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

On the basis of previous work demonstrating nitric oxide (NO)-mediated inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, we hypothesized that NO downregulates NF-kappaB-dependent interleukin-1beta (IL-1beta) production in an ANA-1 macrophage model of lipopolysaccharide (LPS) stimulation. In the presence of LPS (100 ng/ml), levels of IL-1beta protein and mRNA were significantly upregulated with NO synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1beta gene transcription and IL-1beta promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-kappaB long terminal repeat-reporter plasmid exhibited similar findings, suggesting an NO-mediated effect on NF-kappaB activity. Gel shift studies showed that LPS-associated NF-kappaB 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-1beta protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-kappaB DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1beta gene transcription through S-nitrosation of NF-kappaB.