Neutrophil-derived elastase induces TGF-beta1 secretion in human airway smooth muscle via NF-kappaB pathway.
Neutrophil-derived elastase induces TGF-beta1 secretion in human airway smooth muscle via NF-kappaB pathway.
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中性粒细胞来源的弹性蛋白酶通过 NF-kappaB 途径诱导人气道平滑肌分泌 TGF-β1。
DOI：
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发表时间：
2006
影响因子：
6.4
通讯作者：
H. Kuo
中科院分区：
文献类型：
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作者：
Kang;S. Ho;Horng;Shu;Chien‐Ying Liu;Chien;Chun;K. Chung;H. Kuo
慢性、重度哮喘患者气道中有中性粒细胞浸润，意义不确定。气道平滑肌（ASM）除了具有收缩特性外，还通过产生炎症介质参与哮喘的发病机制。在本研究中，我们从tgf - β 1释放的角度研究了中性粒细胞衍生弹性蛋白酶（NE）对ASM的影响，并探讨了其潜在机制。原代ASM细胞在用NE （0.01 ~ 0.5 μ g/ml）刺激前血清饥饿24 h。ELISA法检测上清tgf - β 1， real-time RT-QPCR法定量mRNA。Western blotting和kappaB-2 dEGFP报告基因试验检测NF-kappaB核易位和活化。用免疫共沉淀法研究IL-1受体相关激酶（IRAK）与MyD88的相关性，用FACS扫描和Western blotting检测toll样受体4 （TLR4）。我们证明NE以时间依赖性的方式增强tgf - β 1的释放。放线菌素D （5mm）、环己亚胺（5mm）和NF-kappaB抑制剂（包括吡罗烷二硫代氨基甲酸酯（PDTC, 1mm）、阿司匹林（2.5 mM）和水杨酸钠（2.5 mM））可抑制这种诱导。NE刺激后，IRAK与MyD88结合，IkappaBalpha磷酸化，p65核易位，并增加了核活化活性。我们还发现，在NE治疗后，TLR4水平降低。这些数据表明，NE通过My88/IRAK/NF-kappaB通路上调tgf - β 1基因的表达和释放，可能是通过激活TLR4，并揭示了中性粒细胞在哮喘发病中的潜在作用。
Neutrophils are infiltrated in airways of individuals with more severe and chronic asthma, with uncertain significance. Airway smooth muscle (ASM), apart from its contractile properties, is critically involved in the pathogenesis of asthma by producing inflammatory mediators. In the present study, we investigated the impact of neutrophil-derived elastase (NE) on ASM in terms of TGF-beta1 release, and we explored the underlying mechanisms. Primary ASM cells were serum starved for 24 h before stimulation with NE (0.01-0.5 microg/ml). TGF-beta1 in supernatant was determined by ELISA and mRNA quantified by real-time RT-QPCR. NF-kappaB nuclear translocation and activation was examined by Western blotting and kappaB-2 dEGFP reporter gene assay. Association of IL-1 receptor-associated kinase (IRAK) with MyD88 was studied by co-immunoprecipitation and Toll-like receptor 4 (TLR4) determined by FACS scan and Western blotting. We demonstrated that NE enhanced TGF-beta1 release in a time-dependent manner. This induction was inhibited by actinomycin D (5 mM), cycloheximide (5 mM), and NF-kappaB inhibitors, including pyrrolidine dithiocarbamate (PDTC, 1 mM), aspirin (2.5 mM), and sodium salyicylate (2.5 mM). Stimulation with NE was rapidly followed by association of IRAK with MyD88, phosphorylation of IkappaBalpha, and nuclear translocation of p65 with increased transactivation activity. We also found that TLR4 levels were reduced upon NE treatment. These data suggest that NE upregulates TGF-beta1 gene expression and release via My88/IRAK/NF-kappaB pathway, possibly through activation of TLR4, and shed light on a potential role of neutrophils in the pathogenesis of asthma.