MECHANISMS FOR KERATINOCYTE LIPOPOLYSACCHARIDE RESPONSES

角质细胞脂多糖反应机制

基本信息

批准号：
6534451
负责人：
Christopher Lee Reardon
金额：
$ 18.76万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-30 至 2004-08-31
项目状态：
已结题

项目摘要

Through a unique form of innate immunity, keratinocytes from humans and mice respond to the bacterial product, lipopolysaccharide (LPS), present in the cell walls of Gram-negative bacteria by secreting inflammatory mediators. The overall objective of the proposed research herein is to define the keratinocyte LPS receptors, LPS-associated functions and signaling mechanisms that occur in this innate keratinocyte anti-bacterial defense system so that patients, who develop a functional loss of this skin-defense system leading up to recurrent bacterial skin injections, may be helped to avoid progressions to bacterial cellulitis and sepsis. We propose: (1) To identify LPS receptors on human and murine keratinocytes in vitro, in situ and in vivo and to determine what agents modulate their expression. Preliminary data shown that the membrane- bound LPS receptors, CD143, CD11a/CD18, CD11b/CD18 and CD11c/CD18 are expressed on human and mouse keratinocytes similar to macrophages. We will use primary keratinocytes and cell lines to further characterize and define the control of the expression of various LPS receptors with several agents and cytokines. We will induce and modulate the expression of LPS receptors in skin organ cultures and in live skin. Keratinocytes from different LPS receptor knock-out mice will be used for comparison. (2) To characterize cellular functions induced by LPS activation of human and murine keratinocytes by determining what cytokines by determining what cytokines and inflammatory mediators are specifically made in response to bacterial product and to define how these functions can be controlled with agents that modulate the LPS receptors or affect their interactions with LPS. With various methods, we will define the array of inflammatory cytokines and mediators, such as nitric oxide, that are produced following keratinocyte activation with LPS. Keratinocyte functions will be monitored following cell treatments that affect LPS receptor expression to help evaluate which LPS receptors are the most important for the gene promoter transcriptional activators, NF- kappaB and NF-IL-6, and up-regulation of CD14-associated type-2 Toll- like receptors following LPS receptor triggering in human and murine keratinocytes and how these events can be controlled through LPS receptor modulation. Cell activation by LPS leads to induction of calcium mobilization across the cell membrane, up-regulation of Toll-like receptor 2 (TLR2) molecules which trigger nuclear translocation of transcriptional activators, such as NF-kappaB and NF-IL-6, which in turn bind cytokine gene promoters. We will attempt to show with gel-shift assays. Western blots and quantitative RT-PCR that these signaling agents play an important role in LPS-induced activation of keratinocytes.

通过独特形式的先天免疫，人类和小鼠的角质形成细胞通过分泌炎症介质对革兰氏阴性细菌细胞壁中存在的细菌产物脂多糖（LPS）做出反应。本文提出的研究的总体目标是定义角质形成细胞 LPS 受体、LPS 相关功能和这种先天角质形成细胞抗菌防御系统中发生的信号机制，以便导致这种皮肤防御系统功能丧失的患者反复进行细菌皮肤注射可能有助于避免进展为细菌性蜂窝组织炎和败血症。我们建议：（1）在体外、原位和体内鉴定人和鼠角质形成细胞上的LPS受体，并确定哪些药物调节其表达。初步数据显示，膜结合 LPS 受体 CD143、CD11a/CD18、CD11b/CD18 和 CD11c/CD18 在人和小鼠角质形成细胞上的表达与巨噬细胞类似。我们将使用原代角质形成细胞和细胞系来进一步表征和定义几种药物和细胞因子对各种 LPS 受体表达的控制。我们将诱导和调节皮肤器官培养物和活体皮肤中 LPS 受体的表达。来自不同LPS受体敲除小鼠的角质形成细胞将用于比较。 (2) 通过确定哪些细胞因子和炎症介质专门针对细菌产物产生反应来确定哪些细胞因子，从而表征由 LPS 激活人和鼠角质形成细胞诱导的细胞功能，并确定如何用调节 LPS 的试剂来控制这些功能受体或影响它们与 LPS 的相互作用。通过各种方法，我们将定义一系列炎症细胞因子和介质，例如用 LPS 激活角质形成细胞后产生的一氧化氮。在影响 LPS 受体表达的细胞处理后，将监测角质形成细胞的功能，以帮助评估哪些 LPS 受体对于基因启动子转录激活因子 NF-κB 和 NF-IL-6 以及 CD14 相关型的上调最重要。 2 人类和小鼠角质形成细胞中 LPS 受体触发后的 Toll 样受体以及如何通过 LPS 受体调节来控制这些事件。 LPS 激活细胞，诱导钙跨细胞膜动员，上调 Toll 样受体 2 (TLR2) 分子，从而触发转录激活因子（例如 NF-κB 和 NF-IL-6）的核转位，从而转结合细胞因子基因启动子。我们将尝试用凝胶迁移测定来展示。 Western blots和定量RT-PCR表明这些信号转导剂在LPS诱导的角质形成细胞活化中发挥重要作用。