Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration.
Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration.
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DOI：
10.4049/jimmunol.181.8.5702
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发表时间：
2008-10-15
期刊：
影响因子：
--
通讯作者：
Parkos CA
中科院分区：
文献类型：
--
作者：
Chin AC;Lee WY;Nusrat A;Vergnolle N;Parkos CA
中性粒细胞（PMN）浸润和丝氨酸蛋白酶的相关释放有助于在粘膜病症如炎性肠病的活动期期间的上皮损伤。先前的研究已经证明，PMN与肠上皮细胞的基底外侧表面在化学引诱物的存在下接触导致屏障功能的破坏，即使没有迁移。类似地，已显示丝氨酸蛋白酶介导的上皮蛋白酶激活受体（PAR）的激活增加渗透性。在这项研究中，我们评估了迁移的中性粒细胞是否可以通过上皮PAR激活调节屏障功能。中性粒细胞与T84细胞单层基底外侧表面接触后，或与中性粒细胞弹性蛋白酶和蛋白酶-3孵育后，跨上皮电阻（TER）显著降低，但与组织蛋白酶G孵育后无显著差异。抑制中性粒细胞丝氨酸蛋白酶，但不选择性抑制弹性蛋白酶或组织蛋白酶G，防止了由中性粒细胞接触引起的TER下降，并阻断了中性粒细胞跨上皮迁移。基底外侧，但不是顶端，PAR-1和-2激活与选择性激动剂也降低TER。PAR-1和PAR-2定位于细胞内和紧密连接下的侧表面附近，并且在克罗恩病患者的结肠粘膜中表达增加。联合而非单独转染靶向上皮PAR-1和PAR-2的小干扰RNA，可阻止由PMN接触诱导的TER下降。此外，基底外侧PAR-1和-2激活诱导肌球蛋白L链激酶和调节肌球蛋白L链的磷酸化。最后，上皮PAR-1和-2敲低降低PMN跨上皮迁移的速率。这些结果表明，蛋白酶介导的上皮PAR-1和-2激活，迁移的中性粒细胞，诱导信号事件，增加上皮通透性，从而促进中性粒细胞跨上皮迁移。
Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn’s disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.
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