INHIBITION OF INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION BY INTERFERON-ALPHA AND INTERFERON-BETA IN BOVINE RETINAL PIGMENTED EPITHELIAL-CELLS

Bovine retinal pigmented epithelial (RPE) cells express an inducible n itric oxide synthase (NOS-2) after activation with interferon (IFN)-ga mma and lipopolysaccharide (LPS). Experiments were performed to invest igate the effects of IFN-alpha and IFN-beta on NOS-2 activity. These t ypes of interferons did not aid LPS in the production of nitrite, but markedly inhibited in a concentration-dependent manner the nitrite rel ease due to LPS/IFN-gamma. Analysis by Western and Northern blots show ed that RPE cells co-stimulated with IFN-alpha or IFN-beta with LPS/IF N-gamma accumulated lower levels of NOS-2 protein and mRNA than in the presence of LPS/IFN-gamma alone. The presence of IFN-alpha or IFN-bet a did not accelerate mRNA degradation, implying that these interferons did not affect NOS-2 mRNA stability, but more probably NOS-2 gene exp ression. Furthermore, IFN-gamma binding studies demonstrated that the inhibitory effect of IFN-alpha and IFN-beta is not caused by a blockin g of IFN-gamma receptors. Analysis of NF-kappa B activation by electro phoretic mobility shift assay demonstrated that LPS/IFN-gamma-induced NF-kappa B binding was not changed by the presence of IFN-alpha. Howev er, similar experiments revealed that the activation of interferon reg ulatory factor-1 (IRF-1) by LPS/IFN-gamma was decreased by IFN-alpha. This phenomenon could be due to the decline of IRF-1 mRNA and the up-r egulation of IRF-2 mRNA, an IRF-1 repressor, by IFN-1. These results s uggest that the inhibitory effect of IFN-alpha and -beta on NOS-2 indu ction could be partially explained by their effect on the induction of the IRFs, which were involved in NOS-2 gene transcription.