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COPD恶化的风险。超细颗粒物也可以携带表面自由基，单独或与臭氧协同作用可以产生这种效果。氧化性气体如超生理氧气和臭氧导致肺细胞释放细胞外ATP，激活关键的生存信号。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PI 3激酶依赖性囊泡胞吐作用而出现，在极化支气管上皮中，主要来自顶面。急性臭氧暴露也会导致ATP释放到肺上皮细胞衬里液在小鼠在15分钟内。在体外，细胞外ATP的酶促去除增加细胞死亡，而ATP，DTP，或不可水解的ATP类似物防止臭氧诱导的细胞凋亡和坏死。保护性激动剂及其被P2和P2 Y受体特异性拮抗剂抑制表明P2 Y受体的作用。细胞外ATP激活ERK 1/2和Akt信号传导。除了上皮损伤，臭氧还导致细胞因子如白细胞介素-8（IL-8）的释放。IL-8甚至在细菌感染之前出现在CF气道中，是最早鉴定的病理事件。我们假设，细胞外ATP保持上皮细胞的能量代谢，气道细胞的存活，并抑制IL-8的释放，由于正常臭氧，受损的ATP释放CF气道引起相反的效果，补充细胞外ATP或类似的激动剂将扭转这些过程CF和正常。我们的具体目标是：（1）测量CF和非CF人气道上皮中响应臭氧的ATP释放、IL-8释放和细胞死亡，（2）定量CFTR突变和非突变小鼠气道中臭氧介导的ATP释放、炎性细胞因子释放和细胞损伤/死亡，（3）评估体外和体内臭氧中CF和非CF气道上皮中ATP、DTP和ATP类似物补充的作用，并确定激活的信号通路和相关的代谢效应。将使用在具有气液界面的可渗透支持物上培养的原代和转化CF突变体和正常气道上皮的极化单层。这些研究将增加我们对CF和正常人肺氧化损伤和修复以及气道上皮正常防御机制的理解。

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.