The LPS Responsiveness of TLR2 an TLR4 in the Neutrophil

描述（由申请人提供） 脓毒症综合征与脓毒症所致急性呼吸窘迫综合征 急性呼吸窘迫综合征（ARDS）是一种重要的临床疾病， 没有可用的特定疗法。 最近发现的Toll- 类受体（TLR），特别是作为LPS受体的TLR 2和TLR 4， 推进了我们对LPS暴露后信号启动的理解。 我们假设TLR 4是LPS的主要受体， 在中性粒细胞中诱导NF-κ B和/或p38活化， 涉及IRAK 2和M、syk和Rac 2的通路。 但是，LPS 信号传导可以通过TLR 2以较少的亲合力发生，并且涉及 其他IRAK亚种、酪氨酸激酶或小G蛋白。 我们在这里展示 人嗜中性粒细胞和PLB-985细胞表达TLRI-6 mRNA， TLR 2蛋白。 此外，我们在PLB-985细胞中显示，IRAK， 酪氨酸激酶syk，小蛋白Rac 2与TLR 2在 基线和LPS暴露后，表明它们参与LPS信号传导 通过TLR 2。 我们建议在中性粒细胞中进行研究，包括人类和小鼠，以及PLB- 985细胞系：1. TLR 2和TLR 4在NF-κ B和p38活化中的作用， 包括它们的相互依赖性，2。与之缔合的大分子复合物 与LPS暴露后的TLR 2或TLR 4，和3. IRAK 2作用和激活 & M，酪氨酸激酶syk和林恩，以及小G蛋白Rac 2和Cdc 42 在LPS信号中。 为了实现这些目标，我们将开发可诱导的 反义逆转录病毒技术，用于产生反义TLR 2、TLR 4和 IRAK表达细胞系，TLR 2、TLR 4和IRAK M显性阴性， 和新型免疫沉淀技术的二维凝胶电泳， 与TLR 2、TLR 4和IRAK相关的大分子复合物 亚种。 我还将开发2D凝胶电泳技术， 质谱法鉴定蛋白质，这将有利于未来 信号通路的研究。 对LPS的识别和信号传导的更好理解 在神经元中由LPS启动的通路对于改善 了解脓毒症综合征和脓毒症的基础病理生理学 诱发ARDS。

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.