Cytokine-induced iNOS expression in C6 glial cells: transcriptional inhibition by ethanol

The effect of cytokines, lipopolysaccharide, and ethanol on inducible nitric-oxide synthase (iNOS) expression was studied in C6 glial cells. Maximal induced activity, measured by the accumulation of nitrite in culture medium, occurred following treatment with lipopolysaccharide and interferon-gamma. Each cytokine alone was ineffective, whereas an optimal combination of interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma was near maximal, indicating synergistic interactions. Other combinations caused submaximal activity. Ethanol is known to suppress iNOS expression in C6 cells induced by a phorbol ester plus lipopolysaccharide. The current work shows ethanol also suppresses cytokine-induced iNOS expression and reduces interleukin-1beta and tumor necrosis factor-alpha potency without affecting interferon-gamma potency. Ethanol-mediated reductions in cytokine-induced iNOS mRNA and immunoreactive protein levels suggested an effect on gene transcription. Therefore, C6 cells stably expressing 1846 and 526 base fragments of the rat iNOS gene promoter fused to a luciferase reporter gene were prepared and characterized and used to study the effect of ethanol on iNOS promoter activity. Promoter activity in stable transfected C6 cells was inhibited by ethanol exposure with a similar concentration dependence as observed for inhibition of nitrite production, indicating that iNOS inhibition by ethanol is transcriptional. Furthermore, ethanol inhibition of the 526 base fragment activity, which lacks interferon-gamma enhancement of lipopolysaccharide-induced luciferase activity, confirmed that interferon-gamma-responsive elements do not participate in acute ethanol-induced inhibition of rat iNOS gene transcription.