Roles of N-glycans on neutrophil beta2 integrins in progression of acute lung injury

项目总结 急性肺损伤(ALI)及其严重形式--急性呼吸窘迫综合征(ARDS)是 呼吸衰竭的常见原因。尽管有所有的医学创新，ARDS的治疗仍然是一个悬而未决的问题 有问题。中性粒细胞是ALI期间第一批渗入肺内的免疫细胞，中性粒细胞在肺内释放 蛋白水解酶、细胞因子和氧化剂，以杀死入侵的微生物，以及损伤肺部。临床资料和 动物模型已经证明，中性粒细胞的募集和功能影响ALI的进展。因此， 对微调的中性粒细胞募集和功能的洞察可能为治疗提供新的方法 阿里的。 整合素b2在调节中性粒细胞募集和功能中起着重要作用。 在炎症部位，细胞内信号上调整合素配体结合亲和力，从而允许配体 来捆绑。在高亲和力状态下，与整合素b2结合的配体可以抑制中性粒细胞，这是 中性粒细胞募集，以及导致整合素簇形成(即整合素价态)以增强中性粒细胞 逮捕。目前，细胞内信号转导是调节整合素配体结合的唯一途径 能力。整合素b2的胞外区被N-连接的糖链修饰。然而，N-是否以及如何- 葡聚糖调控整合素b2-配体结合的研究尚未见报道。此外，先进的受体 肺泡上皮细胞I型糖基化终末产物(RAGE)是炎症的主要介质 肺部的反应。中性粒细胞上的整合素aMb2是RAGE的配基，然而，相互作用的作用 整合素aMb2与RAGE在ALI炎症进展中的作用尚不清楚。 我们的初步研究发现，去除N-糖链会增加整合素aLb2介导的中性粒细胞 黏附和配体结合诱导中性粒细胞反应。此外，去除唾液酸 增加了aMb2对愤怒的绑定。因此，我们假设1)胞外区的N-糖链可以防止 整合素aLb2采用高亲和力状态和/或限制整合素簇的形成；和2)减少 整合素aMb2的糖基化促进其与RAGE的结合，从而可能增加炎症反应 肺部。因此，我们提出了两个目标来检验假设：目标1：确定整合素aLb2上的N-糖链 负调控其配体结合亲和力和价态；以及目标2：确定aMb2上的糖链是否负向 调节RAGE介导的AT1细胞炎症反应。N-糖链上含有整合素b2的突变 承载部位，一种测量整合素亲和力状态的新工具，AT1细胞和ALI的小鼠模型将被 用来检验我们的假设。我们建议的研究将破译N-糖链对中性粒细胞b2整合素的作用 在ALI的发病机制中发挥重要作用，这可能为治疗ALI提供新的治疗方法。

PROJECT SUMMARY Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are the common cause of respiratory failure. Despite all medical innovations, treatment of ARDS remains an unsolved problem. Neutrophils are the first immune cells infiltrated into lungs during ALI, where neutrophils release proteinases, cytokines, and oxidants to kill invading microbes, as well as to injure lungs. Clinical data and animal models have proved that neutrophil recruitment and function influences the progression of ALI. Thus, insights into the fine-tuned neutrophil recruitment and function may provide a novel approach for the treatment of ALI. Integrin b2 plays an important role in the regulation of neutrophil recruitment and neutrophil functions. At inflamed sites, the intracellular signaling upregulates the integrin ligand-binding affinity, which allows ligands to bind. Ligand binding to integrin b2 in the high-affinity state arrests neutrophils, a prerequisite step for neutrophil recruitment, as well as causes integrin cluster formation (i.e. integrin valency) to enhance neutrophil arrest. Currently, the intracellular signaling is the only approach to regulate the integrin ligand-binding capability. The extracellular domain of integrin b2 is decorated with N-linked glycans. Yet, whether and how N- glycans regulate the integrin b2-ligand binding has not been studied. In addition, the receptor for advanced glycation end-products (RAGE) on alveolar epithelial cell type I (AT1) is a major mediator for inflammatory responses in lungs. Integrin aMb2 on neutrophils is a ligand for RAGE, however, the role of the interaction of integrin aMb2 with RAGE in the progression of inflammation in ALI is unknown. Our preliminary studies found that removing N-glycans increased integrin aLb2-mediated neutrophil adhesion and ligand binding-induced neutrophil cellular responses. Furthermore, removing sialic acids increased aMb2 binding to RAGE. Thus, we hypothesize that 1) N-glycans on the extracellular domain prevent integrin aLb2 to adopt the high-affinity state and/or limit the integrin cluster formation; and 2) reduced glycosylation of integrin aMb2 facilitates its binding to RAGE that may increase inflammatory responses in lungs. Thus, we propose two Aims to test the hypotheses: Aim 1: Determine if N-glycans on integrin aLb2 negatively regulate its ligand-binding affinity and valency; and Aim 2: Determine if glycans on aMb2 negatively regulate RAGE-mediated inflammatory responses in AT1 cells. Integrin b2 containing mutations on N-glycan bearing sites, a novel tool to measuring integrin affinity states, AT1 cells, and mouse models of ALI will be used to test our hypotheses. Our proposed study will decipher the role of N-glycans on neutrophil b2 integrins in the pathogenesis of ALI, which may lead to the development of a new therapy for ALI.