title: Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils.
abstract: Bacterial DNA containing unmethylated CpG motifs is emerging as an important regulator of functions of human neutrophil granulocytes (polymorphonuclear leukocytes (PMN)). These motifs are recognized by TLR-9. Recent studies indicate that peroxynitrite (ONOO-) may function as an intracellular signal for the production of IL-8, one of the key regulators of leukocyte trafficking in inflammation. In this study we investigated whether bacterial DNA (CpG-DNA) could induce ONOO- signaling in human PMN. Human whole blood, isolated PMN (purity, >95%), and high purity (>99%) PMN respond to CpG-DNA, but not to calf thymus DNA, with secretion of IL-8 and, to a lesser extent, IL-6 and TNF. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. The endosomal acidification inhibitors, bafilomycin A and chloroquine, inhibited CpG-DNA-induced cytokine release from PMN. CpG-DNA-induced IL-8 mRNA expression and release was also blocked by the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester. CpG-DNA evoked concomitant increases in intracellular superoxide and NO levels, leading to enhanced ONOO- formation and, consequently, nuclear accumulation of c-Fos and NF-kappaB. Pharmacological inhibition of NF-kappaB activation attenuated approximately 75% of CpG-DNA-evoked IL-8 release. These results identify ONOO- -dependent activation of NF-kappaB and c-Fos as an important mechanism that mediates PMN responses, including IL-8 gene expression and release, to bacterial DNA and unmethylated CpG motifs in particular. Enhanced ONOO- formation represents a mechanism by which bacterial DNA may contribute to prolongation and amplification of the inflammatory response.
authors.affiliation: Research Center, Maisonneuve-Rosemont Hospital, University of Montreal, Montreal, Quebec, Canada.
authors.forename: Levente
authors.initials: L
authors.lastname: József
authors.forename: Tarek
authors.initials: T
authors.lastname: Khreiss
authors.forename: Driss
authors.initials: D
authors.lastname: El Kebir
authors.forename: János G
authors.initials: JG
authors.lastname: Filep
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002470:Cell Survival; D004247:DNA / Q000276:immunology; D004269:DNA, Bacterial / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D018375:Neutrophil Activation; D009504:Neutrophils / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics; D030421:Peroxynitrous Acid / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: C472452:CpG-DNA, E coli; D004269:DNA, Bacterial; D015850:Interleukin-6; D016209:Interleukin-8; D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D004247:DNA; D019001:Nitric Oxide Synthase