responses. Although potential modulatory function has been attributed to monocytes from patients with Chagas' disease, a systematic phenotypic and functional analysis of these cells has not been performed. In this work, we analyzed the ex vivo expression of important surface molecules (CD11b and HLA-DR) and immunoregulatory cytokines (interleukin-10 [IL-10], IL-12 and tumor necrosis factor alpha [TNF-alpha]) in CD14(+) and CD14(-) monocytes from Chagas' disease patients with polar clinical forms of the disease: indeterminate or severe cardiac. We also evaluated the influence of in vitro infection with T. cruzi in the expression of such molecules. We observed that monocytes from indeterminate-disease patients display lower levels of HLA-DR than those from noninfected individuals both ex vivo and after in vitro infection with T. cruzi. Although ex vivo expression of CD11b was similar among the groups, in vitro infection led to decreased expression of this molecule by monocytes from Chagas' disease patients but not from noninfected individuals. Analysis of the expression of immunoregulatory cytokines showed that while monocytes from indeterminate-disease patients are committed to IL-10 expression, a higher percentage of monocytes from cardiac-disease patients express TNF-alpha after exposure to live parasites. These results suggest that monocytes from indeterminate-disease patients display modulatory characteristics related to low HLA-DR and high IL-10 expression whereas monocytes from cardiac-disease, patients may be committed to induction of inflammatory responses related to high TNF-alpha expression.

FIG. 1.

(A to C) Evaluation of infectivity of adherent cells from noninfected (A), indeterminate-disease (B), and cardiac-disease (C) individuals by T. cruzi by using CFSE labeling. Cells were infected with labeled parasites and stained for surface markers as described in Materials and Methods. Infection of monocytes (CD11b⁺), T cells (CD3⁺), and B cells (CD19⁺) from noninfected individuals and indeterminate-disease or cardiac-disease patients are shown in representative dot plots, as indicated in the figure. The column on the right (CD11b) shows the staining with CD11b in cells incubated with unlabeled parasites. (D) Representative histograms for the mean intensity of CFSE fluorescence in each cell population for each group analyzed.

FIG. 2.

Analysis of surface molecules in monocytes from patients with indeterminate (I) and cardiac (C) Chagas' disease and noninfected individuals (N) after in vitro infection with T. cruzi or exposure to parasite antigen. Cells were doubly stained for the monocyte marker CD14 and CD11b or HLA-DR surface molecules and analyzed by flow cytometry, as described in Materials and Methods. White, hatched, and black bars represent the average values obtained by analysis of nontreated cells, cells infected in vitro with T. cruzi, and cells exposed to parasite antigen, respectively. Results are expressed as average values for double-positive cells as well as mean intensity of expression of each molecule within CD14⁺ cells. Identical symbols above the bars indicate statistical significance using the Tukey-Kramer test. Error bars indicate standard deviation.

FIG. 3.

Analysis of cytokines expressed by CD14⁺ monocytes from patients with indeterminate (I) and cardiac (C) Chagas' disease and noninfected individuals (N) after in vitro infection with T. cruzi or exposure to parasite antigen. Cells were doubly stained for the monocyte marker CD14 and cytokines and analyzed by flow cytometry, as described in Materials and Methods. Clear, white, cross-hatched, and black bars represent the average values obtained by analysis of nontreated cells, cells infected in vitro with T. cruzi, and cells exposed to parasite antigen, respectively. Results are expressed as average values for double-positive cells, as indicated. Identical symbols above the bars indicate statistical significance between groups/treatments using the Tukey-Kramer test. Error bars indicate standard deviation.