Eosinophil integrins: interactions with VCAM and fibronectin

(申请人摘要)嗜酸性粒细胞(EOS)优先聚集在 哮喘患者的呼吸道与呼吸道高反应性有关 和气流阻塞。土壤EOS选择性回收的机制 哮喘可能包括：(A)启动循环中的EOS；(B)局部上调 IL-4、IL-13、Th-2细胞因子对血管细胞黏附分子-1(VCAM)的影响； (C)启动循环EOS在表达VCAM的肺组织上的滚动和停滞 内皮细胞；(D)受阻的EOS向组织内的迁移 趋化因子或其他G蛋白偶联受体激动剂；(E) 尚未定义的趋化、趋化和趋化事件 Eos与VCAM和纤维连接蛋白(Fn)在支气管壁相互作用；(F) EOS进入气道腔的运动；和(G)延长EOS的存活期 GM-CSF驱动的气道壁和管腔。Eos表达2个整合素， 与VCAM和FN、Alpha4beta1和Alpha4beta7交互，以及第三， αDbeta2，与VCAM相互作用，但不与Fn相互作用。VCAM和FN各自以 几种不同的拼接形式。我们假设拼接 变异改变了VCAM和FN对整合素的特异性和价态 以及EOS对整合素结扎的反应。我们的目标是 两重。首先，我们将确定字母4β1如何相互作用， 具有VCAM和FN的差异剪接形式的α4beta7和alphaDbeta2 调节EOS的黏附、迁移和活性。第二，我们将确定 不同形态VCAM和FN在正常及哮喘患者体内的分布 航空公司。为了实现这些目标，我们将(1)表达差异拼接 EOS在输血过程中可能遇到的VCAM和FN的形式 (2)制备复制HSP70表达模式的细胞系 血液和呼吸道EOS上的整合素，并作为EOS的模型，从 哮喘发作前后的循环和肺；(3) 表征血液EOS、呼吸道EOS和模型细胞的能力 与VCAM和FN的剪接形式在结合、黏附、迁移和 生存分析；(4)表征细胞因子和gpr激动剂的作用。 关于整合素与配体的相互作用；以及(5)制备抗整合素-配体相互作用的单抗 VCAM和FN的不同剪接形式并确定其在 正常肺和哮喘肺。

(Applicant's Abstract) Eosinophils (EOS) preferentially accumulate in the airways of patients with asthma and contribute to airway hyperresponsiveness and airflow obstruction. The mechanisms for selective recuitment of EOS in asthma likely include: (a) priming of circulating EOS; (b) local upregulation of vascular cell adhesion molecule-1 (VCAM) by IL-4 and IL-13 Th-2 cytokines; (c) rolling and arrest of primed circulating EOS on VCAM-expressing pulmonary endothelial cells; (d) migration of arrested EOS into tissues in response to chemokines or other agonists of G-protein coupled receptors (GPCRs); (e) yet-to-be-defined chemotactic, chemokinetic, and chemostatic events in which EOS interact with VCAM and fibronectin (FN) in the bronchial wall; (f) movement of EOS into the airway lumen; and (g) prolonged survival of EOS of the airway wall and lumen driven by GM-CSF. EOS express 2 integrins that interact with VCAM and FN, alpha4beta1 and alpha4beta7, and a third, alphaDbeta2, that interacts with VCAM but not FN. VCAM and FN each exist as several differentially spliced forms. We hypothesize that the splice variations change the specificities and valencies of VCAM and FN for integrins on EOS and hence the responses of EOS to integrin ligation. Our aims are twofold. First, we will establish how interactions of alpha4beta1, alpha4beta7, and alphaDbeta2 with differentially spliced forms of VCAM and FN regulate adhesion, migration, and viability of EOS. Second, we will determine the distribution of various forms of VCAM and FN in normal and asthmatic airways. To accomplish these aims, we will (1) express differentially spliced forms of VCAM and FN that likely are encountered by EOS in transit from blood to airway; (2) prepare cell lines that replicate the expression patterns of integrins on blood and airway EOS and serve as models for EOS obtained from the circulation and lung before and after an asthma exacerbation; (3) characterize the abilities of blood EOS, airway EOS, and model cells to interact with splice forms of VCAM and FN in binding, adhesion, migration, and survival assays; (4) characterize the effects of cytokines and GPCR agonists on integrin-ligand interactions; and (5) prepare monoclonal antibodies to differentially spliced forms of VCAM and FN and determine distribution in normal and asthmatic lung.