Inhibition of cytokine-induced nitric oxide synthase expression by gene transfer of adenoviral IκBα

Background: Nitric oxide is overexpressed in nearly every organ during sepsis and it has profound biologic effects. Previously, we showed that maximal inducible nitric oxide synthase (iNOS) expression is up-regulated by a combination of cytokines and that this effect is mediated by the transcription factor NF-κB. Therefore the purpose of this study was to establish whether gene transfer of the inhibitory molecule IκB would result in the abrogation of cytokine-induced iNOS expression. Methods: Cultured hepatocytes were infected with an adenoviral vector containing the IκBα gene (Ad5IκB) and after an 18-hour recovery period were stimulated with the cytokine mixture of tumor necrosis factor-α (500 U/mL) plus interleukin 1β (200 U/mL) plus interferon gamma (100 U/mL). Results: As expected, cytokine mixture induced significant hepatocyte nitrite (NO 2 –) and iNOS messenger RNA production. Cells infected with the IκBα gene showed a dose-dependent decrease in NO 2 – and iNOS messenger RNA levels. Western blot analysis showed a marked decrease in iNOS protein levels in the presence of Ad5IκBα. Gel shift assays of nuclear extracts demonstrated that Ad5IκBα decreased the cytokine-induced DNA binding activity for NFκB. Conclusions: NFκB is an important regulator of cytokine-induced NO expression. These results identify a novel therapeutic approach where gene transfer of the inhibitory molecule IκBα can be used to down-regulate cytokine-induced iNOS expression as well as other NFκB-dependent genes that are up-regulated during the inflammatory response. (Surgery 1999;126:142-7.)