Role of beta 2 integrins in macrophage fusion

 描述(申请人提供)：白细胞整合素αMβ2(Mac-1)在正常的保护性炎症反应和病理性炎症中发挥关键作用。它也是许多疾病的潜在治疗靶点，在这些疾病中，炎症起着至关重要的作用，包括心血管疾病。Mac-1的不同功能和活性源于它与多种配体结合的能力。我们最近发现了Mac-1的一项新功能。特别是，我们发现Mac-1参与了巨噬细胞的融合，这是许多疾病中慢性炎症的一个显著特征，包括动脉粥样硬化和其他血管病理。巨噬细胞融合需要特定的促炎环境，并导致形成多核巨细胞(MGCS)。虽然mGCs在19世纪中叶首先在结核样肉芽肿中被发现，此后在许多炎症性疾病中也被发现，但融合将两种膜结合在一起的分子机制仍不清楚。在我们的初步研究中，我们发现ICAM-1，Mac-1的反向受体，也促进巨噬细胞融合。然而，ICAM-1的缺失仅导致融合的部分减少，这不能解释Mac-1缺失的巨噬细胞中观察到的强烈的融合障碍。这些结果表明，与其他Mac-1配体相比，Mac-1参与了融合。我们发现巨噬细胞表面表达上调的受体MfR(巨噬细胞融合受体/Sirpα)与Mac-1相互作用。和ICAM-1一样，MFR是跨膜受体免疫球蛋白超家族的成员，也是参与巨噬细胞融合的分子之一。基于这一发现和一些初步数据，我们假设Mac-1是巨噬细胞融合所必需的，在此过程中它与MFR相互作用。具体目标1是检验这一假设。重组技术、蛋白质相互作用的研究、rna干扰、骨髓移植和组合多肽库将被用来阐明mac-1如何与mfr结合，并阐明这种相互作用在 核聚变。《特定目标2》将检验这一假说，即丝状伪足和微棘突是巨噬细胞融合的融合中间产物，而Mac-1是形成这些结构所必需的。这一假说是基于我们在巨噬细胞融合过程中发现了这些迄今未知的结构。使用高分辨率电子显微镜、共聚焦免疫荧光显微镜和TIRF的超微结构研究将用于表征这些融合中间体。具体目标3是验证这样一种假说，即mGCS分泌强大的弹性分解酶，有助于动脉粥样硬化的进展。在ApoE-/-和LDLR-/-小鼠以及与Mac-1杂交的小鼠中，将研究动脉粥样硬化斑块中mGCS的形成和Mac-1的作用。总体而言，这些研究将有助于加深对巨噬细胞融合的分子机制的了解，识别以前未考虑过的参与两层膜融合的结构，对Mac-1的生物学有新的见解，并可能揭示意外的治疗靶点。

 DESCRIPTION (provided by applicant): Leukocyte integrin αMβ2 (Mac-1) plays a pivotal role in normal protective inflammatory response and pathological inflammation. It is also a potential therapeutic target in many diseases in which inflammation plays an essential role, including cardiovascular diseases. The diverse functions and activities ascribed to Mac-1 arise from its ability to bind a multitude of ligands. We have recently discovered a new function of Mac-1. In particular, we found that Mac-1 is involved in macrophage fusion, a salient feature of chronic inflammation in many diseases, including atherosclerosis and other vascular pathologies. Macrophage fusion requires a specific proinflammatory environment and leads to the formation of multinucleated giant cells (MGCs). Although MGCs have been first discovered in the middle of the 19th century in tuberculoid granuloma and since then were found in numerous inflammatory diseases, the molecular mechanisms of fusion bringing two membranes together remain unknown. In our preliminary studies we found that ICAM-1, a counter-receptor of Mac-1, also promotes macrophage fusion. However, deficiency of ICAM-1 resulted only in a partial decrease of fusion which could not be accounted for the strong impairment of fusion observed in Mac-1- deficient macrophages. These results suggested than other Mac-1 ligands are involved in fusion. We have found that MFR (macrophage fusion receptor/SIRPα), a receptor upregulated on the surface of fusing macrophage, interacts with Mac-1. Like ICAM-1, MFR is a member of the immunoglobulin superfamily of transmembrane receptors and one of the molecules implicated in macrophage fusion. Based on this discovery and some preliminary data, we hypothesize that Mac-1 is required for macrophage fusion during which it interacts with MFR. Specific Aim 1 is to test this hypothesis. Recombinant techniques, studies of protein-protein interactions, RNA interference, bone marrow transplantations, and combinatorial peptide libraries will be used to elucidate how Mac-1 binds MFR and clarify the role of this interaction in fusion. Specific Aim 2 will test the hypothesis that filopodia and microspikes are fusion intermediates in macrophage fusion and that Mac-1 is required for the formation of these structures. The hypothesis is based on our discovery of these hitherto unknown structures during macrophage fusion. Ultrastructural studies using high-resolution electron microscopy, confocal immunofluorescence microscopy, and TIRF will be used to characterize these fusion intermediates. Specific Aim 3 is to test the hypothesis that MGCs, which secrete potent elastolytic enzymes, contribute to the progression of atherosclerosis. The formation of MGCs in atherosclerotic plaques and the role of Mac-1 will be examined in ApoE-/- and LDLR-/- mice and the mice crossbred with Mac-1. Overall, these studies will lead to an increased understanding of the molecular mechanisms of macrophage fusion, identify previously unconsidered structures involved in the fusion of two membranes, give new insights into the biology of Mac-1, and potentially reveal unsuspected therapeutic targets.