Guillot Loïc, Medjane Samir, Le-Barillec Karine, Balloy Viviane, Danel Claire, Chignard Michel, Si-Tahar Mustapha
Unité de Défense Innée et Inflammation, INSERM E336, France.
J Biol Chem. 2004 Jan 23;279(4):2712-8. doi: 10.1074/jbc.M305790200. Epub 2003 Nov 4.
Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing. It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved. In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria. The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease.
由于呼吸的原因，肺上皮细胞持续面临微生物的挑战。已知免疫髓样细胞表达Toll样受体（TLR），其在检测微生物和启动先天免疫反应中起主要作用。相比之下，关于肺上皮细胞本身TLR的表达、其在细胞内的分布、功能以及涉及的信号通路知之甚少。在这项研究中，我们通过逆转录聚合酶链反应和/或免疫印迹证明，TLR4和辅助分子MD-2在不同的人肺泡和支气管上皮细胞中组成性表达。我们进一步通过流式细胞术、生物素化/沉淀和共聚焦显微镜对这些细胞中TLR4的细胞内定位进行了表征。尽管TLR4在细胞内存在分区，但肺上皮细胞对TLR4激活剂脂多糖（LPS）有反应，LPS是一种与革兰氏阴性菌相关的强效分子模式。使用从TLR4基因敲除小鼠和野生型小鼠分离的呼吸道上皮细胞，我们证明TLR4是这些细胞中LPS的实际激活受体。此外，我们表明这种细胞对LPS的反应涉及一个信号复合物，包括激酶白细胞介素-1受体相关激酶（IRAK）、p38、Jnk和ERK1/2。此外，使用表达MyD88和TRAF6显性负性形式的载体，我们确定LPS诱导的呼吸道上皮细胞激活在很大程度上依赖于TLR4信号中间体。总之，这些数据表明TLR4是肺上皮细胞对革兰氏阴性菌衍生分子反应的关键要素。TLR4在肺上皮细胞中的细胞内定位有望在预防慢性炎症性疾病的发展中发挥重要作用。
