LPS Responsiveness of TLR2 and TLR4 in the Neutrophil

中性粒细胞中 TLR2 和 TLR4 的 LPS 反应

• 批准号: 6320966
• 负责人: PATRICK G ARNDT
• 金额: $ 12.37万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6320966
• 关键词: CD14 molecule; antisense nucleic acid; biological signal transduction; blood toxicology; cell line; gel electrophoresis; human tissue; immunoprecipitation; laboratory mouse; lipopolysaccharides; mass spectrometry; neutrophil; nuclear factor kappa beta; protein structure function; receptor; receptor expression

DESCRIPTION (provided by applicant) The sepsis syndrome and sepsis induced Acute Respiratory Distress Syndrome (ARDS), associated with expo- sure to LPS, are important clinical entities without available specific therapies. The recent identification of the Toll- like receptors (TLRs), in particular TLR2 and TLR4 as LPS receptors has advanced our understanding of the initiation of signaling after LPS exposure. We hypothesize that TLR4 is the predominant receptor responsible for LPS induced NF-KB and/or p38 activation in the neutrophil with the signaling pathway involving IRAK 2 and M, syk and Rac2. Although, alternatively, LPS signaling may occur through TLR2 with less avidity and with involvement of other IRAK sub-species, tyrosine kinases, or small G proteins. We show here that human neutro- phils and PLB-985 cells express mRNA for TLRI-6 and express TLR2 protein. In addition, we show here in PLB-985 cells, that IRAK, the tyrosine kinase syk, and the small protein Rac2 associate with TLR2 at baseline and after LPS exposure, suggesting their involvement in LPS signaling through TLR2. We propose to investigate in neutrophils, both human and murine, and the PLB- 985 cell line: 1. the role of TLR2 and TLR4 in NF-KB and p38 activation, including their interdependence, 2. the macromolecular complex which associate with TLR2 or TLR4 after LPS exposure, and 3. the role and activation of IRAK 2 & M, the tyrosine kinases syk and lyn, and the small G proteins Rac2 and Cdc42 in LPS signaling. To accomplish these goals, we will develop inducible antisense retroviral techniques for the creation of antisense TLR2, TLR4, and IRAK ex- pressing cell lines, dominant negatives for TLR2, TLR4, and IRAK M, and novel immunoprecipitation techniques for 2D gel electrophoresis to examine the macromolecular complexes associated with TLR2, TLR4, and the IRAK subspecies. I will also develop techniques in 2D gel electrophoresis and protein identification by mass spec- trometry which will benefit future investigations into signaling pathways. An improved understanding of the recognition of LPS, and the signaling pathways initiated by LPS, in the neu- trophil are important to improve the understanding of the underlying pathophysiology of sepsis syndrome and sepsis induced ARDS.

描述（由申请人提供） 败血症综合征和败血症引起的急性呼吸窘迫综合征 （ARDS）与博览会有关，是重要的临床实体 没有可用的特定疗法。 最近对通行费的标识 像受体（TLR）一样，尤其是TLR2和TLR4，因为LPS受体具有 提高了我们对LPS暴露后信号传导启动的理解。 我们假设TLR4是负责LPS的主要受体 诱导的NF-KB和/或P38在中性粒细胞中激活，并带信号传导 涉及Irak 2和M，Syk和Rac2的路径。 虽然，或者是LPS 信号传导可能通过TLR2发生较少的TLR2发生，并且参与 其他IRAK亚种，酪氨酸激酶或小G蛋白。 我们在这里显示 人类中心菲尔和PLB-985细胞表达TLRI-6的mRNA，并表达 TLR2蛋白。 此外，我们在这里显示在PLB-985细胞中，irak， 酪氨酸激酶SYK和小蛋白Rac2与TLR2搭配 基线和LPS暴露后，表明它们参与LPS信号传导 通过TLR2。 我们建议在人类和鼠类中的中性粒细胞中进行研究，以及PLB- 985细胞系：1。tlr2和tlr4在NF-KB和p38激活中的作用， 包括它们的相互依存关系2。 LPS暴露后使用TLR2或TLR4，3。 ＆M，酪氨酸激酶Syk和Lyn，以及小的G蛋白Rac2和Cdc42 在LPS信号中。 为了实现这些目标，我们将发展诱导的 反义逆转录病毒技术，用于创建反义TLR2，TLR4和 IRAK施加细胞系，TLR2，TLR4和IRAK M的主要负面因素， 和2D凝胶电泳的新型免疫沉淀技术检查 与TLR2，TLR4和IRAK相关的大分子复合物 亚种。 我还将在2D凝胶电泳和 通过质量表格鉴定蛋白质，这将使未来受益 对信号通路的调查。 对LP的识别和信号的认识有了改进的理解 由LPS引发的途径，在神经膜上对于改善的途径很重要 了解败血症和败血症的潜在病理生理学 诱导的弧。