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Abstract

Intracellular replication of the protozoan parasite Trypanosoma cruzi inside macrophages is essential for the production of the disease and the development of the parasite. Two CD4+ T cell lines, A10 and A28, were established from T. cruzi-infected BALB/c mice which specifically proliferated to parasite antigens. The trypanocidal activity of BALB/c macrophages was induced upon culture with the A10, but not with the A28 T cell line. The cell-free supernatant from this A10 line, as well as from immune spleen cells stimulated with specific antigen or concanavalin A, but not from the A28 T cell line also activated the trypanocidal activity of peritoneal macrophages or of the J774 macrophage-like cell line. When the lymphokine content of the supernatants from both cell lines was analyzed, it was found that the A10 T cell line secreted interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin 2, whereas the A28 line did not secrete IFN-γ upon stimulation. Furthermore, the trypanocidal-inducing ability of A10 supernatant was completely abrogated by neutralizing anti-IFN-γ antibodies and partially abrogated by neutralizing anti-TNF-α antibodies. When recombinant cytokines were added to J774 cells, IFN-γ was able to induce significant trypanocidal activity whereas TNF-α was almost ineffective. However, TNF-α or lipopolysaccharide (LPS) showed a synergistic effect with IFN-γ on macrophage activation. IFN-γ triggered nitric oxide (NO) synthesis by J774 cells whereas TNF-α was almost ineffective. TNF-α and LPS were also synergistic with IFN-γ in the NO production. Both the NO production and the trypanocidal activity in J774 cells induced by T cell supernatants or lymphokine combinations were inhibited by N-monomethyl-L-arginine, a competitive inhibitor of NO synthase activity. A good correlation between the levels of NO production and trypanocidal activity induced by different lymphokine preparations was found. Those results suggest that IFN-γ and TNF-α, secreted by T. cruzi-immune T cells, are involved in the activation of the trypanocidal activity of mouse macrophages through an NO-dependent mechanism.