Chen Hao-Cheng, Lin Horng-Chyuan, Liu Chien-Ying, Wang Chun-Hua, Hwang Tritium, Huang Tzu-Ting, Lin Chien-Huang, Kuo Han-Pin
Department of Thoracic Medicine II, Chang Gung Memorial Hospital, 199 Tun Hwa N. Road, Taipei 10507, Taiwan, ROC.
J Biomed Sci. 2004 Jan-Feb;11(1):49-58. doi: 10.1007/BF02256548.
The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced microvascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01-0.5 microg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 microg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-kappaB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-kappaB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.
中性粒细胞在肺内的隔离以及包括中性粒细胞弹性蛋白酶在内的促炎介质的释放，是脓毒症诱导的微血管通透性和肺泡上皮细胞损伤的原因。为了评估潜在机制，在有或没有抑制剂存在的情况下，将人中性粒细胞弹性蛋白酶（0.01 - 0.5微克/毫升）加入培养的A549上皮细胞中。通过ELISA或RT - PCR分析IL - 8以测量IL - 8合成能力。通过蛋白质印迹分析检测丝裂原活化蛋白激酶（MAPK）活性。中性粒细胞弹性蛋白酶剂量依赖性地增加培养的A549上皮细胞中IL - 8的释放。用特异性弹性蛋白酶抑制剂弹性蛋白酶抑制剂II（0.5、5和50微克/毫升）预处理，剂量依赖性地抑制中性粒细胞弹性蛋白酶诱导的IL - 8释放。中性粒细胞弹性蛋白酶上调了MAPK、p38和细胞外信号调节激酶（ERK）的活性。核转录因子 - κB（NF - κB）和活化蛋白1（AP - 1）也被激活。这些反应被弹性蛋白酶抑制剂II显著抑制。p38 MAPK的特异性抑制剂（SB203580）和NF - κB抑制剂（吡咯烷二硫代氨基甲酸盐），但不是ERK抑制剂（PD 98059），显著抑制中性粒细胞弹性蛋白酶诱导的IL - 8释放和mRNA表达。特异性酪氨酸激酶抑制剂染料木黄酮和蛋白激酶C（PKC）抑制剂Ro 31 - 8220也抑制IL - 8释放和mRNA表达以及p38和NF - κB活化。蛋白激酶A抑制剂H - 89对中性粒细胞弹性蛋白酶诱导的IL - 8合成或p38 MAPK活化没有显著影响。我们的结果表明，中性粒细胞弹性蛋白酶激活p38 MAPK，其上调NF - κB和AP - 1活性，从而诱导IL - 8 mRNA表达和蛋白质合成。酪氨酸激酶和PKC参与中性粒细胞弹性蛋白酶对MAPK途径的激活。
