title: Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha.
abstract: Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.
authors: Department of Cellular Immunology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
authors: S J
authors: SJ
authors: Green
authors: R M
authors: RM
authors: Crawford
authors: J T
authors: JT
authors: Hockmeyer
authors: M S
authors: MS
authors: Meltzer
authors: C A
authors: CA
authors: Nacy
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001120:Arginine / Q000502:physiology*; D003602:Cytotoxicity, Immunologic; D007371:Interferon-gamma / Q000494:pharmacology; D007895:Leishmania tropica / Q000276:immunology*; D007896:Leishmaniasis / Q000276:immunology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008809:Mice, Inbred C3H; D009573:Nitrites / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides; D009573:Nitrites; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D001120:Arginine