Cytosolic myeloid related protein 8/14 (cytosolic MRP8/14) is a critical regulator of beta2 integrin outside-in signaling during leukocyte recruitment in vivo

髓样相关蛋白8/14（MRP 8/14，S100 A8/A9，Calprotectin）属于钙离子结合蛋白S100家族。释放后，该蛋白质作为具有有效免疫调节特性的警报蛋白发挥作用。细胞外MRP 8/14水平升高存在于广泛的炎性疾病中，如炎性肠病、类风湿性关节炎、皮炎或心血管疾病，从而使该分子有资格作为临床中可靠的血清生物标志物。最近，我们能够证明中性粒细胞与发炎内皮的相互作用诱导MRP 8/14释放，导致β 2整联蛋白的依赖性激活，导致白细胞缓慢滚动和白细胞牢固粘附在发炎内皮上。关于MRP 8/14在中性粒细胞中的细胞内功能知之甚少，尽管二聚体占这些细胞中胞浆蛋白含量的40%。然而，一些研究提出了一个额外的细胞内功能的蛋白质，这是独立于其细胞外的作用，作为一个报警。迄今为止发表的数据表明胞质MRP 8/14在质膜和细胞骨架的Ca 2+依赖性相互作用中的作用。细胞骨架重排是白细胞募集过程中白细胞停滞后修饰的先决条件，因此对于有效的白细胞迁移是必不可少的。在这个项目中，我们的目的是确定细胞内的MRP 8/14在体内白细胞募集的功能。基于我们的初步研究结果，我们假设胞质MRP 8/14在所谓的“由外向内”信号传导中的作用，导致有缺陷的粘附加强，从而导致从滚动到牢固粘附的有缺陷的过渡。为了证明这一假设，我们将使用TNF-α诱导的小鼠提睾肌炎症模型进行体内实验，并使用C57 BL/6和Mrp 14-/-小鼠（功能性MRP 8/14双敲除小鼠）进行离体流动室测定。我们将通过不同的功能测定研究胞质MRP 8/14在由内向外信号传导和由外向内信号传导中的作用。最后，我们将阐明MRP 8/14依赖由外向内信号传导的潜在分子机制，通过可视化刺激依赖性粘附簇和Ca 2+定位，并通过分析C57 B1/6和Mrp 14-/-中性粒细胞中细胞内分子的磷酸化模式和GT3活化。

Myeloid related protein 8/14 (MRP8/14, S100A8/A9, Calprotectin) belongs to the family of Ca2+ binding S100 proteins. Upon release, the protein acts as an alarmin with potent immune modulatory properties. Elevated levels of extracellular MRP8/14 are present in a broad spectrum of inflammatory disorders, like inflammatory bowel disease, rheumatoid arthritis, dermatitis or cardio-vascular diseases, thereby qualifying the molecule as a reliable serum biomarker in the clinics. Recently, we were able to show that the interaction of neutrophils with inflamed endothelium induces MRP8/14 release, leading to dependent activation of beta2 integrins, resulting in slow leukocyte rolling and firm leukocyte adhesion on inflamed endothelium. Little is known about the intracellular function of MRP8/14 in neutrophils, although the dimer represents 40% of the cytosolic protein content in these cells. However, a few studies propose an additional intracellular function of the protein, which is independent on its extracellular role as an alarmin. The so far published data suggest a role of cytosolic MRP8/14 in in the Ca2+ dependent interplay of the plasma membrane and the cytoskeleton. Cytoskeletal rearrangement is a prerequisite for post arrest modifications in leukocytes during leukocyte recruitment and hence indispensable for efficient leukocyte migration. Within this project, we intent to identify the function of intracellular MRP8/14 in leukocyte recruitment in vivo. Based on our preliminary findings we assume a role of cytosolic MRP8/14 in the so-called “outside-in” signalling, leading to a defective adhesion strengthening and thus resulting in a defective transition from rolling to firm adhesion. In order to prove this hypothesis we will perform in vivo experiments using the TNF- induced inflammation model of the mouse cremaster muscle and ex vivo flow chamber assays utilizing C57BL/6 und Mrp14-/- mice (functional MRP8/14 double knock out mice). We will investigate the role of cytosolic MRP8/14 in inside-out signaling and outside-in signaling via different functional assays. Finally, we will elucidate the underlying molecular mechanism of MRP8/14 dependent outside-in signalling by visualizing stimulation dependent adhesion clusters and Ca2+ localization and by analyzing phosphorylation patterns of intracellular molecules and GTPase activation in C57Bl/6 and Mrp14-/- neutrophils.