Institute of Clinical Biochemistry, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany.
Cytokine. 2011 Aug;55(2):195-201. doi: 10.1016/j.cyto.2011.04.002. Epub 2011 Apr 29.
Proinflammatory cytokines produced by immune cells destroy pancreatic beta cells in type 1 diabetes. The aim of this study was to investigate the cytokine network and its effects in insulin-secreting cells. INS1E cells were exposed to different combinations of proinflammatory cytokines. Cytokine toxicity was estimated by MTT assay and caspase activation measurements. The NFκB-iNOS pathway was analyzed by a SEAP reporter gene assay, Western-blotting and nitrite measurements. Gene expression analyses of ER stress markers, Chop and Bip, were performed by real-time RT-PCR. Cytokines tested in this study, namely IL-1β, TNFα and IFNγ, had deleterious effects on beta cell viability. The most potent toxicity exhibited IL-1β and its combinations with other cytokines. The toxic effects of IL-1β towards cell viability, caspase activation and iNOS activity were dependent on nitric oxide and abolished by an iNOS blocker. IL-1β was the strongest inducer of the NFκB activation. An iNOS blocker inhibited IL-1β-mediated NFκB activation in the first, initial phase of cytokine action, but did not affect significantly NFκB activation after prolonged incubation. Interestingly iNOS protein expression was induced predominantly by IL-1β and decreased in the presence of an iNOS blocker in the case of a short time exposure. The changes in the expression of ER stress markers were also almost exclusively dependent on the IL-1β presence and counteracted by iNOS blockade. Thus cytokine-induced beta cell death is primarily IL-1β mediated with a NO-independent enhancement by TNFα and IFNγ. The deleterious effects on cell viability and function are crucially dependent on IL-1β-induced nitric oxide formation.
免疫细胞产生的促炎细胞因子会破坏 1 型糖尿病患者的胰岛β细胞。本研究旨在研究细胞因子网络及其对胰岛素分泌细胞的影响。将 INS1E 细胞暴露于不同的促炎细胞因子组合中。通过 MTT 测定和半胱天冬酶激活测定来评估细胞因子毒性。通过 SEAP 报告基因测定、Western blot 和亚硝酸盐测定来分析 NFκB-iNOS 途径。通过实时 RT-PCR 分析内质网应激标志物 Chop 和 Bip 的基因表达。本研究中测试的细胞因子，即 IL-1β、TNFα 和 IFNγ，对β细胞活力具有有害影响。IL-1β 及其与其他细胞因子的组合表现出最强的毒性。IL-1β 对细胞活力、半胱天冬酶激活和 iNOS 活性的毒性作用依赖于一氧化氮，并且可以被 iNOS 抑制剂消除。IL-1β 是 NFκB 激活的最强诱导剂。iNOS 抑制剂在细胞因子作用的初始阶段抑制 IL-1β 介导的 NFκB 激活，但在延长孵育后对 NFκB 激活的抑制作用不明显。有趣的是，iNOS 蛋白表达主要由 IL-1β 诱导，并且在存在 iNOS 抑制剂的情况下，在短时间暴露时会降低。内质网应激标志物表达的变化也几乎完全依赖于 IL-1β 的存在，并通过 iNOS 阻断得到拮抗。因此，细胞因子诱导的β细胞死亡主要是由 IL-1β 介导的，TNFα 和 IFNγ 以非依赖于 NO 的方式增强。对细胞活力和功能的有害影响主要取决于 IL-1β 诱导的一氧化氮形成。
