Counter-regulation mechanism of IL-4 and IFN-α signal transduction through cytosolic retention of the pY-STAT6:pY-STAT2:p48 complex

IFN-alpha and IL-4 induce Th1 and Th2 responses, respectively, and often display antagonistic actions against each other. To elucidate the molecular mechanism of counter-regulation, we have investigated the signal interception by IFN-alpha and IL-4, employing a human B-cell line Ramos, sensitive to both cytokines. In these cells, IFN-alpha effectively inhibited IL-4-induced Fc epsilon receptor II (CD23) expression, whereas IL-4 suppressed IFN-alpha-mediated IRF7 expression. The counter-regulatory action by IL-4 and IFN-alpha proceeded with a delayed kinetics requiring 4h. Notably, IFN-alpha did not affect the IL-4-induced tyrosine phosphorylation of STAT6, but induced a time-dependent cytoplasmic accumulation of phosphotyrosine(pY)-STAT6 and a corresponding decrease in nuclear pY-STAT6. By confocal analysis and co-immunoprecipitation assays, we demonstrated the colocalization and molecular interaction of IL-4-induced pY-STAT6 with IFN-alpha-induced pY-STAT2:p48 in the cytosol. In addition, the over-expression of STAT2 or STAT6 induced the concomitant cytosolic accumulation of pY-STAT6 or pY-STAT2, leading to the suppression of IL-4-induced CD23 or IFN-alpha-induced IRF7 gene expression, respectively. Our data suggest that the signals ensued by IFN-alpha and IL-4 induce cytoplasmic sequestration of IL-4-activated STAT6 and IFN-alpha-activated STAT2:p48 in B cells through the formation of pY-STAT6:pY-STAT2:p48 complex, which provides a novel mechanism by which IFN-alpha and IL-4 cross-regulate their signaling into the nucleus.