Ozone ALTERS Airway Smooth Muscle FUNCTION IN ASTHMA

描述（由申请人提供）：尽管臭氧（O3）可诱导哮喘恶化、乙酰甲胆碱气道高反应性（AHR）和与TNF α和IL-1 β水平升高相关的嗜酸性气道炎症，但在哮喘和过敏原诱导的AHR中介导臭氧效应的机制仍不清楚。初步数据显示，臭氧和过敏原的组合显着增加气道中性粒细胞，基线气道紧张度，AHR和氯化钾诱导的气道平滑肌（ASM）的力量产生，这表明在一定程度上对ASM兴奋-收缩耦合的直接影响。我们推测，O3通过促进嗜水气单胞菌介导的气道炎症和直接增加激动剂诱导的ASM力的产生来增强小鼠过敏原诱导的AHR。为了检验这些假设，在目标1中，中性粒细胞、TNF α和IL-1 β在介导臭氧增强的过敏性AHR和气道炎症中的作用将使用消耗或增加气道中性粒细胞和/或细胞因子的策略来确定。肺阻力和Penh将测量AHR，气道炎症将在支气管肺泡灌洗中通过表征细胞谱和细胞因子和趋化因子水平来评估。在目标2中，使用人类ASM细胞和小鼠气管环过敏原和臭氧处理的野生型和基因敲除小鼠，臭氧对关键的ASM兴奋-收缩耦合途径的影响将被定义。这些基本途径包括臭氧对激动剂诱导的钙反应、磷酸肌醇代谢和RhoA/RhoA激酶激活和肌球蛋白轻链磷酸酶抑制的钙致敏过程的影响。将确定每种激动剂诱导的信号传导事件在介导臭氧和过敏原对ASM细胞功能或气管环力产生的影响中的充分性和必要性。这些研究将确定臭氧增强过敏原诱导的AHR的机制，并为预防哮喘急性发作和降低哮喘发病率和死亡率提供新的治疗靶点。

DESCRIPTION (provided by applicant): Although ozone (O3) induces asthma exacerbations, methacholine airway hyperresponsiveness (AHR) and neutrophilic airway inflammation associated with increases in levels of TNFalpha and IL-1beta, the mechanisms that mediate ozone effects in asthma and in allergen-induced AHR remain unknown. Preliminary data shows that ozone and allergen in combination markedly increased airway neutrophils, baseline airway tone, AHR and KCl-induced airway smooth muscle (ASM) force generation, suggesting in part a direct effect on ASM excitation-contraction coupling. We postulate that O3 enhances allergen-induced AHR in mice by promoting neutrophil-mediated airway inflammation and by directly increasing agonist-induced ASM force generation. To test these hypotheses, in Aim 1, the role of neutrophils, TNFalpha and IL-1beta in mediating ozone-enhanced allergic AHR and airway inflammation will be determined using strategies to deplete or augment airway neutrophils and/or cytokines. Lung resistance and Penh will measure AHR and airway inflammation will be assessed in bronchoalveolar lavage by characterizing cell profiles and levels of cytokines and chemokines. In Aim 2, using human ASM cells and murine tracheal rings from allergen-and ozone-treated wild type and knockout mice, the effects of ozone on critical ASM excitation-contraction coupling pathways will be defined. These essential pathways include ozone effects on agonist-induced: calcium responses, phosphoinositide metabolism and calcium sensitization processes of RhoA/RhoA kinase activation and of myosin light chain phosphatase inhibition. The sufficiency and necessity of each agonist-induced signaling event in mediating ozone and allergen effects on ASM cell function or on tracheal ring force generation will be determined. These studies will identify the mechanisms by which ozone enhances allergen-induced AHR and provide insight into new therapeutic targets to prevent asthma exacerbations and decrease asthma morbidity and mortality.