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

Schroeder, Rebecca A., Charles Cai, and Paul C. Kuo. Endotoxin-mediated nitric oxide synthesis inhibits IL-1b 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 (NO)mediated inhibition of nuclear factor-kB (NF-kB) DNA binding, we hypothesized that NO downregulates NF-kB-dependent interleukin-1b (IL-1b) production in an ANA-1 macrophage model of lipopolysaccharide (LPS) stimulation. In the presence of LPS (100 ng/ml), levels of IL-1b protein and mRNA were significantly upregulated with NO synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1b gene transcription and IL-1b 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-1b 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-1b gene transcription through Snitrosation of NF-kB.