Inhibition of inducible nitric oxide synthase expression by interferons α and β in bovine retinal pigmented epithelial cells

Abstract

Bovine retinal pigmented epithelial (RPE) cells express an inducible nitric oxide synthase (NOS-2) after activation with interferon (IFN)-γ and lipopolysaccharide (LPS). Experiments were performed to investigate the effects of IFN-α and IFN-β on NOS-2 activity. These types of interferons did not aid LPS in the production of nitrite, but markedly inhibited in a concentration-dependent manner the nitrite release due to LPS/IFN-γ. Analysis by Western and Northern blots showed that RPE cells co-stimulated with IFN-α or IFN-β with LPS/IFN-γ accumulated lower levels of NOS-2 protein and mRNA than in the presence of LPS/IFN-γ alone. The presence of IFN-α or IFN-β did not accelerate mRNA degradation, implying that these interferons did not affect NOS-2 mRNA stability, but more probably NOS-2 gene expression. Furthermore, IFN-γ binding studies demonstrated that the inhibitory effect of IFN-α and IFN-β is not caused by a blocking of IFN-γ receptors. Analysis of NF-κB activation by electrophoretic mobility shift assay demonstrated that LPS/IFN-γ-induced NF-κB binding was not changed by the presence of IFN-α. However, similar experiments revealed that the activation of interferon regulatory factor-1 (IRF-1) by LPS/IFN-γ was decreased by IFN-α. This phenomenon could be due to the decline of IRF-1 mRNA and the up-regulation of IRF-2 mRNA, an IRF-1 repressor, by IFN-α. These results suggest that the inhibitory effect of IFN-α and -β on NOS-2 induction could be partially explained by their effect on the induction of the IRFs, which were involved in NOS-2 gene transcription.