LPS SIGNAL TRANSDUCTION IN NEUTROPHILS

描述 （改编自申请人的摘要）刺激中性粒细胞 脂多糖（LPS）是脓毒症发病机制的核心， 成人呼吸窘迫综合征。 LPS 与细胞表面 CD14 结合 蛋白质，导致肌动蛋白组装和粘附。 细胞内的 将 CD14 的 LPS 结合与功能性联系起来的信号通路 细胞反应很大程度上是未知的。 基于化学引诱剂的研究 刺激中性粒细胞和哺乳动物细胞系中的信号事件， 现在存在研究人类细胞内信号转导的框架 通过 LPS 检测中性粒细胞。 存在几个细胞内蛋白质家族 在哺乳动物细胞中，其功能是磷酸化其他蛋白质。 这些 激酶在磷酸化之前处于失活状态，然后能够 依次磷酸化并激活特定的“下游”激酶。 的 申请人假设 LPS 与 CD14 结合后，一个或多个 非受体蛋白酪氨酸激酶 (NRPTK) 被激活。 通过一个 信号通过的一系列连续磷酸化反应 丝裂原激活蛋白 (MAP) 激酶级联的一个分支。 的成员 MAP/ERK 激酶激酶 (MEKK) 家族被激活，进而 磷酸化并激活 MAP/ERK 激酶 (MEK) 家族的成员 然后磷酸化并激活 MAP 激酶家族的成员。 他们最近报道说，MAP 激酶在人类体内被激活 中性粒细胞对 LPS 的反应是 p38 MAP 激酶。 在本提案中，委员 中性粒细胞响应中所利用的 MEK、MEKK 和 NRPTK 家族 LPS刺激将被识别。 这些激酶将被分离 阴离子交换色谱法，测定对 LPS 的激活反应， 并通过特异性抗体和蛋白质测序进行鉴定。 同时，这些信号事件将在小鼠中进行探索 中性粒细胞。 信号蛋白在所有信号蛋白中都具有高度的同源性 哺乳动物细胞，并且预计所利用的信号传导机制 小鼠和人类的中性粒细胞几乎相同。 LPS之间的联系 CD14 的结合、细胞内信号传导以及随后的功能 然后将通过使用特定的抑制剂来建立反应 p38 MAP 激酶、转基因小鼠品系和小鼠模型 肺部中性粒细胞积聚。 对该信号的综合研究 转导途径可能会导致改变 LPS 诱导的新方法 炎症反应。 （摘要结束）

DESCRIPTION (Adapted from applicant's abstract) Stimulation of neutrophils by lipopoly-saccharide (LPS) is central to the pathogenesis of sepsis and the Adult Respiratory Distress Syndrome. LPS binds to CD14, a cell surface protein, resulting in actin assembly and adhesion. The intracellular signaling pathway that links the LPS binding of CD14 with functional cellular responses is largely unknown. Based on studies of chemoattractant stimulation of neutrophils and signaling events in mammalian cell lines, a framework now exists to study intracellular signal transduction in human neutrophils by LPS. Several families of intracellular proteins are present in mammalian cells that function to phosphorylate other proteins. These kinases are inactive until phosphorylated, and then are capable of phosphorylating and activating a specific "downstream" kinase in turn. The applicants hypothesize that following LPS binding to CD14, one or more nonreceptor protein tyrosine kinases (NRPTKs) are activated. Through a series of sequential phosphorylation reactions the signal is passed through a branch of the mitogen-activated protein (MAP) kinase cascade. A member of the MAP/ERK kinase kinase (MEKK) family is activated, which in turn phosphorylates and activates a member of the MAP/ERK kinase (MEK) family which then phosphorylates and activates a member of the MAP kinase family. They have recently reported that the MAP kinase activated in human neutrophils in response to LPS is p38 MAP kinase. In this proposal members of the MEK, MEKK, and NRPTK-families utilized by the neutrophil in response to LPS stimulation will be identified. These kinases will be isolated by anion-exchange chromatography, assayed for activation in response to LPS, and identified by specific antibodies and protein sequencing. Simultaneously, these signaling events will be explored in murine neutrophils. Signaling proteins share a high degree of homology in all mammalian cells, and it is expected that signaling mechanisms utilized by murine and human neutrophils will be nearly identical. The link between LPS binding of CD14, intracellular signaling, and subsequent functional responses will then be established through the use of a specific inhibitor to p38 MAP kinase, genetically modified mice strains, and murine models of pulmonary neutrophil accumulation. The comprehensive study of this signal transduction pathway may lead to new approaches for modifying LPS induced inflammatory responses. (End of abstract)