Synergism between tumor necrosis factor‐α and interferon‐γ on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide‐dependent mechanism

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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.