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

描述（由申请人提供）：臭氧等氧化性空气污染物增加囊性纤维化、哮喘和COPD恶化的风险。超细颗粒物也能承受表面自由基，它们可以单独或与臭氧协同产生这样的效果。超生理氧和臭氧等氧化气体导致肺细胞释放细胞外ATP，激活关键的生存信号。臭氧引起的ATP快速可逆释放（50-200 ppb）发生在气管、支气管、小气道和肺泡上皮细胞中，通过钙、rho激酶和PI 3激酶依赖的囊性胞吐出现，并且在极化支气管上皮中，主要来自顶端表面。急性臭氧暴露也导致ATP在15分钟内释放到小鼠肺上皮衬里液中。在体外，酶去除细胞外ATP会增加细胞死亡，而ATP、DTP或一种不可水解的ATP类似物可以防止臭氧诱导的细胞凋亡和坏死。这些保护激动剂及其对P2和P2Y受体特异性拮抗剂的抑制作用表明它们对P2Y受体起作用。细胞外ATP激活ERK 1/2和Akt信号。除上皮损伤外，臭氧还会引起白细胞介素-8 （IL-8）等细胞因子的释放。IL-8甚至在细菌感染之前就出现在CF气道中，是最早发现的病理事件。我们假设在正常情况下，细胞外ATP保持上皮能量代谢、气道细胞存活并抑制臭氧导致的IL-8释放，而在CF气道中受损的ATP释放会导致相反的效果，补充细胞外ATP或类似的激动剂将逆转CF和正常情况下的这些过程。我们的具体目标是：(1)测量臭氧对CF和非CF气道上皮中ATP释放、IL-8释放和细胞死亡的响应；(2)定量测定臭氧介导的CFTR-突变和非突变小鼠气道中ATP释放、炎症细胞因子释放和细胞损伤/死亡；(3)评估体外和体内臭氧条件下CF和非CF气道上皮中ATP、DTP和ATP类似物补充的影响，确定激活的信号通路和相关的代谢作用。将使用在具有气液界面的可渗透支架上培养的原代和转化的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的理解，以及气道上皮的正常防御机制。

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.