MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

该提案利用了初步调查中开发的技术 在我们的实验室和我们的合作者，以确定机制， 嗜酸性粒细胞引起气道高反应性。 建议开展研究， 从3个层面研究支气管张力的调节：1） 完整的，活化的嗜酸性粒细胞; 2）纯化的 嗜酸性粒细胞蛋白/介质; 3）细胞间相互作用， 上皮/内皮组织由这些介质引起。 先前研究 仅限于离体气道的体外研究， 不允许评估神经循环的相互作用， 嗜酸性粒细胞引起的气道高反应性与气道水肿的关系 proteins. 为了克服先前的局限性，我们开发了一种原位 豚鼠气管的“活外植体”制备， 呼吸道的神经分布和循环。 该制备证明 1)直接的，特征性的收缩反应，主要纯化的 嗜酸性粒细胞蛋白，2）证据的合成增强（而不是 抑制）介质在嗜酸性粒细胞浸润，和3）证据， 与血管内皮的主要收缩相互作用。 研究提出了测试的假设，激活的嗜酸性粒细胞 引起直接气道平滑肌收缩并增强其他收缩性肌纤维 通过这些互动的刺激。 这项工作的主要重点是 定义引起每种嗜酸性粒细胞独特反应性的机制 蛋白质：1）作用位点，2）离子电荷，3）细胞毒性 影响，以及4）RNA酶活性;建议使用 嗜酸性粒细胞蛋白的单克隆抗体，以确定机制， 阻断与蛋白质基质的相互作用所需的浓度， 应用于完整嗜酸性粒细胞的研究。 通过豚鼠 气管准备，单克隆抗体将用于阻断 脱颗粒嗜酸性蛋白的影响，从而评估 特别是哪些蛋白质是导致气道高反应性的原因 在活化的嗜酸性粒细胞中。 最后一系列的实验被提出来， 进一步检查完整的活化嗜酸性粒细胞在引起 气道高反应性 在初步研究中， 允许选择性灌注大鼠的支气管循环， 开发了 该开路制备将用于评估 1)循环活化嗜酸性粒细胞在肺功能增强中作用 大鼠传导气道中的阻力反应; 2） 正常和低密度嗜酸性粒细胞在这一过程中; 3）特定的蛋白质 介质参与; 4）嗜酸性粒细胞诱导的水肿的作用 支气管高反应性激活; 5）LFA-1 α的作用 和Mac-1受体在促进嗜酸性粒细胞迁移、活化和 随后气道反应性上调。 初步研究是 完成拟议研究所需的技术方面。 数据 从这些研究中得出的结果表明， 气道高反应性

This proposal utilizes technologies developed in preliminary investigations in our laboratory and by our collaborators to define mechanisms by which eosinophils cause airway hyperresponsiveness. Studies are proposed to investigate the regulation of bronchomotor tone at 3 levels: 1) the role of intact, activated eosinophils; 2) specific effects of purified eosinophil proteins/mediators; 3) cell-cell interactions between epithelial/endothelial tissues caused by these mediators. Prior studies have been limited to in vitro investigations from excised airways and did not permit assessment of either neurocirculatory interactions nor the role of airway edemagenesis in airway hyperresponsiveness caused by eosinophil proteins. To obviate prior limitations, we have developed an in situ "living explant" preparation of guinea pig trachea, which preserves innervation and circulation to the airway. This preparation demonstrates 1) direct, characteristic contractile responses to the major purified eosinophil proteins, 2) evidence for synthesis of augmenting (rather than inhibitory) mediators during eosinophil infiltration, and 3) evidence for major contractile interactions with underlying vascular endothelium. Studies are proposed to test the hypothesis that activated eosinophils cause direct airway smooth muscle contraction and augment other contractile stimuli through these interactions. A major emphasis of this work is defining the mechanism causing the unique responsiveness of each eosinophil protein with respect to 1) site of action, 2) ion charge, 3) cytotoxic effect, and 4) RNase activity; additional studies are proposed using monoclonal antibodies to eosinophil-proteins to determine the mechanism and concentrations required to block the interactions with protein matrices for application to studies of intact eosinophils. Using the guinea pig tracheal preparation, monoclonal antibodies will be used to block the effects of degranulated eosinophilic proteins and thus to assess specifically which proteins are responsible for airway hyperresponsiveness in activated eosinophils. A final series of experiments is proposed to examine further the response of intact activated eosinophils in causing airway hyperresponsiveness. In preliminary studies, a new preparation permitting selective perfusion of the bronchial circulation of the rat has been developed. This open circuit preparation will be utilized to assess 1) the role of circulating activated eosinophils in augmenting lung resistance responses in the conducting airways of the rat; 2) the role of normo- and hypodense eosinophils in this process; 3) the specific protein mediators involved; 4) the role of edemagenesis induced by eosinophil activation in bronchial hyperresponsiveness; and 5) the role of LFA-1alpha and Mac-1 receptors in promoting eosinophil migration, activation and consequent upregulation of airway responsiveness. Preliminary studies are completed for necessary technical aspects of the proposed studies. Data derived from these studies suggest therapeutic approaches for disorders of airway hyperresponsiveness.