Requirement of tumor necrosis factor at and nuclear factor-kappa B in the induction by IFN-gamma of inducible nitric oxide synthase in macrophages

IFN-gamma induces NO production, inducible NO synthase (iNOS) protein, and promoter expression in mouse macrophage cells. Mutation of IFN regulatory factor 1 responsive element, gamma-activated site, as well as NF-kappa B elements in the murine iNOS promoter strongly reduced IFN-gamma-induced iNOS transcriptional activity. The role of NF-kappa B activation in iNOS induction by IFN-gamma was corroborated by overexpression of the NF-kappa B inhibitory protein I kappa B alpha, which inhibited iNOS promoter activity induced by IFN-gamma. In addition, IFN-gamma treatment induced p65 binding to the iNOS promoter by chromatin immunoprecipitation assay and NF-kappa B binding to DNA by EMSA, although with a delayed kinetics, suggesting an indirect autocrine role for another cytokine produced in response to IFN-gamma. It is interesting that we found that IFN-y induced TNF-alpha secretion, and the induction of iNOS expression by IFN-gamma was abolished in primary peritoneal macrophages from TNF-alpha-deficient (TNF-alpha(-/-)) mice or in RAW 264.7 cells treated with anti-TNF-alpha neutralizing antibodies. Moreover, exogenons addition of recombinant mouse TNF-alpha restored iNOS expression induced by IFN-gamma in TNF-alpha(-/-) mice. It is intriguing that NF-kappa B binding to DNA in response to IFN-gamma treatment was absent in TNF-alpha(-/-) mice. Taken together, our data suggest that the TNF-alpha produced in response to IFN-gamma is required for iNOS induction by activating NF-kappa B transcription factor.