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.     

Activation of the murine interleukin-12 p40 promoter by functional interactions between NFAT and ICSBP

Interleukin (IL)-12 is a heterodimeric cytokine that is critical for the development of a T-helper-1 immune response and immunity against intracellular pathogens. The IL-12 p40 gene product, expressed specifically in macrophages and dendritic cells, heterodimerizes with p35 to form bioactive IL-12, and heterodimerizes with p19 to comprise the cytokine IL-23. Regulation of the murine IL-12 p40 promoter is complex. Multiple cis-acting elements have been characterized that are involved in activation by bacterial products. However, molecular mechanisms through which interferon (IFN)-gamma and bacterial products synergistically activate IL-12 p40 gene expression are less clear. In this study, a composite NFAT/ICSBP binding site at -68 to -54 is identified that is functionally important for p40 promoter activation by lipopolysaccharide (LPS) and LPS plus IFN-gamma. DNA binding of NFAT and ICSBP is demonstrated on the endogenous promoter by chromatin immunoprecipitation. NFAT is required for ICSBP binding to this region. Overexpression of NFAT and ICSBP synergistically activates the p40 promoter. A dominant negative NFAT molecule attenuates LPS- and IFN-gamma-activated endogenous IL-12 p40 mRNA expression. A physical association between NFAT and ICSBP in the absence of DNA is detected by co-immunoprecipitation of endogenous proteins. Three NFAT domains are required for ICSBP interaction. Finally, in LPS- and IFN-gamma-activated RAW-264.7 cells, the association between NFAT and ICSBP is abrogated by IL-10 priming.     

Inhibition of IFN-gamma-induced janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide

The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, down-regulate IL-12 p40 and inducible NO synthase expression in LPS/IFN-gamma-stimulated macrophages. We showed previously that VIP/PACAP inhibit NF-kappaB nuclear translocation through the stabilization of IkappaB and reduce IFN regulatory factor-1 (IRF-1) binding to the regulatory elements found in the IL-12 p40 and inducible NO synthase promoters. In this paper we studied the molecular mechanisms involved in the VIP/PACAP regulation of IRF-1 transactivating activity. Our studies indicate that the inhibition in IRF-1 binding correlates with a reduction in IRF-1 protein and mRNA in IFN-gamma-treated Raw 264.7 macrophages. In agreement with the described Janus kinase (Jak)1/Jak2/STAT1/IRF-1 activation pathway, VIP/PACAP inhibit Jak1/Jak2, STAT1 phosphorylation, and the binding of STAT1 to the GAS sequence motif in the IRF-1 promoter. The effects of VIP/PACAP are mediated through the specific VIP/PACAP receptor-1 and the cAMP/protein kinase A (PKA) transduction pathway, but not through the induction of suppressor of cytokine signaling-1 or suppressor of cytokine signaling-3. Because IFN-gamma is a major stimulator of innate immune responses in vivo, the down-regulation of IFN-gamma-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response by endogenous neuropeptides.