Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O-2 during phagocytosis of opsonized promastigotes. Addition of the O-2(-) scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O-2(-) enhances macrophage leishmanicidal activity. The importance of NO. produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO. in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor N-G-monomethyl-L-arginine inhibited IFN-gamma-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-gamma. These data suggest that O-2(-) and NO. each contribute to intracellular killing of L. chagasi inhuman and murine macrophages.