LPS Responsiveness of TLR2 and TLR4 in the Neutrophil

描述(由申请人提供) 脓毒症综合征及败血症所致的急性呼吸窘迫综合征 与内毒素相关的不良反应(ARDS)是重要的临床实体 没有可用的特殊疗法。最近对通行费的识别- 类受体(TLRs)，尤其是作为内毒素受体的TLR2和TLR4 进一步加深了我们对脂多糖暴露后信号启动的理解。 我们推测TLR4是内毒素的主要受体 通过信号转导诱导中性粒细胞中的核因子-kB和/或p38活化 途径涉及IRAK 2和M、SYK和RAC2。尽管，作为替代，内毒素 信号可能通过TLR2发生，但亲和力较低，并参与 其他IRAK亚种，酪氨酸激酶或小G蛋白。我们在这里展示 人中性粒细胞和PLB-985细胞表达TLRI-6的研究 TLR2蛋白。此外，我们在这里展示了在PLB-985细胞中，IRAK， 酪氨酸激酶SYK和与TLR2相关的小蛋白rac2在 基线和内毒素暴露后，表明它们参与了内毒素信号转导 通过TLR2。 我们建议研究中性粒细胞，包括人和小鼠，以及原核细胞- 985细胞系：1.TLR2和TLR4在核因子-kB和p38活化中的作用 包括它们之间的相互依赖关系，2.相互作用的大分子复合体 3.IRAK-2的作用和激活 &M，酪氨酸激酶SYK和Lyn，以及小G蛋白rac2和CDc42 在内毒素信号中。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TLR2、TLR4和 IRAK表达细胞系，TLR2、TLR4和IRAK M显性阴性， 和新的免疫沉淀技术用于2D凝胶电泳检测 与TLR2、TLR4和IRAK相关的大分子络合物 亚种。我还将开发2D凝胶电泳技术和 蛋白质的质谱学鉴定将对未来有所裨益 对信号通路的研究。 提高对内毒素的识别和信号转导的理解 在中性粒细胞中由内毒素启动的通路对于改善 对脓毒症综合征和脓毒症的基本病理生理的认识 诱发急性呼吸窘迫综合征。

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.