gp91 phox Function in VCAM-1-dependent Lung Eosinophilia

描述(申请人提供)：哮喘反应是由环境氧化剂和过敏原引起的。哮喘反应的一个组成部分是肺嗜酸性粒细胞增多症。嗜酸性粒细胞增多症合并哮喘是慢性阻塞性肺疾病死亡的危险因素。实验性哮喘患者肺内嗜酸性粒细胞的浸润依赖于内皮细胞上的黏附分子血管细胞黏附分子-1(VCAM-1)。我们发现了VCAM-1的一条信号转导途径。VCAM-1激活内皮细胞产生低水平的ROS的NADPH氧化酶。在VCAM-1依赖的白细胞体外迁移过程中，这些ROS是定位内皮细胞回缩所必需的。因此，VCAM-1不仅仅是白细胞迁移的支架，它还激活了内皮细胞的“门样”功能。我们建议在体内检测VCAM-1依赖的信号，并对VCAM-1的细胞质结构域进行结构/功能分析。为了测试这些VCAM-1信号在体内是否起作用，我们将检测实验性哮喘中VCAM-1依赖的嗜酸性粒细胞增多。我们推测，在实验性哮喘中，内皮细胞gp91Phox是肺嗜酸粒细胞增多所必需的。为了研究哮喘的发病机制，我们将使用NADPH氧化酶缺陷模型，该模型由带有野生型白细胞的嵌合小鼠和gp91Phox缺陷的非造血细胞组成。在特定的目标1-2中，我们将确定卵蛋白攻击的嵌合小鼠是否表现出肺组织的变化：1)白细胞浸润，2)调节嗜酸性粒细胞增多的黏附分子、细胞因子和趋化因子的表达，以及3)呼吸道高反应性。将确定是否使用转基因和过继细胞转移方法挽救内皮细胞gp91Phox介导的肺部炎症变化。为了研究VCAM-1胞浆区的结构/功能，我们将使用野生型和嵌合型VCAM-1分子对VCAM-1信号进行体外检测。VCAM-1的13个氨基酸的胞浆结构域在小鼠和人中是相同的，这表明它具有重要的功能。该结构域含有潜在的磷酸化位点。我们假设与VCAM-1结合的配体激活了VCAM-1胞浆结构域的磷酸化，从而刺激内皮细胞ROS的产生和内皮细胞肌动蛋白的重组。生化、药理学和遗传学方法将被用来解决这一假说。在目标3中，将确定VCAM-1胞浆区的丝氨酸和酪氨酸突变是否会改变VCAM-1对NADPH氧化酶的激活。在目标4中，将确定与VCAM-1结合的配体是否激活VCAM-1细胞质区域中丝氨酸和酪氨酸的磷酸化。VCAM-1调节肺嗜酸性粒细胞增多的机制的确定将为调节肺嗜酸性粒细胞增多提供新的见解，并为提出干预哮喘的依赖于VCAM-1的嗜酸性粒细胞增多成分提供基础。

DESCRIPTION (provided by applicant): Asthmatic responses are induced by environmental oxidants and allergens. A component of the asthmatic response is lung eosinophilia. Eosinophilia with asthma is a risk factor for mortality from chronic obstructive pulmonary disease. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. We have discovered a signal transduction pathway for VCAM-1. VCAM-1 activates endothelial cell NADPH oxidase production of low levels of reactive oxygen species (ROS). These ROS are required for localized endothelial cell retraction during VCAM- 1-dependent leukocyte migration in vitro. Thus, VCAM-1 is not simply a scaffold for leukocyte migration but activates an endothelial cell 'gate-like' function. We propose to examine VCAM-1-dependent signals in vivo and structure/function analysis of the cytoplasmic domain of VCAM-1. To test whether these VCAM-1 signals function in vivo, VCAM-1-dependent eosinophilia in experimental asthma will be examined. We hypothesize that in experimental asthma, endothelial cell gp91 phox is required for lung eosinophilia. To study the pathogenesis of asthma, we will use a NADPH oxidase deficient model consisting of chimeric mice with wild type leukocytes and gp91 phox deficient nonhematopoietic cells. In specific aims 1-2, we will determine whether ovalbumin-challenged chimeric mice exhibit alterations in lung 1) leukocyte infiltration, 2) expression of adhesion molecules, cytokines and chemokines that regulate eosinophilia, and 3) airway hyperresponsiveness. It will be determined whether endothelial cell gp91 phox-mediated changes in the lung inflammation are rescued using transgenic and adoptive cell transfer approaches. To examine the structure/function of the cytoplasmic domain of VCAM-1, VCAM-1 signals will be examined in vitro using wild type and chimeric VCAM-1 molecules. The 13 amino acid cytoplasmic domain of VCAM-1 is identical in mice and humans, suggesting that it has an important function. This domain contains potential phosphorylation sites. We hypothesize that ligand binding to VCAM-1 activates phosphorylation of the VCAM-1 cytoplasmic domain for stimulation of endothelial cell ROS generation and endothelial cell actin restructuring. Biochemical, pharmacologic and genetic approaches will be used to address this hypothesis. In aim 3, it will be determined whether mutations in serines and tyrosine in the cytoplasmic domain of VCAM-1 alters VCAM-1 activation of NADPH oxidase. In aim 4, it will be determined whether ligand binding to VCAM-1 activates phosphorylation of serines and tyrosine in the cytoplasmic domain of VCAM-1. The identification of mechanisms for VCAM-1 modulation of lung eosinophilia will provide new insights into regulation of lung eosinophilia as well as provide a basis towards proposing interventions in the VCAM-1-dependent eosinophilia component of asthma.