CYTOKINES, ASTHMA AND AIRWAY SMOOTH MUSCLE

对支气管收缩激动剂的气道高反应性， 对支气管扩张剂β-激动剂低反应性 人类哮喘的特征 目前的证据倾向于 哮喘的气道炎症和气道反应性改变。 哮喘气道中释放的细胞因子可能提供了这种联系。 这些 细胞因子不仅具有调节补体和水平的能力， 气道中免疫/炎症细胞的激活，但也出现 来改变气道对激动剂的反应 例如，每一个“亲- 哮喘”细胞因子IL-1 β、TNF α、IL-2、IL-4和IL-5， 研究，在哮喘气道中的含量增加， 已显示在动物体内导致气道高反应性。 它 我们的假设是这些细胞因子对气道平滑有直接作用， 肌肉反应导致对扩张器刺激的反应降低， 对收缩刺激的反应增强。 我们建议研究这个问题， 假设，并确定这些细胞因子的作用机制 使用培养的人气道平滑肌（HASM）细胞。 一种新型 技术，磁扭转细胞仪，提供了一个机械指数， 平滑肌反应，细胞骨架硬度，将与 测量Ca++或cAMP，提供一个系统，允许 确定受体活化途径中的位点 平滑肌收缩/舒张，这可能会被细胞因子改变。 培养系统还允许产生足够量的 功能性细胞，与其进行生物化学或分子生物学 分析以进一步检查细胞因子的作用机制。 将追求四个具体目标：1）测量剂量和时间相关 “促哮喘”细胞因子对HASM细胞刚度和cAMP的影响 响应于异丙肾上腺素和前列腺素E2（PGE 2）的形成; 2） 通过以下方式检验细胞因子降低β-肾上腺素能反应的假设： 增加前列腺素G/H合酶（PGHS）表达，导致 增加PGE 2形成，G1的EP 3前列腺素受体活化， 通过测量PGHS-2和Gia表达抑制腺苷酸环化酶，和 通过使用PGHS-2和Gi抑制剂以及EP 3受体激动剂; 3）测量 “促哮喘”细胞因子的剂量和时间相关效应 在激动剂诱导的HASM细胞硬度和细胞内钙 被认为在哮喘病变中是重要的，即组胺， 乙酰胆碱和P物质，以及4）测试细胞因子 通过增加HASM细胞对收缩激动剂刚度反应， 增加磷脂酶C（PLC）的活化，通过测量IP 3 通过收缩激动剂和PLC活性形成。 论证 细胞因子在导致β-肾上腺素能 受体功能障碍可能为改善安全性提供新的途径 以及这些药剂的有效使用。 示范作用和 细胞因子增强气道平滑肌的作用机制 对收缩激动剂的反应可能使我们能够开发新的模型， 干预以防止气道高反应性的诱导，以及 从而限制了气道在哮喘期间变窄的能力 插曲。

Airway hyperresponsiveness to bronchoconstrictor agonists and hyporesponsiveness to bronchodilator beta-agonists are characteristic features of human asthma. Current evidence favors a causal link between the airway inflammation and altered airway responsiveness of asthma. Cytokines released in the asthmatic airway may provide this link. These cytokines not only have the capacity to modulate the complement and level of activation of immune/inflammatory cells in the airways, but also appear to alter airway responses to agonists. For example, each of the "pro- asthmatic" cytokines IL-1beta, TNFalpha, IL-2, IL-4, and IL-5, proposed for study, is present in increased quantities in the asthmatic airway and has been shown to result in airway hyperresponsivemness in animals in vivo. It is our hypothesis that these cytokines have direct effects on airway smooth muscle responses resulting in decreased responses to dilator stimuli and increased responses to contractile stimuli. We propose to examine this hypothesis and to determine the mechanism of action of these cytokines using human airway smooth muscle (HASM) cells in culture. A novel technique, magnetic twisting cytometry, that provides a mechanical index of smooth muscle response, cytoskeletal stiffness, will be combined with measurements of Ca++ or cAMP, providing a system that allows for determination of the site in the pathway leading from receptor activation to smooth muscle contraction/relaxation that may be altered by cytokines. A culture system also allows for generation of sufficient quantities of functional cells with which to perform biochemical or molecular biological analyses in order to further examine the mechanism of action of cytokines. Four specific aims will be pursued; 1) Measure the dose and time related effects of "pro-asthmatic" cytokines on HASM cell stiffness and cAMP formation in response to isoproterenol and prostaglandin E2 (PGE2); 2) Test the hypothesis that cytokines decrease beta-adrenergic responses by increasing prostaglandin G/H synthase (PGHS) expression leading to increased PGE2 formation, EP3 prostanoid receptor activation of G1 and inhibition of adenylyl cyclase by measuring PGHS-2 and Gia expression, and by use of PGHS-2 and Gi inhibitors, and EP3 receptor agonists; 3) Measure the dose and time related effects of "pro-asthmatic" cytokines on changes in HASM cell stiffness and intracellular calcium induced by agonists believed to be of importance in the asthmatic lesion, namely histamine, acetylcholine, and substance P, and 4) Test the hypothesis that cytokines increase HASM cell stiffness responses to contractile agonists by increasing the activation of phospholipase C (PLC), by measuring IP3 formation by contractile agonists and PLC activity. Demonstration of the role and mechanism of action of cytokines in leading to beta-adrenergic receptor dysfunction may provide new avenues for improvements in the safe and efficacious use of these agents. Demonstration of the role and mechanism of action of cytokines in augmenting airway smooth muscle responses to contractile agonist may allow us to develop new models of intervention to prevent the induciton of airway hyperresponsiveness, and thereby limit the capacity of the airways to narrow during an asthmatic episode.