Trypanosoma cruzi-induced tyrosine phosphorylation in murine peritoneal macrophages.

Trypanosoma cruzi, the etiological agent of Chagas' disease, binds to and invades macrophages and other cells (fibroblasts, muscle cells) via a complicated set of interactions, but the changes induced by parasite-to-cell interactions are largely unknown. This report investigates the ability of T. cruzi to elicit a tyrosine kinase pathway in immature and mature resident murine peritoneal macrophages (MPM) that differ in their susceptibility to parasite infection. T. cruzi stimulated the phosphorylation of tyrosine residues in several endogenous substrates (proteins of 40-42, 53-56, 66, 75, 80, 90, 95, 100, and 112 kDa), but only in immature MPM. Mature MPM had high levels of spontaneous tyrosine phosphorylation. Upstream tyrosine kinases, such as src-like tyrosine kinases, were not responsible for the differential patterns of tyrosine phosphorylation since they were present in both mature and immature MPM. We suggest that the tyrosine phosphorylation patterns stimulated by T. cruzi reflect most of the biochemical events that occur in parasite host-cell interactions.

Impaired protein catabolism in Trypanosoma cruzi-infected macrophages: possible involvement in antigen presentation.

The effect of Trypanosoma cruzi infection on the ability of mature and immature murine peritoneal macrophage (MPM) subpopulations to catabolize the bacteriophage lambda repressor cI protein (cI) has been investigated. The capacity of infected MPM to present the cI and to stimulate various CD4+, I-Ad- or I-Ed-restricted T-cell hybridomas specific for cI was also assessed. Our results show that the radioiodinated cI uptake and catabolism decreased sharply after infection of MPM with T. cruzi. A cI presentation deficiency appeared in mature and immature MPM infected with T. cruzi trypomastigotes. The ability of infected MPM to bind immunogenic cI (12-26) peptides to the plasma membrane Ia molecules was also altered, especially in immature MPM, as shown with paraformaldehyde prefixed MPM, suggesting that these MPM only have a few functional Ia molecules on their membrane. The reduced capacity of cI presentation to the I-Ed-restricted B26.1 hybridomas by infected MPM subpopulations was comparable to that of the I-Ad-restricted B24.4 and B26.2 T cells. The percentage of major histocompatibility complex (MHC) class II-positive MPM was also reduced after T. cruzi infection. The percentage of positive interleukin-2 receptor (IL-2R) MPM was sharply lowered in infected cells, even with a pre- or a post-interferon-gamma (IFN-gamma) activation. Finally, inhibition of prostaglandin with indomethacin, or of nitric oxide with N-monomethyl-L-arginine, or of tumour necrosis factor-alpha (TNF-alpha) with specific monoclonal antibodies did not restore the cI presentation capacities of the MPM subpopulations. Taken together, these results suggest that T. cruzi infection induces a reduced capacity for macrophages to take up and catabolize antigen, resulting in a deficient antigen processing and presentation of the derived immunogenic peptides to specific CD4+ T-helper type-1 cell hybridomas. The decreased cI presenting capacity was a function of the cell's burden and maturity.

Immunophenotyping of murine peritoneal macrophages and lymphocytes by flow cytometry.

Peritoneal cells include mainly macrophages and both T and B lymphocytes. We describe a simple procedure to analyze by flow cytometry the antigens expressed on macrophage and/or lymphocyte membranes. F4/80 and CR3 macrophage antigens were detected with anti-F4/80 and anti-Mac-1 monoclonal antibodies (mAb) respectively, CD4 and THY1.2 T lymphocyte antigens with GK1.5 and J1j mAbs respectively, B CD5 lymphocytes were identified with LY1.2 mAb and Fc gamma RII/Fc gamma RIII were detected with 2.4G2 mAb.

Quantification of bacterial phagocytosis by flow cytometry and spectrofluorimetry.

Bacterial phagocytosis is a cardinal function of phagocytes. We describe a simple procedure to easily quantify this function using fluoresceinated bacteria. Non-ingested bacteria and those adsorbed to the cell membrane are eliminated by an enzymatic procedure. Only macrophages with ingested fluorescent bacteria are detected, thereby permitting an accurate quantification of the phagocytic process by both spectrofluorimetric measurement and flow cytometric analysis.

Separation of murine peritoneal macrophages using Percoll density gradients.

Macrophages harvested from the murine peritoneal cavity are functionally and morphologically heterogeneous. Here, we describe a procedure which permits the determination of specific cell densities using a continuous density gradient of Percoll (analytical step). Subsequently, discontinuous density gradients are used in routine (preparative step) to isolate all the cell subpopulations according to their actual specific density. This procedure has been successfully used for both mouse and rat peritoneal macrophages.

Interferon-gamma-activated immature macrophages exhibit a high Trypanosoma cruzi infection rate associated with a low production of both nitric oxide and tumor necrosis factor-alpha.

Murine peritoneal macrophages (MPM) can be subdivided into two subpopulations of mature and immature macrophages. In contrast to mature macrophages, immature ones were highly susceptible to Trypanosoma cruzi infection. This highly susceptibility was associated with a low production of alpha 2-macroglobulin. Interferon-gamma (IFN-gamma)-activated immature macrophages also exhibited a higher infection rate than did IFN-gamma-activated mature ones. This higher rate of infection was associated with a low production of both nitric oxide (N = O) and tumor necrosis factor-alpha (TNF-alpha). In contrast, mature MPM showed a lower rate of infection and produced higher levels of N = O and TFN-alpha. Taken together, these results show a clear-cut difference in the course of T. cruzi infection in relation to the macrophage maturation state.

Mouse peritoneal macrophages: characterization of functional subsets following Percoll density gradients.

Mouse resident peritoneal cells were separated into twelve fractions on Percoll gradients according to their specific density and were thoroughly characterized by Giemsa staining, some biochemical assays, immunophenotyping and phagocytic tests. Among these fractions, the macrophages were mainly represented in 7 subsets of 1.073 to 1.104 g/ml densities. The results of this study emphasize that resident peritoneal macrophages of primo-explantation can be divided into two distinct subpopulations with separate functions, related to the stage of cell maturity. In fact, our results show that one macrophage subpopulation is rich in immature cells, characterized by their peroxidative activity, the expression of F4/80 antigen, Mac-1 and Fc receptors, in correlation with their high specific density; the second subpopulation contains mature macrophages (lower percentage of peroxidase-positive cells) with lower densities and a lower level of expression of the above-mentioned molecules. Antibody-dependent and antibody-independent bacterial phagocytosis, the phagocytic index and Fc gamma RII rosetting increased together with the cell density, and were elevated in the immature cell subpopulation. T and B lymphocytes were also identified in all the macrophage subsets, but in a low proportion.