Lung epithelial cell survival signaling in ozone in cystic fibrosis and normals

描述(由申请人提供)：氧化性空气污染物，如臭氧，会增加囊性纤维化、哮喘和慢性阻塞性肺病恶化的风险。也可以承载表面自由基的超细颗粒，无论是单独还是与臭氧协同作用，都可以产生这样的效果。超生理氧气和臭氧等氧化性气体会导致肺细胞释放细胞外ATP，激活关键的生存信号。由臭氧(50-200 ppb)引起的迅速、可逆的ATP释放发生在气管、支气管、小气道和肺泡上皮细胞中，由于钙、Rho激酶和PI3-激酶依赖的囊泡胞吐而出现，在极化的支气管上皮中，主要来自顶端表面。急性臭氧暴露还可导致小鼠在15分钟内将ATP释放到肺上皮衬底液中。在体外，酶去除细胞外的ATP会增加细胞的死亡，而ATP、DTP或非水解性的ATP类似物可以防止臭氧诱导的细胞凋亡和坏死。保护性激动剂，以及它们被P2和P2Y受体特异性拮抗剂抑制，表明了P2Y受体的作用。胞外ATP激活ERK1/2和Akt信号。除了上皮损伤外，臭氧还会导致白细胞介素8(IL-8)等细胞因子的释放。IL-8甚至在细菌感染之前就已经在CF呼吸道中出现，是最早被发现的病理事件。我们假设，在正常人中，细胞外的ATP保护上皮细胞的能量代谢、呼吸道细胞的存活并抑制臭氧引起的IL-8的释放，而细胞外的ATP的损伤释放会导致相反的效果，补充细胞外的ATP或类似的激动剂将逆转在CF和正常人中的这些过程。我们的具体目标是：(1)检测CFTR突变和非突变小鼠气道上皮细胞对臭氧的反应，释放ATP、IL-8和细胞死亡；(2)定量检测臭氧介导的突变和非突变小鼠气道ATP释放、炎性细胞因子释放和细胞损伤/死亡；(3)评估在体外和体内补充ATP、DTP和ATP类似物对CFR和非CFF呼吸道上皮细胞的影响，并确定激活的信号通路和相关的代谢效应。将使用培养在具有气液界面的可渗透支架上的原代和转化的CF突变和正常呼吸道上皮的偏振单分子层。这些研究将增加我们对肺组织氧化损伤和修复的了解，以及对呼吸道上皮正常防御机制的了解。

DESCRIPTION (provided by applicant): Oxidant air pollutants like ozone increase risk for exacerbation of cystic fibrosis, asthma, and COPD. Ultrafine particulates, which also can bear surface free radicals, can have such effects, alone or in synergy with ozone. Oxidant gases like supraphysiologic oxygen and ozone cause extracellular ATP release from lung cells, activating critical survival signals. Prompt, reversible ATP release due to ozone (50-200 ppb) occurs in tracheal, bronchial, small airway, and alveolar epithelial cells, appears due to calcium-, rho kinase-, and PI 3-kinase-dependent vesicular exocytosis, and, in polarized bronchial epithelium, is mainly from the apical surface. Acute ozone exposure also causes ATP release into lung epithelial lining fluid in mice within 15 min. In vitro, enzymatic removal of extracellular ATP increases cell death, while ATP, DTP, or a nonhydrolyzable ATP analog prevent ozone-induced apoptosis and necrosis. The protecting agonists, and their inhibition by P2 and P2Y receptor-specific antagonists, indicate a role for P2Y receptors. Extracellular ATP activates ERK 1/2 and Akt signaling. Besides epithelial injury, ozone causes release of cytokines like interleukin-8 (IL-8). IL-8 appears in CF airways even before bacterial infection, being the earliest identified pathologic event. We hypothesize that extracellular ATP preserves epithelial energy metabolism, airway cell survival and inhibits IL-8 release due to ozone in normals, that impaired ATP release in CF airways causes opposite effects, and that supplemental extracellular ATP or similar agonists will reverse these processes in CF and normals. Our specific aims are: (1) measure ATP release, IL-8 release, and cell death in CF and non-CF airway human epithelium in response to ozone, (2) quantitate ozone-mediated ATP release, inflammatory cytokine release, and cell injury/death in airways of CFTR- mutant and non-mutant mice, (3) assess effects of ATP, DTP and ATP analog supplementation in CF and non-CF airway epithelium in ozone in vitro and in vivo, and determine activated signaling pathways and related metabolic effects. Polarized monolayers of primary and transformed CF-mutant and normal airway epithelium cultured on permeable supports with an air-liquid interface will be used. These studies will increase our understanding of lung oxidant injury and repair in CF and normals, and of normal defense mechanisms of airway epithelium.