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

Abstract

IFN-a 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-a and IL-4, employing a human B-cell line Ramos, sensitive to both cytokines. In these cells, IFN-a effectively inhibited IL-4-induced Fc epsilon receptor II (CD23) expression, whereas IL-4 suppressed IFN-amediated IRF7 expression. The counter-regulatory action by IL-4 and IFN-a proceeded with a delayed kinetics requiring 4 h. Notably, IFN-a 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-a-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-a-induced IRF7 gene expression, respectively. Our data suggest that the signals ensued by IFN-a and IL-4 induce cytoplasmic sequestration of IL-4-activated STAT6 and IFN-a-activated STAT2:p48 in B cells through the formation of pY-STAT6:pY-STAT2:p48 complex, which provides a novel mechanism by which IFN-a and IL-4 cross-regulate their signaling into the nucleus.Keywords: Counter-regulation . Cytosolic retention . IFN-a signaling . IL-4 signaling pY-STAT6:pY-STAT2:p48 complex Supporting Information available online IntroductionIn the innate and the adaptive immune system, cytokines act as key regulators for the activation, proliferation, differentiation, development, and death of immune cells [1,2]. IL-4 is an immunomodulatory cytokine secreted by activated Th2 lymphocytes, basophils, and mast cells [3]. Its pleiotropic functions include the differentiation of Th2 cells, B-cell activation, immunoglobulin isotype switching, the inhibition of Th1 differentiation, and the development of allergic diseases. In hematopoietic cells, responses to IL-4 are mediated by the receptor complex composed of IL-4 receptor (IL-4R) a and common g-chain (gc). Once these receptor chains are heterodimerized upon IL-4 binding, the receptor-associated Jaks (Jak 1/3) are activated, inducing phosphorylation of a tyrosine residue within the cytoplasmic tail of IL-4Ra [3]. The phosphotyrosine (pY) 461 motif generated on the receptor then acts as a docking site to recruit STAT6, leading to the tyrosine phosphorylation of STAT6 by Jaks. Subsequently, phosphorylated STAT6 departs from the receptor, dimerizes, and translocates into the nucleus, where it turns on the expression of IL-4 target genes [3,4]. The IL-4-induced STAT6 activity is shown to be essential for the establishment of distal promoter activity for GATA3 transcription in developing Th2 cells [5]. IFNs are widely expressed cytokines with multiple biological act...

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