Inhibition of cytokine-induced nitric oxide synthase expression by gene transfer of adenoviral I kappa B alpha

Background. Nitric oxide is overexpressed in nearly every organ during seps is and it has profound biologic effects. Previously, we showed that maximal inducible nitric oxide synthase (iNOS) expression is up-regulated by a com bination of cytokines and that this effect Is mediated by the transcription factor NF-kappa B. Therefore the purpose of this study was to establish wh ether gene transfer of the inhibitory molecule I kappa B would result in th e abrogation of cytokine-induced iNOS expression. Methods. Cultured hepatocytes were infected with an adenoviral vector conta ining the I kappa B alpha gene (Ad5I kappa B) and after an 18-hour recovery period were stimulated with the cytokine mixture of tumor necrosis factor- alpha (500 U/mL) plus interleukin 1 beta (200 U/mL) plus interferon gamma ( 100 U/mL). Results. As expected cytokine mixture induced significant hepatocyte nitrit e (NO2-) and iNOS messenger RNA production. Cells infected with the I kappa B alpha gene showed a dose-dependent decrease in NO2- and iNOS messenger R NA levels. Western blot analysis showed a marked decrease in iNOS protein l evels in the presence of Ad5I kappa B alpha. Gel shift assays of nuclear ex tracts demonstrated that Ad5I kappa B alpha decreased the cytokine-induced DNA binding activity for NF kappa B. Conclusions. NF kappa B is an important regulator of cytokine-induced NO ex pression. These results identify a novel therapeutic approach where gene tr ansfer of the inhibitory molecule I kappa B alpha can be used to down-regul ate cytokine-induced iNOS expression as well as other NF kappa B-dependent genes that are up-regulated during the inflammatory response.