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.
Highlights
Nitric oxide (NO), a diffusible gas, plays an important role in many physiological and diverse pathophysiological conditions [1, 2]
Several lines of evidence presented in this manuscript clearly support the conclusion that the ligation of CD40 markedly stimulates the expression of inducible nitric-oxide synthase (iNOS) in activated microglial cells
The stimulation of IFN-␥-induced expression of iNOS by CD40 ligation was greater in cells transfected with the CD40 cDNA expression construct as compared with that in cells transfected with the empty vector
Summary
Nitric oxide (NO), a diffusible gas, plays an important role in many physiological and diverse pathophysiological conditions [1, 2]. NO, which is derived in excessive amounts from the activation of inducible nitric-oxide synthase (iNOS) in glial cells (microglia and astrocytes), is assumed to contribute to oligodendrocyte degeneration in demyelinating diseases and neuronal death during neurodegenerative diseases [3,4,5,6,7]. Analysis of cerebrospinal fluid from MS patients has shown increased levels of nitrite and nitrate compared with normal controls [11]. Increased levels of nitrotyrosine have been found in demyelinating lesions of MS brains as well as in spinal cords of mice with EAE [12, 13]. We report that CD40 ligation markedly stimulates the expression of iNOS by augmenting the activation of NF-B in IFN-␥-treated microglial cells
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