INTERLEUKIN 1-BETA-STIMULATED PRODUCTION OF NITRIC-OXIDE IN RAT HEPATOCYTES IS MEDIATED THROUGH ENDOGENOUS SYNTHESIS OF INTERFERON-GAMMA

The multiple interlocking regulatory mechanisms that underlie inductio n 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 product ion, investigators have recently shown that interleukin-lp (IL-1 beta) alone can initiate iNOS expression. In contrast, interferon gamma (IF N-gamma) serves as the sole initiating factor in other cell systems. O n the basis of the known ability of IL-1 beta to induce transcription and translation of the IFN family of genes, we hypothesized that IL-1 beta-mediated hepatocyte expression of iNOS is dependent on endogenous IFN-gamma synthesis. In a system of rat hepatocytes in primary cultur e, IL-1 beta induced production of both NO and IFN-gamma. Using in sit u hybridization and immunoblot analysis, IFN-gamma messenger RNA (mRNA ) and protein were detected in hepatocytes exposed to IL-1 beta. Inhib ition of NO synthesis using the competitive substrate inhibitor N-mono methyl-L-arginine (100 mu mol/L) did not alter the extent of IL-1 beta -mediated IFN-gamma synthesis. In contrast, anti-IFN-gamma antibody or inhibition of IFN-gamma mRNA translation by addition of antisense IFN -gamma oligodeoxynucleotide probes resulted in undetectable levels of NO metabolites and iNOS protein. Repletion of IFN-gamma to the system restored NO production to levels noted in the presence of IL-1 beta al one. Transient transfection analysis using a rat hepatocyte iNOS promo ter-reporter gene plasmid construct showed that IL-1 beta-induced prom oter activation was abolished in the presence of anti-IFN gamma or ant isense IFN-gamma. Again, addition of IFN-gamma to the system restored activity of the iNOS promoter. Parallel experiments examining IL-1 bet a-mediated endogenous hepatocyte IL-1 beta and TNF-alpha synthesis ind icated no role for these cytokines in the induction of iNOS expression by IL-1 beta. It is concluded that IL-1 beta-mediated hepatocyte synt hesis of NO is dependent on the simultaneous endogenous synthesis of I FN-gamma.