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 i n the expression of inducible nitric-oxide synthase (iNOS) in mouse BV-2 mi croglial cells and primary microglia. Ligation of CD40 alone by either cros s-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-overexpr essed BV-2 microglial cells suggests that a signal transduced by the ligati on of CD40 alone is not sufficient to induce NO production. However, CD40 l igation markedly stimulated interferon-gamma (IFN-gamma)-mediated NO produc tion. Ligation of CD40 in CD40-overexpressed cells further stimulated IFN-g amma -induced production of NO. This stimulation of NO production was accom panied by stimulation of the iNOS protein and mRNA In addition to BV-2 glia l cells, CD40 ligation also stimulated IFN-gamma -mediated NO production in mouse primary microglia and peritoneal macrophages. To understand the mech anism of induction/stimulation of iNOS, we investigated the roles of nuclea r factor kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBP beta), transcription factors responsible for the induction of iNOS. IFN-gam ma alone was able to induce the activation of NF-kappaB as well as C/EBP be ta. However, CD40 ligation alone induced the activation of only NF-kappaB b ut not of C/EBP beta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of iNOS and that t he activation of C/EBP beta is also necessary for the expression of iNOS. C onsistently, dominant-negative mutants of p65 (Delta p65) and C/EBP beta (D eltaC/EBP beta) inhibited the expression of iNOS in BV-2 microglial cells t hat were stimulated with the combination of IFN-gamma and CD40 ligand. Stim ulation of IFN-gamma -mediated activation of NF-kappaB but not of C/EBP bet a by CD40 ligation suggests that CD40 ligation stimulates the expression of iNOS in IFN-gamma -treated BV-2 microglial cells through the stimulation o f NF-kappaB activation. This study illustrates a novel role for CD40 ligati on in stimulating the expression of iNOS in microglial cells, which may par ticipate in the pathogenesis of neuroinflammatory diseases.