Transcellular Communication in Airway Inflammation and Airway Hyperresponsiveness

描述(申请人提供)：建议进行研究，以检测上皮细胞(EPI)、巨噬细胞(MO)和气道平滑肌细胞(ASMC)被激活以传递信号的机制，这些信号为粒细胞a)黏附/迁移，b)LTC4的产生，以及c)将正常反应的呼吸道转变为高反应的呼吸道。中心假说是，由刺激的EPI、MO或ASMC分泌的内源性14 kDa V组PLA2(GVPLA2)是一种细胞间信使蛋白，在哮喘人的呼吸道中高表达，并在小鼠EPI、MO和ASMC的免疫致敏过程中被诱导。推测gVPLA2通过一种不依赖于cPLA2激活的新机制，使嗜酸性粒细胞富含磷脂酰胆碱的外膜产生LTC4。在目标1中，研究旨在验证这一假设，即[32-整合素介导的嗜酸性粒细胞黏附是由gVPLA2通过一种不依赖于MAPK介导的cPLA2磷酸化的新机制来调节的。研究表明，从呼吸道停留细胞分泌的gVPLA2传递信号，诱导嗜酸性粒细胞中的(32-整合素)黏附。建议进一步研究，以阐明gVPLA2在生理条件下诱导黏附的潜在途径。目的2，研究内源性gVPLA2在跨细胞通讯过程中介导LTC4分泌增加的细胞进入和细胞内作用。进一步的研究是通过使用抗gVPLA2或TAT-dN-cPLA2的中和性mAb来测试gVPLA2的特异性。目的3，研究gVPLA2和cPLA2在急性和慢性哮喘小鼠模型中调节气道炎症和高反应性(AHR)的机制。在初步数据的基础上，提出了一项研究，以检验gVPLA2内源性分泌介导免疫致敏小鼠AHR的假设。我们将评估抗gVPLA2单抗MCL-3G1对乙酰甲胆碱或gVPLA2攻击前后小鼠AHR的阻断作用。将使用免疫致敏的gVPLA2基因敲除和cPLA2基因敲除小鼠进行进一步的研究，以确定这些酶在AHR和体内炎症细胞迁移中的特定作用。来自这些研究的数据应该阐明一种新的跨细胞通讯的内源性机制，该机制启动了呼吸道炎症和AHR。

DESCRIPTION (provided by applicant): Studies are proposed to examine the mechanisms by which epithelial cells (EPI), macrophages (MO) and airway smooth muscle cells (ASMC) are activated to communicate signals for granulocyte a) adhesion /migration, b) generation of LTC4, and c) conversion of normally reactive airways into hyperresponsive airways. The central hypothesis is that the endogenous 14 kDa group V PLA2 (gVPLA2) secreted from stimulated EPI, MO or ASMC serves as an intercellular messenger protein that is highly expressed in asthmatic human airways and is inducible during immune sensitization in mouse EPI, MO and ASMC. It is hypothesized that gVPLA2 hydrolyzes the phosphatidylcholine-rich outer plasma membrane of eosinophils to cause subsequent LTC4 secretion by a novel mechanism that is independent of cPLA2 activation. In Aim 1, studies are proposed to examine the hypothesis that [32-integrin-mediated eosinophil adhesion is regulated by gVPLA2 by a novel mechanism independent of MAPK-mediated cPLA2 phosphorylation. Studies are proposed to demonstrate that gVPLA2 secreted from airway resident cells transmits the signal for induction of (32-integrin adhesion in eosinophils. Further studies are proposed to elucidate the potential pathway by which adhesion is induced under physiological conditions by gVPLA2. In Aim 2, studies are proposed to examine the cellular entry and intracellular action of endogenous gVPLA2 in mediating augmented secretion of LTC4 during transcellular communication. Further studies are proposed to test the specificity of gVPLA2 by using a neutralizing mAb against gVPLA2 or TAT-dn-cPLA2. In Aim 3, studies are proposed to examine the mechanisms of gVPLA2 and cPLA2 in the mediation of airway inflammation and airway hyperresponsiveness (AHR) in acute and chronic murine models of asthma. Based upon preliminary data, studies are proposed to test the hypothesis that endogenous secretion of gVPLA2 mediates AHR in immune sensitized mice. The effect of MCL-3G1, a mAb against gVPLA2, in blocking AHR to methacholine or gVPLA2 challenge in pre/post OA-challenge mice will be assessed. Further studies using immune-sensitized gVPLA2-knockout and cPLA2-knockout mice will be generated to establish the specific role of these enzymes in AHR and inflammatory cell migration in vivo. Data derived from these studies should elucidate a new endogenous mechanism of transcellular communication that initiates airway inflammation and AHR.