IL-2 Together With SDF-1α Induces A Persistent And Selective Activation Of Syk Kinase Resulting In Th1 Polarization

Objective: To address which signaling pathways might be involved in the on-switch of Th cells by IL-2 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-2 and SDF-1α/CXCL12; the blocking effects of PNA Syk antisenses on the activation of Syk 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-2 and stromal cell-derived factor (SDF)-1α/CXCL12 for 8 days, the cells have been switched to Thlpattern (84 % IFN-y positive); RT-PCR showed that there are approximately 7.3 x 102 copie for IFN-y and 2.1 * 103 copies for IL-4 mRNA in freshly isolated CB CD4+ T cells, 1.0 x 104 copies for IFN-y mRNA and 3.6 x I02 copies for IL-4 mRNA in the cells co-stimulated with IL-2 and SDF-la/CXCL12, respectively. The immune complex kinase assay (KA) and immunoblotting (IB) showed that IL-2 and SDF-la/CXCL12 together have induced a weak phosphorylation of Syk kinase at 30 min. A strong and persistent phosphorylation of Syk kinase and Cbl-b protein have been seen within 8day co-stimulation with IL-2 and SDF-lot/CXCL12, whereas no detectable phosphorylation of ZAP-70 kinase has been seen in these cells. IL-2 and SDF-la/CXCL12 together has induced a strong phosphorylation of Cbl about 30 min, whereas an attenuating pattern of activity of Cbl protein has been seen in 4day and 8day; PNA showed that Syk PNA antisense significantly inhibit kinase activity and protein expression of Syk in non-stimulated or co-sitmulated CB CD4+ T cells in culture within 8 days, respectively. In parallel, Syk PNA antisense has selectively inhibited IFN-y mRNA and intracellular [FN-y expression induced by IL-2 and SDF-la/CXCL12; EMSA showed that NFAT has been detected in nuclei extracts of CB CD4~ T cells co-stimulated with IL-2 + SDF-la/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-2 and SDF-loc/CXCL12 selectively activate NFAT1 in CB CD4" T cells, whereas neither NFAT3 nor NFAT4 has been activated in these cells.Conclusions: After stimulation with IL-2 and SDF-la/CXCL12, CB CD4+ T cells have been switched to Thl pattern, a persistent and selective activation of Syk kinase have been induced, and Syk PNA antisenses significantly inhibit kinase activity and protein expression of Syk and block the polarization of Thl cell, implying persistent and selective activation of Syk kinase is essential for Thl polarization; An attenuating Cbl...