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

The effect of cytokines, lipopolysaccharide, and ethanol on inducible nitri c-oxide synthase (iNOS) expression was studied in C6 glial cells. Maximal i nduced 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 int erleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma was near maximal, indicating synergistic interactions. Other combinations caused su bmaximal 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 inter leukin-1 beta and tumor necrosis factor-alpha potency without affecting int erferon-gamma potency. Ethanol-mediated reductions in cytokine-induced iNOS mRNA and immunoreactive protein levels suggested an effect on gene transcr iption. Therefore, C6 cells stably expressing 1846 and 526 base fragments o f the rat iNOS gene promoter fused to a luciferase reporter gene were prepa red and characterized and used to study the effect of ethanol on iNOS promo ter activity. Promoter activity in stable transfected C6 cells was inhibite d by ethanol exposure with a similar concentration dependence as observed f or inhibition of nitrite production, indicating that iNOS inhibition by eth anol is transcriptional. Furthermore, ethanol inhibition of the 526 base fr agment activity, which lacks interferon-gamma enhancement of Iipopolysaccha ride-induced luciferase activity, confirmed that interferon-gamma -responsi ve elements do not participate in acute ethanol-induced inhibition of rat i NOS gene transcription.