gp91 phox Function in VCAM-1-dependent Lung Eosinophilia

描述（由申请人提供）：哮喘反应由环境氧化剂和过敏原诱导。哮喘反应的一个组成部分是肺嗜酸性粒细胞增多。嗜酸性粒细胞增多症伴哮喘是慢性阻塞性肺疾病死亡的危险因素。实验性哮喘中嗜酸性粒细胞向肺内的浸润依赖于内皮细胞上的粘附分子血管细胞粘附分子-1（VCAM-1）。我们已经发现了VCAM-1的信号转导途径。VCAM-1激活内皮细胞NADPH氧化酶产生低水平的活性氧（ROS）。这些ROS是体外VCAM- 1依赖性白细胞迁移过程中局部内皮细胞收缩所必需的。因此，VCAM-1不仅是白细胞迁移的支架，而且激活了内皮细胞的“门样”功能。我们建议检查VCAM-1依赖的信号在体内和VCAM-1的胞质结构域的结构/功能分析。为了测试这些VCAM-1信号是否在体内起作用，将检查实验性哮喘中的VCAM-1依赖性嗜酸性粒细胞增多症。我们假设在实验性哮喘中，内皮细胞gp 91 phox是肺嗜酸性粒细胞增多所必需的。为了研究哮喘的发病机制，我们将使用由野生型白细胞和gp 91 phox缺陷型非造血细胞嵌合小鼠组成的NADPH氧化酶缺陷型模型。在具体目标1-2中，我们将确定卵清蛋白攻击的嵌合小鼠是否表现出肺1）白细胞浸润，2）调节嗜酸性粒细胞增多的粘附分子、细胞因子和趋化因子的表达，和3）气道高反应性的改变。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gp 91 phox介导的肺部炎症变化。为了检查VCAM-1的胞质结构域的结构/功能，将使用野生型和嵌合VCAM-1分子在体外检查VCAM-1信号。VCAM-1的13个氨基酸的胞质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。我们假设配体与VCAM-1的结合激活了VCAM-1胞质结构域的磷酸化，以刺激内皮细胞ROS的产生和内皮细胞肌动蛋白的重组。生物化学，药理学和遗传学的方法将被用来解决这个假设。在目的3中，将确定VCAM-1的胞质结构域中的丝氨酸和酪氨酸的突变是否改变NADPH氧化酶的VCAM-1活化。在目的4中，将确定与VCAM-1结合的配体是否激活VCAM-1的胞质结构域中丝氨酸和酪氨酸的磷酸化。鉴定VCAM-1调节肺嗜酸性粒细胞增多的机制将为肺嗜酸性粒细胞增多的调节提供新的见解，并为提出哮喘VCAM-1依赖性嗜酸性粒细胞增多组分的干预措施提供基础。

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.