Ligation of CD40 Stimulates the Induction of Nitric-oxide Synthase in Microglial Cells

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-γ (IFN-γ)-mediated NO production. Ligation of CD40 in CD40-overexpressed cells further stimulated IFN-γ-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-γ-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 κB (NF-κB) and CCAAT/enhancer-binding protein β (C/EBPβ), transcription factors responsible for the induction of iNOS. IFN-γ alone was able to induce the activation of NF-κB as well as C/EBPβ. However, CD40 ligation alone induced the activation of only NF-κB but not of C/EBPβ, suggesting that the activation of NF-κB alone by CD40 ligation is not sufficient to induce the expression of iNOS and that the activation of C/EBPβ is also necessary for the expression of iNOS. Consistently, dominant-negative mutants of p65 (Δp65) and C/EBPβ (ΔC/EBPβ) inhibited the expression of iNOS in BV-2 microglial cells that were stimulated with the combination of IFN-γ and CD40 ligand. Stimulation of IFN-γ-mediated activation of NF-κB but not of C/EBPβ by CD40 ligation suggests that CD40 ligation stimulates the expression of iNOS in IFN-γ-treated BV-2 microglial cells through the stimulation of NF-κB 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.