Traces of bacterial lipopolysaccharide suppress IFN-gamma-induced nitric oxide synthase gene expression in primary mouse macrophages.

A nitric oxide synthase (iNOS) inducible by cytokines and microbial products contributes to the cytotoxic and antimicrobial activity of mouse macrophages. Bacterial LPS interacts synergistically with IFN-gamma to induce iNOS when both stimuli are added together. In contrast, we show here that pre-exposure of peritoneal macrophages to low concentrations of LPS suppresses the induction of iNOS when IFN-gamma is added subsequently. Suppression required pretreatment with LPS for at least 8 h and was optimal with LPS concentrations in the range of 50 to 200 pg/ml. Suppression was exerted by smooth and rough forms of LPS from Escherichia coli and by lipid A from Salmonella minnesota, but not by a biologically inactive lipid A from Rhodobacter sphaeorides. Suppression of nitrite accumulation and iNOS enzyme activity by prior exposure of macrophages to LPS could be explained by their markedly decreased content of iNOS protein, as revealed by immunoblot with monospecific anti-iNOS IgG. Messenger RNA for iNOS was affected in a biphasic manner by pretreatment with LPS. Five hours after addition of IFN-gamma, iNOS mRNA levels were unaltered or even enhanced by pretreatment with LPS, but by 24 to 48 h, expression of iNOS mRNA was inhibited strongly enough to account for the reduced levels of iNOS protein. Suppression by LPS did not appear to be mediated by endogenous prostaglandins, transforming growth factor-beta, or TNF-alpha, even though pretreatment with exogenous TNF-alpha was also suppressive. These findings suggest that preactivation of pathways normally contributing to synergistic induction of iNOS may deplete macrophages of factors needed for its expression. Regulation of iNOS in vivo may depend on the relative tempo with which the inflammatory and immune responses evolve.