Ligation of CD40 stimulates the induction of nitric-oxide synthase in microglial cells

J Biol Chem. 2001 Nov 30;276(48):44527-33. doi: 10.1074/jbc.M106771200. Epub 2001 Sep 10.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Northern
  • CCAAT-Enhancer-Binding Protein-beta / biosynthesis
  • CD40 Antigens / biosynthesis
  • CD40 Antigens / metabolism*
  • Cells, Cultured
  • DNA / metabolism
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Activation
  • Genes, Dominant
  • Genes, Reporter
  • Humans
  • Immunoblotting
  • Interferon-gamma / biosynthesis
  • Interferon-gamma / metabolism
  • Ligands
  • Macrophages / metabolism
  • Macrophages, Peritoneal / metabolism
  • Mice
  • Microglia / cytology*
  • Microglia / enzymology*
  • Mutation
  • NF-kappa B / metabolism
  • Nitric Oxide Synthase / metabolism*
  • Nitric Oxide Synthase Type II
  • Protein Binding
  • RNA, Messenger / metabolism
  • Signal Transduction
  • Time Factors
  • Transcription Factor RelA
  • Transcription Factors
  • Transcription, Genetic
  • Transfection


  • CCAAT-Enhancer-Binding Protein-beta
  • CD40 Antigens
  • DNA, Complementary
  • Ligands
  • NF-kappa B
  • RNA, Messenger
  • Transcription Factor RelA
  • Transcription Factors
  • Interferon-gamma
  • DNA
  • NOS2 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse