The Roles Of PKC-?-GATA3 Signaling Pathway In Morphine Induced Th2 Shift

BackgroundPolarization of naive cells to the Th2 phenotype is an immunosuppressive phenomenon.Naive CD4+Th0 cells can bi-potentially divide into two major subsets based on the environmental signals.The Th1/Th2 paradigm was then proposed useful for initial categorization based upon the selective production of cytokines,IFN-?and IL-4,respectively.These two subsets of Th cells reciprocally inhibit each other and keep a relative balance during differentiation.In the initial stage of immune response,once the immune system choose a response based on a certain subset,the Th cells would positively enhance own response and inhibit the differentiation to the other subset,resulting to a shift of the Th1/Th2 balance toward a certain dominance.Th cell differentiation into Th2 effecter cell populations contributes to impaired cellular immunity after surgery,increases the risk of postoperative infection,and probably results to tumor metastasis and recurrence in cancer patients.It has been reported that application of morphine be associated with Th2 shift after surgery.As known to all,morphine is a potent analgesic used in a board clinical range,which is still the first choice for the treatment of post-operative acute pain.However,morphine has been recently proved to directs T cells toward Th2 differentiation in vitro and vivo,and the?opioid receptor antagonist naltrexone inhibits morphine-induced Th2-dominant shift in a dose-dependent manner.Given the morphine-induced Th2 shift impairs the cell-mediated immune function,it is of great significance to explore the mechanism involved in Th differentiation.The role of GATA3 in Th2 cells is well known as the master regulator to guide the differentiation of Th2.GATA3 can directly binds to the IL-4 gene locus at various regions to affect IL-4 expression through regulating epigenetic modifications at the Th2cytokine gene locus.In addition to the function of promoting in Th2 cells,The production of high levels of IL-4 and the auto-activation of GATA3 expression build a positive-feedback loop that further induces the expression of GATA3 and the differentiation of Th2.GATA3 also inhibits the expression of IFN-?and directly transactivates IL-5 and IL-1.PKC-?activates NF-?B,AP-1 and NFAT,which are all required for T cell activation,proliferation and differentiation.Thus,activation of PKC-?is considered as an upstream event of GATA3 activation.Based on the above results,we hypothesis that morphine affect the phosphorylation of PKC-?by activating the?opioid receptor on the surface of T cells,thereby promoting the expression and activity of GATA3 gene and inducing the skewing of Th2cells.This effect of morphine can be reversed by?receptor antagonist and PKC-?inhibitor.The present study was firstly to immunoassay the dose-effect relationship using different concentrations of morphine inducing naive CD4+T cell.In the second part,?receptor antagonist and PKC-?inhibitor were added into the model of morphine-induced differentiation of naive CD4+T cells,then investigate the role of PKC-?-GATA3 signaling pathway and elucidate the mechanism of Th cell differentiation in vitro.Objectives1.To investigate the dose-effect relationship of Th2 shift induced by morphine in naive CD4~+T cells.2.To study the role of?receptor-PKC-?-GATA3 signaling pathway in the differentiation of naive CD4+T cells,and elucidate the underlying molecular mechanisms of Th cell specification in vitro,then investigate the possible intervention targets for regulating the balance of Th cells after operation.Methods1.Naive CD4~+T cells of pretreatments with different concentrations of morphine were stimulated by IL-2 and CD28 for 24 hours.Flow cytometry was performed to distinguish Th1 and Th2 cell.RT-PCR was used to detect the expression levels of IL-4and IFN-?.The concentrations of IL-4 and IFN-?in culture supernatant were examined by ELISA.The optimum concentration of morphine-induced Th2 shift was explored.2.The role of?opioid receptor activation/blockade in Th2 cell differentiation signaling pathway was investigated through detecting PKC-?level by Western blot,the expression levels of IL-4 and GATA3 by RT-PCR,and activities of DATA3 by EMSA.3.The role of PKC-?activation/blockade in Th2 cell differentiation signaling pathway was investigated through detecting PKC-?level by Western blot,the expression levels of IL-4 and GATA3 by RT-PCR,and activities of GATA3 by EMSA.Results1.The expression levels of IL-4 by RT-PCR and the concentrations of IL-4 in culture supernatant in morphine 50nmol/ml group were higher than those of other groups.The ratio of Th2 by flow in morphine 50nmol/ml group was also significantly higher.2.The levels of PKC-?at T538 and S676,the expression levels of IL-4 and GATA3,and the activities of GATA3 were significantly increased in morphine group compared with control,and decreased in morphine combined with naloxone group.3.The levels of PKC-?at T538 and S676,the expression levels of IL-4 and GATA3,and the activities of GATA3 were significantly decreased in morphine combined with AEB071 group when compared with morphine group.Conclusions1.Morphine induces naive CD4~+T cell to a Th2-dominant shift with an optimal efficiency at the concentration of 50nmol/ml.2.PKC-?levels as well as GATA3 expression and activity increase in morphine-induced Th2 shift model and could be revised by?opioid receptor antagonist,suggesting morphine induces Th2 shift via?opioid receptor on the surface of T cell.3.Treatment with AEB071 inhibits morphine-induced Th2 shift and decreases PKC-?levels and the expression and activity of GATA3,which suggesting that PKC-?plays a key role in morphine-induced Th2 shift signaling pathway.