| Objective:High mobility group box 1 protein(HMGB1) as a late-acting cytokine mediates lethality of sepsis and systemic inflammation.The present study was performed to determine the effect of HMGB 1 on regulatory T cells(Tregs) of spleen in mice,and to clarify the potential mechanism of immunosuppression related to stimulation with HMGB1, aiming at a possible method for preventing the development of sepsis and multiple organ dysfunction syndrome following serious injure or inflammation.Methods:①CD4~+CD25~+Tregs and CD4~+CD25~-T cells were isolated from the spleens of male BABL/c mice by magnetic beads.The purity of these cells were detected.②CD4~+CD25~+Tregs were seeded on 96-well(1×10~5 cells /well) cell culture plates supplemented with PHA(20μg/ml),Con A(5mg/ml) or coated with anti-CD3(1μg/ml). After stimulated with different stimulus for various length of time,the expressions of cytotoxic T lymphocyte-associated antigen 4(CTLA-4) and forkhead/winged helix transcription factor 3(Foxp3) molecules of Tregs were determined.The time-dependent responses between different stimulus and CTLA-4 as well as Foxp3 were analyzed by means of flow cytometry.③CD4~+CD25~+Tregs were seeded on 96-well(1×10~5 cells/well) cell culture plates coated with anti-CD3(1μg/ml) and soluble anti-CD28(1μg/ml),and the cells were stimulated with HMGB1 for various length of time or in different concentrations.After being stimulated,the expressions of CTLA-4 and Foxp3 molecules of Tregs were determined.The time-dependent and dose-dependent responses between HMGB1 and CTLA-4 and Foxp3 were analyzed by means of flow cytometry,the IL-10 secretion collected in the cell suspension was determined by means of ELISA.④CD4~+CD25~+Treg and CD4~+CD25~-T cells were isolated separately.When cultured with Treg in ratio of 1:1,1:5,1:10,and 1:20,respectively,the proliferation of CD4~+CD25~-T cells was analyzed by MTT test.⑤After being stimulated for 72 h in concentration of 1000ng/ml HMGB1,Tregs were cultured with CD4~+CD25~-T.cells together in the cell-ratio of 1:1 and 1:5.Proliferation of CD4~+CD25~-T was analyzed by MTT test.⑥CD4~+CD25~+Tregs were seeded and stimulated with HMGB1 for various length of time or in different concentrations on 96-well(1×10~5 cells /well) cell culture plates coated with anti-CD3(1μg/ml) and soluble anti-CD28(1μg/ml).After being stimulated,the time-dependent and dose- dependent responses between HMGB1 and Foxp3 mRNA were analyzed by means of quantatative PCR of SYBR GREEN.⑦By collecting the cells suspension,the different kinds of cytokines were examined by means of ELISA,including IL-2,IL-10,IL-4 and interferon(IFN)-γ.Results:1.The purity of CD4~+CD25~+Tregs and CD4~+CD25~-T cells were 91.74%～98.14%and 88.73%～93.85%respectively after isolated twice by magnetic beads, and the activity of Treg exceeded 97%.2.After stimulation with PHA,the CTLA-4 expressions on surface of mice splenic Tregs and intranuclear Foxp3 molecules were unchanged(P＞0.05).In Con A-treated group,the expression of CTLA-4 was significantly enhanced at 24 h(P＜0.01),while the CTLA-4 expression returned to the control range at 48 h and 72 h.The expression of Foxp3 was not markedly different between ConA-treated group and controls.When treated with plate-bound anti-CD3,both CTLA-4 and Foxp3 expressions were significantly up-regulated at 24 h to 72 h(P＜0.05 or P＜0.01),especially at 24 h and 48 h(P＜0.01).3.After stimulation with HMGB1,the CTLA-4 expressions on surface of mice splenic Tregs and intranuclear Foxp3 molecules and Foxp3mRNA were markedly decreased at 24 h to 72 h(P＜0.05 or P＜0.01),and the expression levels of CTLA-4 and Foxp3 and Foxp3mRNA were lowest at 72 h(P＜0.01).When Tregs were cultured in the presence of 10ng/ml,100ng/ml,and 1000ng/ml HMGB1 for 72 h, expressions of the CTLA-4 and Foxp3 molecules and Foxp3mRNA were significantly down-regulated(P＜0.05 or P＜0.01),and values of them were lowest in 1000ng/ml HMGB1-treated group(P＜0.01).4.The suppressive activity of proliferation of CD4~+CD25~-T cells was exceeded 90%when the proportion of Tregs to CD25T cells was 1:1,meanwhile,the suppressive activity of Tregs was markedly decreased when stimulated with HMGB1.5.The secretion of IL-2 of Tregs stimulated by HMGB1 was not markedly changed(P＞0.05),while a dose-dependent decrease between IL-10 and HMGB1 was obviously(P＜0.05 or P＜0.01).When CD4~+CD25~-T cells were cultured with stimulated Tregs,in comparsion with unstimulated-Treg group,the levels of IL-2 and IFN-γwere elevated following the increased concentration of HMGB1(P＜0.05 or P＜0.01).Meanwhile the secretion of IL-4 and IL-10 were significantly decreased when cultured with stimulated Tregs(P＜0.05 or P＜0.01).Conclusion:1.The CD4~+CD25~+Tregs isolated by magnetic beads were pure and suitable for the subsequent experiments.2.Among different stimulus,anti-CD3 appears to be an effective immunoregulatory signal that influences the activation of mice splenic Tregs.3.HMGB1 stimulation can result in significantly down-regulatory expressions of suppressive molecules CTLA-4 and Foxp3 as well as cytokine IL-10 of splenic Tregs in mice,by inhibiting the Foxp3mRNA expression.4.HMGB1 cannot break down the anergy of Tregs in vitro,but can significantly down-regulate the immunosuppression of mice splenic Tregs,thereby influences the proliferation of effector T cells,secretion of IL-2 and cells- polarization.
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