IL-4 Together With SDF-1α Induces A Persistent And Selective Activation Of ZAP-70 Kinase Resulting In Th2 Polarization

Objective: To address which signaling pathways might be involved in the on-switch of Th cells by IL-4 + stromal cell-derived factor (SDF)-1α/CXCL12, we have detected expression of intracellular Th1 and Th2 cytokine, and next examined activation of important factors or protein of signaling pathways.Methods: Intracellular Th1 and Th2 cytokines and cytokine mRNA were detected by flow cytometry and the real time quantitative RT-PCR, respectively; The activation of Syk and ZAP-70 kinase and its downstream substrates Cbl or Cbl-b protein were detected by the immune complex kinase assay (KA) and immunoblotting(IB) assay, respectively, in the cord blood (CB) CD4~+ T cells which were either freshly isolated or co-stimulated with IL-4 and SDF-1α/CXCL12; the blocking effects of PNA ZAP-70 antisenses on the activation of ZAP-70 kinase were detected by the immune complex kinase assay (KA) and immunoblotting(IB) assay, respectively, in CB CD4~+ T cell; NFAT family activation was assessed by EMSA in nuclei extracts and the whole cells extracts of CB CD4~+ T cells co-stimulated with IL-2 and SDF-1α/CXCL12.Results: The results of the FACS showed that after stimulation with IL-4 and SDF-1α/CXCL12 for 8 days, the cells have been switched to Th2 pattern (90.3 % IL-4positive); RT-PCR showed that there are approximately 7.3 × 10~2 copies for IFN-γ and 2.1 × 10~3 copies for IL-4 mRNA in freshly isolated CB CD4~+ T cells, 1.3 × 10~4 copies for IL-4 mRNA and 7.8 × 10~1copies for IFN-y mRNA in the cells co-stimulated with IL-4 and SDF-1α/CXCL12, respectively. The immune complex kinase assay(KA) and immunoblotting(IB) showed that IL-4 and SDF-1α/CXCL12 together have induced a weak phosphorylation of ZAP-70 kinase at 30 min. A strong and persistent phosphorylation of ZAP-70 kinase and Cb1-b protein have been seen within 8-day co-stimulation with IL-4 and SDF-1α, whereas no detectable phosphorylation of Syk kinase has been seen in these cells. IL-4 and SDF-1α/CXCL12 together has induced an increasing pattern of activity of Cb1 protein has been seen in 8-day; PNA showed that ZAP-70 PNA antisense significantly inhibit kinase activity and protein expression of ZAP-70 in non-stimulated or co-sitmulated CB CD4~+ T cells in culture within 8 days, respectively. In parallel, ZAP-70 PNA antisense has dramatically inhibited IL-4 mRNA and intracellular IL-4 expression induced by IL-4 and SDF-1α/CXCL12, whereas IFN-y expression has not significantly changed in these cells; EMSA showed that NFAT has been detected in nuclei extracts of CB CD4~+ T cells stimulated with IL-4 + SDF-1α/CXCL12 in an increasing and persistent manner within 30 min and 8 days. In contrast, there are almost equal amounts of NFAT protein seen in whole cell extracts of these cells either freshly isolated or co-stimulated cells. In addition, IL-4 and SDF-α/CXCL12 selectively activate NFAT2 in CB CD4~+ T cells, whereas neither NFAT3 nor NFAT4 has been activated in these cells.Conclusions: After co-stimulation with IL-4 and SDF-1α/CXCL12, CB CD4~+ T cells have been switched to Th2 pattern, a persistent and selective activation of ZAP-70 kinase have been induced, and ZAP-70 PNA antisenses significantly inhibit kinase activity and protein expression of ZAP-70 and block the polarization of Th2 cell, implying persistent and selective activation of ZAP-70 kinase is essential for Th2...