Tumor necrosis factor alpha augments nitric oxide-dependent macrophage cytotoxicity against Entamoeba histolytica by enhanced expression of the nitric oxide synthase gene.
Nitric oxide (NO measured as nitrite, NO2-) is the major effector molecule produced by activated macrophages for in vitro cytotoxicity against Entamoeba histolytica trophozoites. In this study, we determine whether tumor necrosis factor alpha (TNF-alpha) produced by activated bone marrow-derived macrophages (BMM) is involved in the induction of the inducible NO synthase gene (mac-NOS) for NO-dependent amebicidal activity. TNF-alpha alone did not directly induce macrophage NO2- production to kill amebae; however, in combination with increasing concentrations of TNF-alpha and gamma interferon (IFN-gamma), BMM amebicidal activity and NO2- production progressively increased and showed a significant linear correlation. Antiserum to TNF-alpha and the NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA) inhibited the synergistic effects of TNF-alpha and IFN-gamma. BMM activated with increasing concentrations of lipopolysaccharide (LPS) and IFN-gamma showed a significant linear correlation between TNF-alpha release and NO2- production. Antiserum to TNF-alpha suppressed TNF-alpha release, NO2- production, and amebicidal activity by 93, 53, and 86%, respectively. L-NMMA diminished NO2- production by 74% and macrophage amebicidal activity by 83% but had no effect on TNF-alpha release. Quantification by Northern (RNA) blot analyses demonstrated that IFN-gamma in combination with TNF-alpha or LPS increased markedly the accumulation of mac-NOS and TNF-alpha mRNAs in a time-dependent manner with a concomitant increase in NO and TNF-alpha production. Peak induction of mac-NOS occurred after 24 h, whereas TNF-alpha mRNA was rapidly expressed after 4 h and remained stable for 48 h. Taken together, these data argue that TNF-alpha augments NO-dependent macrophage cytotoxicity against E. histolytica via elevated levels of mac-NOS mRNA expression which may be associated with the accumulation of TNF-alpha mRNA.
Lin
J Y
JY
Institute of Parasitology of McGill University, Ste.-Anne de Bellevue, Québec, Canada.
Seguin
R
R
Keller
K
K
Chadee
K
K
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000818:Animals; D003602:Cytotoxicity, Immunologic; D004748:Entamoeba histolytica / Q000276:immunology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases