IL-4-activated STAT-6 inhibits IFN-gamma-induced CD40 gene expression in macrophages/microglia

The antagonism between the cytokines IFN-gamma and IL-4 is well documented, but the mechanism by which IL-4 inhibits IFN-gamma -induced gene expressio n is not clearly understood. CD40 is a type I transmembrane protein that is critical for proper functioning of the immune system. We have previously s hown that IFN-gamma is the most potent inducer of CD40 expression by macrop hages and microglia, In this report, we describe the molecular mechanisms b y which IL-4 inhibits IFN-gamma -induced CD40 expression. IL-4 suppresses I FN-gamma -induced CD40 gene expression in both macrophages and microglia, a nd such inhibition is dependent on the activation of STAT-6. Nuclear run-on and transfection studies indicate that IL-4-mediated repression is at the transcriptional level. Furthermore, IL-4 inhibition of IFN-gamma induced CD 40 expression is specific, since IL-4 does not inhibit IFN-gamma -induced I FN-responsive factor-1 gene expression. Site directed mutagenesis studies d emonstrate that two STAT binding sites, named proximal and distal IFN-gamma -activated sequences, in the human CD40 promoter are important for IL-4 in hibition of IFN-gamma -induced CD40 promoter activity, Moreover, EMSAs indi cate that IL-4-activated STAT-6 binds to these two STAT binding sites. Thes e results suggest that IL-4 inhibition of IFN-gamma -induced CD40 gene expr ession is mediated by direct STAT-6 binding to the CD40 promoter.