Endotoxin-mediated nitric oxide synthesis inhibits IL-1β gene transcription in ANA-1 murine macrophages

Endotoxin-mediated nitric oxide synthesis inhibits IL-1 beta gene transcription in ANA-1 murine macrophages. Am. J. Physiol. 277 (Cell Physiol. 46): C523-C530, 1999.-On the basis of previous work demonstrating nitric oxide;dde (NO)mediated inhibition of nuclear factor-KB (NF-KB) DNA binding, we hypothesized that NO downregulates NF-KB-dependent interleukin-lp (IL-1 beta) production in an ANA-1 macrophage model of lipopolysaccharide (LPS) stimulation. In the presence of LPS (100 ng/ml), levels of IL-lp protein and mRNA were significantly upregulated with NO synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1 beta gene transcription and IL-1 beta promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-KB long terminal repeat-reporter plasmid exhibited similar findings, suggesting an NO-mediated effect on NF-KB activity. Gel shift studies showed that LPS-associated NF-KB DNA binding was increased, both in the setting of NO synthase inhibition and in a reducing environment. Repletion of NO by addition of an S-nitrosothiol restored IL-1 beta protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-KB DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1 beta gene transcription through S-nitrosation of NF-kappa B.