Interleukin 1β-stimulated production of nitric oxide in rat hepatocytes is mediated through endogenous synthesis of interferon gamma

Abstract

The multiple interlocking regulatory mechanisms that underlie induction of hepatocyte inducible nitric oxide synthase (iNOS) expression are largely unknown. Although previous work has indicated the requirement for multiple proinflammatory cytokines to induce hepatocyte NO production, investigators have recently shown that interleukin-1β (IL-1β) alone can initiate iNOS expression. In contrast, interferon gamma (IFN-γ) serves as the sole initiating factor in other cell systems. On the basis of the known ability of IL-1β to induce transcription and translation of the IFN family of genes, we hypothesized that IL-1β-mediated hepatocyte expression of iNOS is dependent on endogenous IFN-γ synthesis. In a system of rat hepatocytes in primary culture, IL-1β induced production of both NO and IFN-γ. Using in situ hybridization and immunoblot analysis, IFN-γ messenger RNA (mRNA) and protein were detected in hepatocytes exposed to IL-1β. Inhibition of NO synthesis using the competitive substrate inhibitor N-monomethyl-L-arginine (100 μmol/L) did not alter the extent of IL-1β-mediated IFN-γ synthesis. In contrast, anti-IFN-γ antibody or inhibition of IFN-γ mRNA translation by addition of antisense IFN-γ oligodeoxynucleotide probes resulted in undetectable levels of NO metabolites and iNOS protein. Repletion of IFN-γ to the system restored NO production to levels noted in the presence of IL- 1β alone. Transient transfection analysis using a rat hepatocyte iNOS promoter-reporter gene plasmid construct showed that IL-1β-induced promoter activation was abolished in the presence of anti-IFN-γ or antisense IFN-γ. Again, addition of IFN-γ to the system restored activity of the iNOS promoter. Parallel experiments examining IL-1β-mediated endogenous hepatocyte IL-1β and TNF-α synthesis indicated no role for these cytokines in the induction of iNOS expression by IL-1β. It is concluded that IL-1β- mediated hepatocyte synthesis of NO is dependent on the simultaneous endogenous synthesis of IFN-γ.