| Sepsis is uncontrolled systemic inflammatory response to infection associated usually with multiple organ dysfunction,which is characterized by high mortality.It is clear that immune dysfunction is an important cause of sepsis at present.T lymphocytes are not only regulatory cells,but also effector cells in the host immune system.T lymphocytes-mediated immune suppression associated with sepsis include suppression of proliferative activity,increased apoptosis and the shift of naive T lymphocytes differentiation from helper T cell(Th)1subset to Th2 subset.Several studies have confirmed that the excessive release of HMGB1 directly could induce dysfunction of T-cell mediated immunity,including suppression of proliferative activity of T cells and expression levels of IL-2 as well as IL-2 receptor,thus inducing naive T cells differentiation shift from Thl subset to Th2.Increased HMGB1 secretion is an important cause of Th2 immune response in thermal injured rats.The experiments in vitro have demonstrated that HMGB1 could directly induce naive T cells differentiation shift from Thl subset to Th2.RAGE and TLR-4 are the main receptors for the recognition and binding of HMGB1,and are both expressed in T lymphocytes.However,it has not been reported whether they are directly involved in the pathological process of HMGB1-mediated CD4+T lymphocytes Th1/Th2 differentiation.In this study,the effects of HMGB1 on the Thl/Th2 differentiation of CD4+T lymphocytes and the expression of RAGE and TLR-4 were observed through in vitro experiments.Through the inhibition of RAGE and TLR-4 expression,we explored the role of RAGE and TLR-4 in HMGB1-mediated Thl/Th2 differentiation of CD4+T lymphocytes.Part I The effects of RAGE on HMGB1-mediated Thl/Th2 differentiation of CD4+T lymphocytesObjective To investigate the effects of RAGE on HMGB1-mediated Thl/Th2 differentiation of CD4+T lymphocytes.Method CD4+ T lymphocytes isolated from the spleens of male BALB/C mice by magnetic beads were suspended in RPMI-1640 with 10%FCS at 2×106cell/well on 96-well cell culture plates in vitro.The cells were stimulated by ConA at 3?g/ml for 12 hours,then recombinant HMGB1 in 10 ng/mL,100 ng/mL and 1000 ng/mL concentration or PBS was added to the T cell suspension for 12h,24h,48h respectively.The cell suspension was obtained to detect IL-4 and IFN-y expression by enzyme linked immunosorbent assay(EILSA)and RAGE,CATA-3 expression were detected by western-blot and real-time fluorescent quantitative PCR(RT-PCR).After the CD4+T lymphocytes were interfered with by using RAGE neutralization antibody and Lentrivirus vectors carrying the DNA fragments encoding GFP tagged RAGE siRNA(Lv-RAGE-siRNA),which decreased the expression of RAGE.Then,the expression of IFN-?,IL-4,RAGE and CATA-3 induced by HMGB1 were were detected by ELISA,western-blot and RT-PCR respectively.Results Compared with control group,the treatment with 10ng/mL HMGB1 for 24 h resulted in an up-regulation of IFN-y/IL-4 ratio(P<0.05),and down-regulation of IFN-?/IL-4 ratio in 100ng/mL and 1000 ng/mL groups(P<0.05).When the cells were exposed to 100ng/ml HMGB1 for different times(12,24,48 h),a decrease of IFN-?/IL-4 ratio(P<0.05)were observed in 24h and 48h.Compared with control group,RAGE protein and mRNA,GATA-3 mRNA expression of cells in 100ng/mL HMGB1 treatment for 24h were significantly increased(P<0.05),Compared with HMGB1 group,IFN-y/IL-4 tatio of cell suspension in HMGB1+RAGE neutralization antibody group was increased(P<0.05),and GATA-3 mRNA expression was down-regulated(P<0.05),while IFN-y/IL-4 ratio of cell suspension in HMGB1?Lv-RAGE-siRNA group were significantly decreased(P<0.05).Compared with HMGB1 group,RAGE expression in HMGB1+RAGE neutralization antibody group and HMGB1+Lv-RAGE-siRNA group were decreased(P<0.05).However,compared with control group,RAGE expression in HMGB1+RAGE neutralization antibody group was still up-regulated(P<0.05),while RAGE expression in HMGB1+Lv-RAGE-siRNA group was significantly decreased(P<0.05).Conclusion The Thl/Th2 differentiation of CD4+T lymphocytes induced by HMGB1 in vitro was at least partly related to over-activation of RAGE/GATA-3 signaling pathway.Part II The roles of TLR4 in HMGB1-mediated Thl/Th2 differentiation of CD4+T lymphocytesObjective To explore the effects of TLR4 on HMGB1-mediated Thl/Th2 differentiation of CD4+T lymphocytes.Method CD4+T lymphocytes isolated from the spleens of male BALB/C mice by magnetic beads were cultured in RPMI-1640 with 10%FCS at 2×10bcell/well on 96-well cell culture plates in vitro.The cells were stimulated with ConA at 3?g/ml for 12 hours,then recombinant HMGB1 in 100ng/mL concentration for 24h.TLR4 expression were detected by western-blot and RT-PCR.After the CD4+T lymphocytes were interfered with by TLR4 neutralization antibody and Lentrivirus vectors carrying the DNA fragments encoding GFP tagged TLR4 siRNA(Lv-TLR4-siRNA),which decreased the expression of TLR4.Then,the expression of IFN-?,IL-4 and TLR4 induced by HMGB1 were detected by ELISA,western-blot and RT-PCR respectively.Finally,the Annexinv-FITC cell apoptosis detection kit was used to detect the apoptosis of CD4+T lymphocytes induced by HMGB1.Results Compared with control group,TLR4 protein and mRNA expression of cells in 100ng/mL HMGBI treatment for 24h were significantly increased(P<0.05),while IFN-y/IL-4 ratio of cell suspension in HMGB1+Lv-TLR-4-siRNA group and HMGB1+TLR-4 neutralization antibody group were not significant difference(P>0.05).Compared with the control group,the apoptosis rate of CD4+T lymphocytes in HMGB1 group was significantly increased(P<0.05),but the apoptosis rate of CD4+T lymphocytes in HMGB1+TLR4 neutralization antibody group were significantly decreased compared with HMGB1 group(P<0.05)Conclusion The Thl/Th2 differentiation of CD4+T lymphocytes mediated by HMGB1 in vitro was independent of TLR4 signal pathway activation,but the activation of TLR4 was involved in the apoptosis response of CD4+T lymphocytes induced by HMGB1. |
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