Macrophage Infiltration of Renal Tissue via beta-3 Integrins and Agrin

描述(申请人提供)：肾小球肾炎是最常见的一种自身免疫诱导的炎症事件，巨噬细胞的肾脏浸润导致肾功能丧失，原因是纤维化。我们实验室的长期目标是确定控制巨噬细胞黏附的分子开关，从而调节炎症反应。我们以前发现23个整合素黏附受体的酪氨酸磷酸化是一个这样的开关，并已经证明23磷酸化是巨噬细胞与某些配体黏附所必需的。由于肌动蛋白细胞骨架不能组织成应力纤维，阻断23磷酸化会导致粘附力的丧失。在本申请中，我们提出的证据表明，这种独特的机制是巨噬细胞进入肾组织所必需的。我们假设巨噬细胞通过肾基底膜是通过蛋白多糖集聚蛋白和磷酸化的23整合素之间的相互作用。在这项应用中，我们将评估23个整合素及其磷酸化在实验性肾小球肾炎向肾组织迁移中的作用。我们将通过体外迁移研究来确定肾基底膜上的集聚蛋白是否调节巨噬细胞的渗透。我们将通过活巨噬细胞的TIRF显微镜来表征巨噬细胞足体粘连和伪足的产生，这些是迁移到三维组织中所必需的。我们将从生物化学的角度确定磷酸化23的信号，它控制着巨噬细胞伪足和足体中肌动蛋白的细胞骨架重组。识别器官特异性巨噬细胞免疫转运过程中使用的机制可以为治疗干预提供新的靶点，并补充全球免疫抑制疗法。

DESCRIPTION (provided by applicant): Glomerulonephritis is most commonly an autoimmune-induced inflammatory event wherein infiltration of the kidney by macrophages results in loss of renal function due to fibrosis. The long-term goal of our laboratory is to identify the molecular switches that govern macrophage adhesion and thus regulate the inflammatory response. We previously identified tyrosine phosphorylation of 23 integrin adhesion receptors as one such switch and have demonstrated that 23 phosphorylation is necessary for macrophage adhesion to some ligands. Blockade of 23 phosphorylation results in loss of adhesion due to a failure of the actin cytoskeleton to organize into stress fibers. In this application we present evidence that this unique mechanism is required for macrophage entry into renal tissues. We hypothesize that macrophage passage through renal basement membrane is mediated by an interaction between the proteoglycan agrin and phosphorylated 23 integrins. In this application we will assess the role of 23 integrins and their phosphorylation in migration to renal tissues during experimental glomerulonephritis. We will determine whether agrin in renal basement membrane regulates macrophage infiltration by in vitro transmigration studies. We will characterize the production of macrophage podosomal adhesions and pseudopods that are required for migration into three-dimensional tissues by TIRF microscopy of living macrophages. We will biochemically determine the signaling from phosphorylated 23 that controls actin cytoskeletal reorganization in the macrophage pseudopod and podosome. Identification of mechanisms used during organ-specific macrophage immune trafficking can provide novel targets for therapeutic intervention and complement global immune-suppressive therapies.