The Immunopharmacology Research Group, University of Tampere School of Medicine and Tampere University Hospital, Tampere, Finland.
Respir Res. 2012 Aug 24;13(1):73. doi: 10.1186/1465-9921-13-73.
Eosinophils are critically involved in the pathogenesis of asthma. Nitric oxide (NO) is produced in high amounts in asthmatic lungs and has an important role as a regulator of lung inflammation. NO was previously shown to induce eosinophil apoptosis mediated via c-jun N-terminal kinase (JNK) and caspases. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS) and JNK.
Apoptosis was determined by flow cytometric analysis of relative DNA content, by Annexin-V labelling and/or morphological analysis. Immunoblotting was used to study phospho-JNK (pJNK) expression. Mitochondrial membrane potential was assessed by JC-1-staining and mitochondrial permeability transition (mPT) by loading cells with calcein acetoxymethyl ester (AM) and CoCl2 after which flow cytometric analysis was conducted. Statistical significance was calculated by repeated measures analysis of variance (ANOVA) or paired t-test.
NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) induced late apoptosis in GM-CSF-treated eosinophils. SNAP-induced apoptosis was suppressed by inhibitor of mPT bongkrekic acid (BA), inhibitor of JNK SP600125 and superoxide dismutase-mimetic AEOL 10150. Treatment with SNAP led to late loss of mitochondrial membrane potential. Additionally, we found that SNAP induces early partial mPT (1 h) that was followed by a strong increase in pJNK levels (2 h). Both events were prevented by BA. However, these events were not related to apoptosis because SNAP-induced apoptosis was prevented as efficiently when BA was added 16 h after SNAP. In addition to the early and strong rise, pJNK levels were less prominently increased at 20-30 h.
Here we demonstrated that NO-induced eosinophil apoptosis is mediated via ROS, JNK and late mPT. Additionally, our results suggest that NO induces early transient mPT (flickerings) that leads to JNK activation but is not significant for apoptosis. Thereby, we showed some interesting early events in NO-stimulated eosinophils that may take place even if the threshold for irreversible mPT and apoptosis is not crossed. This study also revealed a previously unknown physiological function for transient mPT by showing that it may function as initiator of non-apoptotic JNK signalling.
嗜酸性粒细胞在哮喘发病机制中起关键作用。一氧化氮（NO）在哮喘患者的肺部中大量产生，并作为肺炎症的调节剂发挥重要作用。先前的研究表明，NO 通过 c-jun N 末端激酶（JNK）和半胱天冬酶诱导嗜酸性粒细胞凋亡。我们的目的是阐明导致粒细胞巨噬细胞集落刺激因子（GM-CSF）处理的人嗜酸性粒细胞中 NO 诱导凋亡的级联反应，重点研究线粒体、活性氧（ROS）和 JNK 的作用。
通过相对 DNA 含量的流式细胞术分析、Annexin-V 标记和/或形态分析来确定凋亡。免疫印迹用于研究磷酸化 JNK（pJNK）表达。通过 JC-1 染色评估线粒体膜电位，通过用 calcein 乙氧甲酯（AM）和 CoCl2 负载细胞后进行流式细胞术分析来评估线粒体通透性转换（mPT）。通过重复测量方差分析（ANOVA）或配对 t 检验计算统计学意义。
NO 供体 S-亚硝基-N-乙酰-D,L-青霉胺（SNAP）诱导 GM-CSF 处理的嗜酸性粒细胞晚期凋亡。mPT 抑制剂 bongkrekic 酸（BA）、JNK 抑制剂 SP600125 和超氧化物歧化酶模拟物 AEOL 10150 抑制 SNAP 诱导的凋亡。SNAP 处理导致线粒体膜电位晚期丧失。此外，我们发现 SNAP 诱导早期部分 mPT（1 h），随后 JNK 水平强烈增加（2 h）。这两个事件都被 BA 阻止。然而，这些事件与凋亡无关，因为当 BA 在 SNAP 后 16 小时添加时，SNAP 诱导的凋亡同样被有效阻止。除了早期和强烈的增加外，pJNK 水平在 20-30 小时时也不那么明显地增加。
在这里，我们证明了 NO 诱导的嗜酸性粒细胞凋亡是通过 ROS、JNK 和晚期 mPT 介导的。此外，我们的结果表明，NO 诱导早期短暂的 mPT（闪烁），导致 JNK 激活，但对凋亡没有重要意义。因此，我们展示了一些有趣的早期事件在 NO 刺激的嗜酸性粒细胞，这可能发生，即使不可逆的 mPT 和凋亡的阈值没有被跨越。这项研究还通过显示短暂的 mPT 可以作为非凋亡 JNK 信号的启动子，揭示了其以前未知的生理功能。
