MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

提议开展研究，以便竞争性地继续开展调查 成 嗜酸性粒细胞引起支气管扩张的机制 在人类哮喘状态下的反应性。 在先前的赠款中， 期间， 研究表明，嗜酸性粒细胞 分泌 白三烯（LT）C_4引起豚鼠的直接收缩， 气管平滑肌 in vivo. 目前的提议验证了分子 嗜酸性粒细胞对内皮和气道基质的粘附 嗜酸性粒细胞分泌并增强气道平滑肌收缩。 这 建议采用新开发的方法来测量 细胞间 显微切片中引起气道收缩反应的相互作用 的 通过NHLBI赞助的 与汉堡Krankenhaus Gro/beta/hansdorf合作， 德国） 其中非常少量的人嗜酸性粒细胞（大于或 平等 50，000/室）。 建议进行研究， 的 嗜酸性粒细胞粘附于人脐静脉的机制 内皮细胞（HUVEC）和基质蛋白纤连蛋白 （FN） 导致支气管活性LTC 4分泌增加，然后导致 增强人支气管移植物的收缩。 机制 通过 其指导β 1-整联蛋白VLA-4与FN的结合， β 2- 整合素或VLA-4与内皮细胞的结合导致合成增加， 分泌 将直接检查嗜酸性粒细胞中的LTC 4。 使用新 开发的视频显微测量系统，分子的直接影响， 人嗜酸性粒细胞与HUVEC或FN的粘附在增强平滑肌细胞的增殖中的作用 肌肉 将检查来自人气道的收缩。 初步 研究已经确定粘附分子连接 原因 增加嗜酸性粒细胞的LTC 4分泌。 进一步的初步 研究表明，连接到FN引起增加的活性， 胞浆PLA 2（cPLA 2）和sPLA 2活性增加， 对应 这些异构体的易位。 利用特异性单克隆 β 2亚基或VLA-4的抗体（mAb），并通过选择性 使用 针对PLA 2亚型的mAb，建议进行研究， 审查 分子粘附增强刺激的假设 收缩 通过增加LTC 4的分泌， 造成 在粘附诱导易位后cPLA 2活性增加， 核膜和sPLA 2至质膜/灌流液。 这些 调查应建立一个机制， 选择性激活人嗜酸性粒细胞，并建立直接 这种机制与人类增强收缩的相关性 气道 平滑肌处于过度反应状态 数据来源于这些 研究应该提出生理学上的直接机制， 相关 治疗人类哮喘的方法。

Studies are proposed for competitive continuation of investigations into mechanisms by which eosinophils cause augmented bronchomotor responsiveness in the human asthmatic state. In the prior grant period, investigations were completed that demonstrated that eosinophil secretion of leukotriene (LT) C4 caused direct contraction of guinea pig trachealis in vivo. The current proposal tests the hypothesis that molecular adhesion of eosinophils to endothelium and airway matrix primes eosinophil secretion and augments airway smooth muscle contraction. This proposal utilizes a newly developed method for measurement of cell-cell interactions causing airway contractile responses in microsections of explanted human airways (obtained through an NHLBI-sponsored collaboration with the Krankenhaus Gro/beta/hansdorf, Hamburg, Germany) in which a very small number of human eosinophils (greater than or equal to 50,000/chamber) is utilized. Studies are proposed to examine the mechanism by which adhesion of eosinophils to human umbilical vein endothelial cells (HUVEC) and to the matrix protein fibronectin (FN) causes augmented secretion of bronchoactive LTC4, which then causes augmented contraction of human bronchial explants. The mechanism by which direct binding of the beta1-integrin, VLA-4, to FN and of beta2- integrin or VLA-4 to endothelium causes augmented synthesis and secretion of LTC4 in eosinophils will be examined directly. Using the newly developed videomicrometry system, the direct effects of molecular adhesion of human eosinophils to HUVEC or FN in augmenting smooth muscle contraction from human airways will be examined. Preliminary investigations have established that adhesion molecule ligation causes augmented secretion of LTC4 from eosinophils. Further preliminary studies indicate that ligation to FN causes increased activity of cytosolic PLA2 (cPLA2) and increased sPLA2 activity with corresponding translocation of these isoforms. Utilizing specific monoclonal antibodies (mAb) to the beta2 subunit or to VLA-4 and by selective use of mAb directed against isoforms of PLA2, studies are proposed to examine the hypothesis that molecular adhesion augments stimulated constriction of human airways through augmented secretion of LTC4, which is caused by increased activity of cPLA2 after adhesion-induced translocation to nuclear membrane and sPLA2 to plasma membrane/perfusate. These investigations should establish one mechanism that accounts for the selective activation of human eosinophils and establish the direct relevance of this mechanism to augmented contraction of human airway smooth muscle in the hyperreactive state. Data derived from these studies should suggest direct mechanisms for physiologically relevant approaches to the treatment of human asthma.