Inhibition of major histocompatibility complex class II gene transcription by nitric oxide and antioxidants

Abstract

Interferon (IFN)-γ facilitates cellular immune response, in part, by inducing the expression of major histocompatibility complex class II (MHC-II) molecules. We demonstrate that IFN-γ induces the expression of HLA-DRA in vascular endothelial cells via mechanisms involving reactive oxygen species. IFN-γ-induced HLA-DRA expression was inhibited by nitric oxide (NO) and antioxidants such as superoxide dismutase, catalase, pyrrolidine dithiocarbamate, and N-acetylcysteine. Nuclear run-on assays demonstrated that NO and antioxidants inhibited IFN-γ-induced HLA-DRA gene transcription. Transient transfection studies using a fully functional HLA-DRA promoter construct ([-300]-DRα.CAT) showed that inhibition of endogenous NO synthase activity by Nω-monomethyl-L-arginine or addition of exogenous hydrogen peroxide (H2O2) augmented basal and IFN-γ-stimulated [-300] DRα.CAT activity. However, H2O2 and Nω-monomethyl-L-arginine could induce HLA-DRA expression suggesting that H2O2 is a necessary but not a sufficient mediator of IFN-γ-induced HLA-DRA expression. Electrophoretic mobility shift assay and Western blotting demonstrated that NO and antioxidants had little or no effect on IFN-γ-induced IRF-1 activation or MHC-II transactivator (CIITA) expression but did inhibit IFN-γ-induced activation of STAT1α (p91) and Y box transcription factors, NF-YA and NF-YB. These results indicate that NO and antioxidants may attenuate vascular inflammation by antagonizing the effects of intracellular reactive oxygen species generation by IFN-γ, which is necessary for MHC-II gene transcription.