Modulation of astrocyte inducible nitric oxide synthase and cytokine expression by interferon beta is associated with induction and inhibition of interferon gamma-activated sequence binding activity

Although interferon (IFN)-beta is firmly established as a therapeutic agent for multiple sclerosis, information regarding its role in astrocyte cytokine production is limited. In primary cultures of human astrocytes, we determined the effects of IFN-beta on astrocyte cytokine [tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6] and inducible nitric oxide synthase (iNOS) expression by ribonuclease protection assay and ELISA. We found that IFN-beta inhibited astrocyte cytokine/iNOS induced by IL-1 plus IFN-gamma, but in the absence of IFN-gamma, IFN-beta enhanced IL-1-induced cytokine/iNOS expression. Electrophoretic mobility shift analysis (EMSA) demonstrated that IFN-gamma induced sustained IFN-gamma-activated sequence (GAS) binding, while IFN-beta induced transient GAS binding. When used together, IFN-beta inhibited IFN-gamma-induced GAS binding activity. Nuclear factor-kappa B (NF-kappaB) activation was not altered by either IFNs, whereas IFN stimulated response element (ISRE) was only activated by IFN-beta and not IFN-gamma. These results suggest that IFN-beta can both mimic and antagonize the effect of IFN-gamma by modulating induction of nuclear GAS binding activity. Our results demonstrating differential regulation of astrocyte cytokine/iNOS induction by IFN-beta are novel and have implications for inflammatory diseases of the human CNS.     

Complex formation of the interferon (IFN) consensus sequence-binding protein with IRF-1 is essential for murine macrophage IFN-gamma-induced iNOS gene expression

This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. An ICSBP binding site in the iNOS promoter region (-923 to -913) was identified using an electrophoretic mobility shift assay and chromatin co-immunoprecipitation. Overexpression of ICSBP greatly enhanced IFN-gamma-induced iNOS promoter activation in RAW264.7 cells, and IFN-gamma-induced iNOS promoter activation was abolished in ICSBP-/- macrophages. Furthermore, transduction of retrovirus-ICSBP in ICSBP-/- macrophages rescued IFN-gamma-induced iNOS gene expression. However, transduction of retrovirus-ICSBP in the absence of IFN-gamma activation did not induce iNOS expression in either RAW264.7 cells or ICSBP-/- macrophages. Interestingly, ICSBP alone transduced into ICSBP-/- macrophages did not bind to IFN-stimulated response element site (-923 to -913) of the iNOS promoter region, although following activation with IFN-gamma, a DNA.protein complex was formed that contains ICSBP and IRF-1. Co-transduction of ICSBP with IRF-1 clearly induces nitric oxide production. In addition, interleukin-4 inhibits IFN-gamma-induced iNOS gene expression by attenuating the physical interaction of ICSBP with IRF-1. Complex formation of ICSBP with IRF-1 is essential for iNOS expression, and interleukin-4 attenuates the physical interaction of ICSBP with IRF-1 resulting in the inhibition of INOS gene expression.