Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways - Evidence for an intracellular compartmentalization of TLR4
Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways - Evidence for an intracellular compartmentalization of TLR4
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DOI：
10.1074/jbc.m305790200
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发表时间：
2004-01-23
影响因子：
4.8
通讯作者：
Si-Tahar, M
中科院分区：
文献类型：
--
作者：
Guillot, L;Medjane, S;Si-Tahar, M
由于呼吸，肺上皮细胞持续暴露于微生物挑战。人们认识到，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性免疫应答中起主要作用。相反，很少有人知道TLR在肺上皮细胞本身的表达，它们在细胞内的分布，它们的功能，以及所涉及的信号通路。在这项工作中，我们证明了逆转录-PCR和/或免疫印迹，TLR 4和辅助分子MD-2组成型表达在不同的人肺泡和支气管上皮细胞。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表征了TLR 4在这些细胞中的细胞内定位。尽管TLR 4的这种细胞内区室化，肺上皮细胞对TLR 4激活剂脂多糖（LPS）（一种有效的革兰氏阴性菌相关分子模式）有反应。使用从TLR 4敲除和野生型小鼠分离的呼吸道上皮细胞，我们证明TLR 4是这些细胞中LPS的实际活化受体。此外，我们发现，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ERK 1/2。此外，使用表达MyD 88和TRAF 6的显性阴性形式的载体，我们确定了LPS诱导的呼吸道上皮细胞的活化在很大程度上依赖于TLR 4信号传导中间体。总之，这些数据表明，TLR 4是肺上皮细胞对来自革兰氏阴性菌的分子的反应中的关键元件。TLR 4在肺上皮细胞中的细胞内定位被认为在预防慢性炎症性疾病的发展中起重要作用。
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.