Role of CD14 and TLR4 in Urothelial Response to LPS.

描述(申请人提供)：尿路感染每年导致约960万人次就诊，20%的女性在一生中会发展为尿路感染。大多数(70%-90%)的尿路感染是由产生脂多糖(LPS)的革兰氏阴性菌引起的，而脂多糖是革兰氏阴性菌细胞壁的组成部分，负责导致疼痛和炎症的细胞反应。各种器官系统(胃肠道、呼吸系统、尿路)的粘膜是抵御革兰氏阴性细菌感染的第一道天然防线；然而，调节尿路上皮细胞对脂多糖的反应的细胞过程尚不清楚。尽管其他研究人员报道了尿路上皮细胞不表达CD14(主要的脂多糖同源细胞受体)，但最近的一篇报道描述了CD14在人膀胱粘膜中的存在，我们实验室的初步研究证明了人尿上皮细胞中存在CD14和蛋白。这些细胞还表达Toll样受体4(TLR4)，它似乎为响应内毒素的信号提供了跨膜途径。尿路上皮细胞具有产生细胞因子的能力，这些细胞因子可以招募和激活白细胞。这项研究的目的是验证CD14和TLR4在尿路上皮细胞对内毒素的反应中起关键作用的假设。利用人类尿路上皮细胞和CD14或TLR4基因突变的小鼠的培养，我们将在体外和体内研究在存在或不存在功能性CD14和TLR4的情况下尿路上皮细胞对内毒素的反应。此外，我们将确定CD14的可溶性形式是否放大了尿路上皮细胞对内毒素的反应。拟议的实验将更好地确定在存在(体内)和不存在(体外)其他天然免疫成分的情况下，这些受体在尿路上皮细胞对内毒素的反应中的参与。我们期望这些研究将为进一步的研究提供基础，以确定可能参与这一过程的其他蛋白质，并剖析将尿路上皮细胞结合的内毒素转化为细胞反应的信号通路。这项研究的长期目标是确定将产生内毒素的革兰氏阴性细菌引起的膀胱炎引起的疼痛和有害细胞影响降至最低的策略。

DESCRIPTION (provided by applicant): Urinary tract infections result in approximately 9.6 million doctor visits annually, and 20% of women will develop a urinary tract infection during their lifetime. A majority (70-90%) of urinary tract infections are the result of gram-negative bacteria that produce lipopolysaccharide (LPS), and LPS is the component of the cell wall of gram-negative bacteria responsible for cellular response resulting in pain and inflammation. The mucosa of a variety of organ systems (GI, respiratory, urinary) functions as the first line of innate defense against infection by gram-negative bacteria; however, cellular processes that regulate the response of urothelial cells to LPS remain unknown. Although other investigators have reported that urothelial cells do not express CD14 (the primary cognate cellular receptor for LPS), a recent report described the presence of message for CD14 in human bladder mucosa, and preliminary investigations in our laboratory demonstrated the presence of message and protein in human urothelial cells. These cells also express Toll-Like Receptor 4 (TLR4), which appears to provide the transmembrane pathway for signaling in response to LPS. Urothelial cells have the capacity to produce cytokines which recruit and activate leukocytes. The goal of this research is to test the hypothesis that CD14 and TLR4 play a crucial role in the response of the urothelium to LPS. Using cultures of human urothelial cells and mice with genetic disruption of either CD14 or TLR4, we will investigate the response of the urothelium in vitro and in vivo to LPS in the presence or absence of functional CD14 and TLR4. Further, we will determine whether or not the soluble form of CD14 amplifies the response of urothelial cells to LPS. The proposed experiments will better define participation of these receptors in the response of urothelial cells to LPS in the presence (in vivo) and absence (in vitro) of other components of innate immunity. We anticipate that these studies will provide the foundation for continued investigations to identify additional proteins that may participate in this process and dissect signaling pathways which translate LPS binding by urothelial cells into cellular response. The long-range goal of this research is to identify strategies to minimize pain and harmful cellular effects associated with cystitis due to LPS-producing gram-negative bacteria.