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目的:研究IL-18是否参与调节T细胞通过直接接触激活的单核细胞的功能及其细胞内机制。方法:采用磁珠分离技术从健康人外周血分离纯化T细胞和单核细胞,被植物血凝素(PHA)预刺激的T细胞用多聚甲醛固定后按4:1与单核细胞共培养。采用ELISA法测定上清中肿瘤坏死因子(TNF)-α和IL-18的含量。采用流式细胞仪分析单核细胞表面IL-18受体α链的表达(IL-18Rα)。结果:PHA刺激48h或72h的T细胞诱导单核细胞产生的TNF-α显著高于未接受PHA刺激的T细胞。单独培养的单核细胞和T细胞几乎不产生TNF-α。T细胞直接接触可诱导单核细胞产生IL-18,且该作用能被核因子(NF)-κB抑制剂N-乙酰基-L-半胱氨酸(NAC)和磷脂酰肌醇(PI)3-激酶抑制剂LY294002分别抑制,但有丝分裂原激活蛋白激酶(MAPK)抑制剂SB203580对之无作用。与T细胞共孵育24h的单核细胞表面IL-18Rα的表达明显上调。单克隆的IL-18中和抗体呈剂量依赖性抑制与T细胞共孵育的单核细胞产生TNF-α。IL-18不能促进单纯的单核细胞产生TNF-α,但呈剂量依赖性地促进由于接触T细胞膜而激活的单核细胞产生TNF-α,且该作用可被NAC和LY294002分别拮抗,但不受SB203580影响。结论:T细胞可通过直接接触诱导单核细胞产生TNF-α和IL-18,上调单核细胞表面的IL-18R,并激活单核细胞内的NF-κB和PI3-激酶。IL-18可增强T细胞接触激活的单核细胞分泌致炎细胞因子的功能,该作用依赖细胞内NF-κB和PI3-激酶途径的激活。
AIM: To investigate whether IL-18 is involved in regulating the function and intracellular mechanism of T cells activated by direct contact with monocytes. Methods: T cells and monocytes were isolated and purified from peripheral blood of healthy people by magnetic bead separation. T cells pre-stimulated by phytohemagglutinin (PHA) were fixed with paraformaldehyde and co-cultured with monocytes at a ratio of 4: 1 . The contents of tumor necrosis factor (TNF) -α and IL-18 in the supernatant were measured by ELISA. The expression of IL-18 receptor α chain (IL-18Rα) on monocyte surface was analyzed by flow cytometry. Results: T cells stimulated by PHA for 48 h or 72 h induced TNF-α production significantly higher than those untreated with PHA-stimulated T cells. Monocytes and T cells cultured in isolation produced almost no TNF-α. The direct contact of T cells can induce monocytes to produce IL-18, and the effect can be blocked by nuclear factor (NF) -κB inhibitor N-acetyl-L-cysteine (NAC) and phosphatidylinositol (PI) 3-kinase inhibitor LY294002, respectively, but the mitogen-activated protein kinase (MAPK) inhibitor SB203580 had no effect on it. IL-18Rα expression was significantly up-regulated on monocytes co-incubated with T cells for 24 h. Monoclonal IL-18 neutralizing antibodies produced TNF-α in a dose-dependent manner in monocytes incubated with T cells. IL-18 failed to promote TNF-α production by naive monocytes but promoted TNF-α production by monocytes activated by exposure to T cell membranes in a dose-dependent manner, and this effect was antagonized by NAC and LY294002 respectively, but not Affected by SB203580. CONCLUSION: T cells induce TNF-α and IL-18 production by monocyte direct contact, up-regulate IL-18R on monocytes and activate NF-κB and PI3-kinase in monocytes. IL-18 enhances the ability of T cells to contact activated monocytes to secrete proinflammatory cytokines that are dependent on the activation of intracellular NF-κB and PI3-kinase pathways.