Nitric Oxide Inhibits LPS-Induced IL-6 Production in Enterocytes

In recent studies, production of interleukin-6 (IL-6) in cultured enterocytes was stimulated by lipolysaccharide (LPS). In other cell types, IL-6 production was inhibited by nitric oxide (NO). We tested the hypothesis that LPS-induced IL-6 production in the enterocyte is regulated, at least in part, by NO. IEC-6 cells (a rat intestinal epithelial cell line) were cultured for 3 days with different combinations of LPS (1-10 μg/ml), the NO synthase inhibitor N-ω-nitro- L-arginine (NNA, 3300 μ M), L -arginine (10 m M), the NO donor sodium nitroprusside (SNP, 0.5-1 μ M), or medium alone as control. IL-6 levels in the culture medium were determined by the B9 murine hybridoma bioassay. Nitrite, a stable end product of NO metabolism, was measured by HPLC. PCR was performed to determine inducible NO synthase (iNOS) mRNA expression in the IEC-6 cells. Treatment of IEC-6 cells with LPS stimulated IL-6 production. LPS-induced IL-6 production was further increased by NNA in a dose-dependent fashion. This effect of NNA was abolished by the addition of L-arginine. SNP caused a dose-dependent decrease in IL-6 production. Nitrite production was increased in a dose-dependent fashion after LPS treatment. PCR revealed an increase in iNOS mRNA expression in IEC-6 cells after administration of 1 μg/ml LPS. The results suggest that NO inhibits LPS-induced IL-6 production in the enterocyte. NO may be an important regulator of intestinal cytokine response during sepsis and endotoxemia.