抗CD40抗体诱导RelB核转位对骨髓源性树突状细胞表型和功能的影响

Effect of RelB nuclear translocation induced by anti-CD40 antibody on phenotype and function of bone marrow-derived dendritic cells

目的:探讨抗CD40抗体对小鼠骨髓源性树突状细胞表型和功能的影响及其可能机制。方法:取野生型雌性健康BALB/c小鼠胫骨和股骨制备骨髓树突状细胞(BMDCs),给予不同浓度抗CD40抗体(0、10或20μg/mL)刺激24 h,采用流式细胞术检测各组BMDCs表面分子MHC-Ⅱ、CD40、CD80和CD86的表达水平;采用ELISA法检测BMDCs培养上清液中IL-6、IL-12p70和TNF-α水平;使用荧光定量PCR法检测BMDCs中RelA和RelB mRNA表达水平。部分实验中,给予抗CD40抗体(20μg/mL)刺激前30 min加入RelB核转位抑制剂SN52(40μg/mL)干预,24 h后检测各组BMDCs表面分子表达及培养上清液细胞因子水平。结果:高浓度抗CD40抗体组(20μg/mL)和低浓度抗CD40抗体组(10μg/mL)BMDCs表面分子MHC-Ⅱ、CD40、CD80和CD86、促炎性细胞因子IL-6、IL-12p70和TNF-α以及RelB mRNA表达水平明显高于空白对照组(0μg/mL)(P<0.05或P<0.01),且该效应具有剂量依赖性,3组间RelA mRNA表达水平比较差异无明显统计学意义(P>0.05)。此外,抗CD40抗体加SN52干预组BMDCs表面MHC-Ⅱ、CD40、CD80和CD86及培养上清液中IL-6、IL-12p70和TNF-α水平明显低于单纯抗CD40抗体组(P<0.01),且与空白对照组比较差异无统计学意义(P>0.05)。结论:抗CD40抗体可能通过诱导RelB核转位促进BMDCs表型和功能成熟,并且该效应具有剂量依赖性,这可能为疾病免疫治疗提供新的思路。

Objective:To investigate the effect of anti-CD40 antibody on the phenotype and function of bone marrow dendritic cells(BMDCs)in mice and its possible mechanism.Methods:BMDCs were prepared from the tibia and femur of wild-type female healthy BALB/c mice and stimulated with dif-ferent concentrations of anti-CD40 antibodies(0,10 or 20μg/mL)for 24 hours.Flow cytometry was used to detect the expression of BMDCs surface molecules MHC-Ⅱ,CD40,CD80 and CD86.The levels of IL-6,IL-12p70 and TNF-αin the culture supernatant of BMDCs were detected by ELISA,and the expression levels of RelA and RelB mRNA in BMDCs were detected by real-time fluores-cence quantitative PCR.In some experiments,RelB nuclear translocation inhibitor SN52(40μg/mL)was added 30 minutes before the stimulation of anti-CD40 antibody(20μg/mL).After 24 hours,the expression of BMDCs surface molecules and the level of cytokines in the culture supernatant were de-tected.Results:The expression levels of BMDCs surface molecules MHC-Ⅱ,CD40,CD80 and CD86,proinflammatory cytokines IL-6,IL-12p70 and TNF-αand RelB mRNA in high anti-CD40 antibody group(20μg/mL)and low anti-CD40 antibody group(10μg/mL)were significantly higher than those in control group(0μg/mL)(P<0.05 or P<0.01),and the effect was dose-dependent.There was no significant difference in RelA mRNA expression among the three groups(P>0.05).In addition,the levels of MHC-Ⅱ,CD40,CD80 and CD86 on the surface of BMDCs and the levels of IL-6,IL-12p70 and TNF-αin the culture supernatant in the anti-CD40 antibody plus SN52 group were significantly lower than those in the anti-CD40 antibody group(P<0.01),and there was no sig-nificant difference between the anti-CD40 antibody plus SN52 group and the control group(P>0.05).Conclusion:Anti-CD40 antibody may promote BMDCs phenotypic and functional maturation by acti-vating RelB nuclear translocation,and the effect is dose-dependent,which may provide a new idea for disease immunotherapy.