Mechanisms of Leukocyte Integrin Signaling

描述(申请人提供)：在炎症性疾病，如类风湿性关节炎和血管炎中，不受控制的中性粒细胞黏附和激活显著地导致组织损伤。整合素是中性粒细胞上调节细胞活化的主要黏附受体。因此，研究中性粒细胞整合素信号转导的基本机制对于我们理解炎症性疾病的发病机制至关重要。这一应用主要集中在诱导中性粒细胞和巨噬细胞激活的整合素信号通路上。在以前的工作中，我们已经证明了由外向内整合素信号是由Src家族和Syk酪氨酸激酶介导的。反过来，这些酶向SLP-76发出信号，导致MAP激酶的下游激活。Src家族激酶、Syk和SLP-76的顺序参与使人想起经典的免疫受体信号通路，如B细胞或T细胞受体通路，这依赖于含有ITAM的适配蛋白。在初步数据中，我们发现来自DAP-12-/-FcRGamma-/-小鼠的中性粒细胞缺乏髓系细胞中存在的两个主要的ITAM适配器，在中性粒细胞整合素信号转导方面也完全缺陷。基于这些观察，我们假设白细胞外-整合素信号通路模仿经典的免疫受体信号通路。我们将通过一系列的遗传和生化实验来检验这一假设。利用逆转录病毒介导的胎肝造血干细胞转导，我们将把一系列Syk和DAP-12的突变版本引入SYK-/-或DAP-12-/-FcRGamma-/-小鼠，以测试ITAM-Syk相互作用是否需要初级中性粒细胞和巨噬细胞的整合素信号。我们还将使用生化方法和基于FRET的显微镜技术来测试Beta2整合素是否与含有ITAM的DAP-12和Syk形成复合体。如果Syk和DAP12/FcRGamma在由外向内的整合素信号转导中起关键作用，那么这些分子的缺失应该会导致体内炎症过程中同样的缺陷。我们将使用两个已知的整合素依赖的疾病模型来测试这一点。如果我们的假设得到证实，整合素外-内信号通过使用ITAM分子模拟免疫受体通路，这将显著改变对中性粒细胞依赖炎症中整合素功能的看法。整合素由外向内信号通路的治疗靶向可能提供新型抗炎药物。

DESCRIPTION (provided by applicant): Uncontrolled neutrophil adhesion and activation contributes to significantly to tissue damage in inflammatory diseases such as rheumatoid arthritis and vasculitis. Integrins are the major adhesive receptors on neutrophils that regulate cellular activation. Investigating the basic mechanisms of neutrophil integrin signaling is therefore critical to our understanding of the pathogenesis of inflammatory disease. This application is focused on the "outside-in" integrin signaling pathway that induces neutrophil and macrophage activation. In previous work, we have demonstrated that outside-in integrin signaling is mediated by Src-family and Syk tyrosine kinases. In turn, these enzymes signal to SLP-76 leading to downstream activation of MAP kinases. The sequential involvement of Src-family kinases, Syk and SLP-76 is reminiscent of classical immunoreceptor signaling, such as B or T-cell receptor pathways, which depends on ITAM-containing adapter proteins. In preliminary data, we have found that neutrophils derived from DAP-12-/- FcRgamma-/- mice, which lack the two major ITAM adapters present in myeloid cells, are also completely defective in neutrophil integrin signaling. Based on these observations we hypothesize that leukocyte outside-in integrin signaling mimics classical immunoreceptor signaling pathways. We will test this hypothesis is a series of genetic and biochemical experiments. Using retroviral-mediated fetal liver hematopoietic stem cell transduction, we will introduce a series of mutant versions of Syk and DAP-12 into syk-/- or DAP-12-/- FcRgamma-/- mice that will test whether ITAM-Syk interactions are required for integrin signaling in primary neutrophils and macrophages. We will also test whether beta2 integrins form a complex with ITAM-containing DAP-12 and Syk, using biochemical methods and FRET-based microscopy techniques. If Syk and DAP12/FcRgamma are critical in outside-in integrin signaling, then deficiency of these molecules should result in equivalent defects in inflammatory processes in vivo. We will test this using two disease models that are known to be integrin dependent. If our hypothesis that integrin outside-in signaling mimics immunoreceptor pathways by use of ITAM molecules is validated, this will significantly change the view of integrin function in neutrophil-dependent inflammation. Therapeutic targeting of the integrin outside-in signaling pathway may provide novel classes of anti-inflammatory drugs.