基质金属蛋白酶2/9促进T细胞CD100脱落在冠状动脉粥样硬化性心脏病中的作用

Matrix metalloproteinase 2/9 promotes CD100 shedding from T cells in coronary atherosclerotic heart disease

目的观察CD100、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)、MMP9在冠心病患者中的变化,评估MMP2/9对T细胞中CD100的调控在冠心病发病中的作用。方法24例稳定性心绞痛(stable angina,SA)、22例不稳定性心绞痛(unstable angina,UA)、31例急性心肌梗死(acute myocardial infarction,AMI)和18例对照者入组本研究。酶联免疫吸附试验(ELISA)检测血浆可溶型CD100(sCD100)、MMP2、MMP9水平,流式细胞术检测T细胞中膜型CD100(mCD100)平均荧光强度(MFI)。分选CD4^(+)和CD8^(+)T细胞,使用CD100、MMP2/9和抗CD100抗体刺激,ELISA法检测上清中细胞因子水平,实时定量PCR法检测CD4^(+)T细胞中转录因子T-bet、GATA-3、FoxP3、RORγt和CD8^(+)T细胞中穿孔素、颗粒酶B mRNA相对表达量。结果AMI组血浆sCD100、MMP2、MMP9水平高于对照组、SA组和UA组,AMI组CD4^(+)和CD8^(+)T细胞中mCD100 MFI低于对照组、SA组和UA组(P<0.05)。CD100刺激后CD4^(+)T细胞分泌干扰素-γ(IFN-γ)、白细胞介素(IL)-17、IL-22水平及T-bet、RORγt mRNA相对表达量升高(P<0.05)。CD100刺激后CD8^(+)T细胞分泌肿瘤坏死因子(TNF-α)、IFN-γ水平及穿孔素、颗粒酶B mRNA相对表达量升高(P<0.05)。MMP2或MMP9单独刺激AMI患者CD4^(+)T细胞,上清sCD100、mCD100 MFI无明显变化,MMP2+MMP9联合刺激后,上清sCD100升高,mCD100 MFI降低,分泌IFN-γ、IL-17、IL-22水平及T-bet、RORγt mRNA相对表达量升高(P<0.05),加入抗CD100抗体后CD4^(+)T细胞中上述指标较联合刺激组降低(P<0.05)。MMP2、MMP9或MMP2+MMP9联合刺激AMI患者CD8^(+)T细胞,上清sCD100升高,mCD100 MFI降低,TNF-α、IFN-γ水平及穿孔素、颗粒酶B mRNA相对表达量升高(P<0.05),加入抗CD100抗体后CD8^(+)T细胞中上述指标较联合刺激组降低(P<0.05)。结论AMI患者中,高表达的MMP2/9促进T细胞mCD100脱落,上调sCD100水平,诱导T细胞活化。

Matrix metalloproteinase(MMP)regulates CD100 shedding in infections and cancers,which contributes to T cell activity.The aim of this study was to investigate the levels of CD100,MMP2 and MMP9 in patients with coronary atherosclerotic heart disease(CAHD),and to assess the regulatory activity of MMP2/9 to CD100 on T cells in the pathogenesis of CAHD.Twenty-four stable angina(SA)patients,22 unstable angina(UA)patients,31 acute myocardial infarction(AMI)patients and 18 controls were enrolled.Soluble CD100(sCD100),MMP2 and MMP9 levels in the plasma were measured by enzyme linked immunosorbent assay(ELISA),while membrane-bound CD100(mCD100)mean fluorescence intensity(MFI)was assessed by flow cytometry on T cells.CD4^(+)T and CD8^(+)T cells were purified,and were stimulated with CD100,MMP2/9 and anti-CD100.Then cytokine expression in the supernatants was measured by ELISA;transcriptional factors T-bet,GATA-3,FoxP3 and RORγt mRNA in CD4^(+)T cells as well as perforin and granzyme B mRNA in CD8^(+)T cells were semi-quantified by real-time PCR.Data sowed that plasma sCD100,MMP2 and MMP9 levels in AMI patients were up-regulated,as compared with control,SA and UA patients,while mCD100 MFI on CD4^(+)and CD8^(+)T cells from AMI patients was down-regulated(P<0.05).Interferon-γ(IFN-γ),interleukin(IL)-17 and IL-22 secretion as well as T-bet and RORγt mRNA levels were elevated in CD100-stimulated CD4^(+)T cells(P<0.05).Tumor necrosis factor-α(TNF-α)and IFN-γsecretion as well as perforin and granzyme B mRNA level were increased in CD100 stimulated CD8^(+)T cells(P<0.05).MMP2 or MMP9 solely stimulation did not affect sCD100 or mCD100 MFI in CD4^(+)T cells from AMI patients,however,MMP2+MMP9 co-stimulation induced sCD100 up-regulation and mCD100 down-regulation in CD4^(+)T cells,also elevated the IFN-γ,IL-17 and IL-22 secretion as well as T-bet and RORγt mRNA levels(P<0.05).Administration of anti-CD100 reduced the above index in CD4^(+)T cells,as compared with co-stimulation group(P<0.05).MMP2,MMP9 or MMP2+MMP9 co-stimulation induced up-regulation of sCD100,TNF-α,IFN-γ,preforin mRNA and granzymeβmRNA,but down-regulation of mCD100 in CD8^(+)T cells from AMI patients(P<0.05).Administration of anti-CD100 reduced the above index of mCD100 in CD8^(+)T cells compared with co-stimulation group(P<0.05).In conclusion,elevated MMP2/MMP9 promotes mCD100 shedding of T cells and the secretion of sCD100 in AMI patients,thus inducing T cell activation.