CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 expression on antigen-presenting cells (including macrophages and microglia) is crucial for T-cell activation. Aberrant expression of CD40 has been associated with autoimmune inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We have recently shown that the cytokine interferon (IFN)-gamma is the most potent inducer of CD40 expression in macrophages and microglia, and this induction is mediated by the IFN-gamma-activated transcription factor STAT-1alpha and constitutively expressed PU.1 and/or Spi-B. In this study, we have discovered that a major component of IFN-gamma-induced CD40 expression involves the endogenous production of the cytokine TNF-alpha. The inclusion of anti-TNF-alpha-neutralizing antibody significantly inhibits IFN-gamma-induced CD40 mRNA and CD40 promoter activity. IFN-gamma-induced CD40 protein expression is attenuated in TNF-alpha-deficient microglia and can be restored with exogenous TNF-alpha. Site-directed mutagenesis studies demonstrate that three of the four NF-kappaB elements in the CD40 promoter are required for IFN-gamma-induced CD40 promoter activity. IFN-gamma treatment leads to the activation of NF-kappaB in a time-dependent manner, which is inhibited in the presence of anti-TNF-alpha-neutralizing antibody. These results indicate that IFN-gamma-induced TNF-alpha production and subsequent NF-kappaB activation are integral parts of the mechanism of IFN-gamma-induced CD40 expression.

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer's disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-gamma induces CD40 expression in macrophages and microglia. IFN-gamma leads to STAT-1alpha activation directly and up-regulation of NF-kappaB activity due to the secretion and subsequent autocrine signaling of TNF-alpha. However, TNF-alpha alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1alpha activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-gamma-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-gamma-induced TNF-alpha secretion, as well as STAT-1alpha and NF-kappaB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-gamma-mediated STAT-1alpha activation, which also then results in suppression of IFN-gamma-induced TNF-alpha secretion and subsequent NF-kappaB activation.