title: IFN-gamma synergizes with LPS to induce nitric oxide biosynthesis through glycogen synthase kinase-3-inhibited IL-10.
abstract: Interferon-gamma (IFN-gamma) plays a crucial role in innate immunity and inflammation. It causes the synergistic effect on endotoxin lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS)/NO biosynthesis; however, the mechanism remains unclear. In the present study, we investigated the effects of glycogen synthase kinase-3 (GSK-3)-mediated inhibition of anti-inflammatory interleukin-10 (IL-10). We found, in LPS-stimulated macrophages, that IFN-gamma increased iNOS expression and NO production in a time-dependent manner. In addition, ELISA analysis showed the upregulation of tumor necrosis factor-alpha and regulated on activation, normal T expressed and secreted, and the downregulation of IL-10. RT-PCR further showed changes in the IL-10 mRNA level as well. Treating cells with recombinant IL-10 showed a decrease in IFN-gamma/LPS-induced iNOS/NO biosynthesis, whereas anti-IL-10 neutralizing antibodies enhanced this effect, suggesting that IL-10 acts in an anti-inflammatory role. GSK-3-inhibitor treatment blocked IFN-gamma/LPS-induced iNOS/NO biosynthesis but upregulated IL-10 production. Inhibiting GSK-3 using short-interference RNA showed similar results. Additionally, treating cells with anti-IL-10 neutralizing antibodies blocked these effects. We further showed that inhibiting GSK-3 increased phosphorylation of transcription factor cyclic AMP response element binding protein. Inhibiting protein tyrosine kinase Pyk2, an upstream regulator of GSK-3beta, caused inhibition on IFN-gamma/LPS-induced GSK-3beta phosphorylation at tyrosine 216 and iNOS/NO biosynthesis. Taken together, these findings reveal the involvement of GSK-3-inhibited IL-10 on the induction of iNOS/NO biosynthesis by IFN-gamma synergized with LPS.
authors.affiliation: Institute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, Taiwan. cflin@mail.ncku.edu.tw
authors.forename: Chiou-Feng
authors.initials: CF
authors.lastname: Lin
authors.forename: Cheng-Chieh
authors.initials: CC
authors.lastname: Tsai
authors.forename: Wei-Ching
authors.initials: WC
authors.lastname: Huang
authors.forename: Chi-Yun
authors.initials: CY
authors.lastname: Wang
authors.forename: Hsiang-Chi
authors.initials: HC
authors.lastname: Tseng
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Jui-In
authors.initials: JI
authors.lastname: Kai
authors.forename: Szu-Wen
authors.initials: SW
authors.lastname: Wang
authors.forename: Yi-Lin
authors.initials: YL
authors.lastname: Cheng
mesh_terms: D000818:Animals; D015536:Down-Regulation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D007371:Interferon-gamma / Q000494:pharmacology*; D016753:Interleukin-10 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D015854:Up-Regulation
chemical_list: D008070:Lipopolysaccharides; D016753:Interleukin-10; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3