title: Ligation of CD40 stimulates the induction of nitric-oxide synthase in microglial cells.
abstract: The present study was undertaken to investigate the role of CD40 ligation in the expression of inducible nitric-oxide synthase (iNOS) in mouse BV-2 microglial cells and primary microglia. Ligation of CD40 alone by either cross-linking antibodies against CD40 or a recombinant CD40 ligand (CD154) was unable to induce the production of NO in BV-2 microglial cells. The absence of induction of NO production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggests that a signal transduced by the ligation of CD40 alone is not sufficient to induce NO production. However, CD40 ligation markedly stimulated interferon-gamma (IFN-gamma)-mediated NO production. Ligation of CD40 in CD40-overexpressed cells further stimulated IFN-gamma-induced production of NO. This stimulation of NO production was accompanied by stimulation of the iNOS protein and mRNA. In addition to BV-2 glial cells, CD40 ligation also stimulated IFN-gamma-mediated NO production in mouse primary microglia and peritoneal macrophages. To understand the mechanism of induction/stimulation of iNOS, we investigated the roles of nuclear factor kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), transcription factors responsible for the induction of iNOS. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone induced the activation of only NF-kappaB but not of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of iNOS and that the activation of C/EBPbeta is also necessary for the expression of iNOS. Consistently, dominant-negative mutants of p65 (Deltap65) and C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of iNOS in BV-2 microglial cells that were stimulated with the combination of IFN-gamma and CD40 ligand. Stimulation of IFN-gamma-mediated activation of NF-kappaB but not of C/EBPbeta by CD40 ligation suggests that CD40 ligation stimulates the expression of iNOS in IFN-gamma-treated BV-2 microglial cells through the stimulation of NF-kappaB activation. This study illustrates a novel role for CD40 ligation in stimulating the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.
authors.affiliation: Department of Oral Biology, University of Nebraska Medical Center, Lincoln, Nebraska 68583, USA.
authors.forename: M
authors.initials: M
authors.lastname: Jana
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Koka
authors.forename: S
authors.initials: S
authors.lastname: Ghosh
authors.forename: T M
authors.initials: TM
authors.lastname: Petro
authors.forename: K
authors.initials: K
authors.lastname: Pahan
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000096:biosynthesis; D019013:CD40 Antigens / Q000096:biosynthesis / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D007371:Interferon-gamma / Q000096:biosynthesis / Q000378:metabolism; D008024:Ligands; D008264:Macrophages / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000166:cytology* / Q000201:enzymology*; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D051996:Transcription Factor RelA; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D019013:CD40 Antigens; D018076:DNA, Complementary; D008024:Ligands; D016328:NF-kappa B; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D014157:Transcription Factors; D007371:Interferon-gamma; D004247:DNA; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse