MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

建议对竞争性继续调查进行研究 vt.进入，进入 嗜酸性粒细胞引起支气管运动增强的机制 人类哮喘状态下的反应性。在之前的拨款中 句号， 完成的调查表明，嗜酸性粒细胞 分泌物 白三烯C4对豚鼠的直接收缩作用 管状线虫 在活体内。目前的提议检验了分子的假设 嗜酸性粒细胞与内皮细胞和气道基质的黏附 嗜酸性粒细胞分泌，增强呼吸道平滑肌收缩。 这 Proposal利用一种新开发的方法来测量 细胞-细胞 在显微切片中引起呼吸道收缩反应的相互作用 的 移植的人类呼吸道(通过NHLBI赞助的 与汉堡Krankenhaus Gro/Beta/Hansdorf合作， (德国) 其中非常少量的人类嗜酸性粒细胞(大于或 相等 至50,000个/室)。建议进行研究以检查 这个 嗜酸性粒细胞与人脐静脉的黏附机制 内皮细胞(HUVEC)与基质蛋白纤维连接蛋白 (FN) 导致支气管活性LTC4分泌增加，进而导致 人支气管外植体的收缩增强。这一机制 通过 它直接将β1-整合素VLA-4与Fn和Of结合 Beta2- 整合素或VLA-4促进内皮细胞合成增强和 分泌物 将直接检测嗜酸性粒细胞中LTC4的含量。使用新的 开发的视频显微测量系统，分子的直接影响 人嗜酸性粒细胞与人脐静脉内皮细胞或纤维连接蛋白的黏附 肌肉 我们将检查人类呼吸道的收缩情况。初步 研究证实，黏附分子的连接 成因 嗜酸性粒细胞分泌LTC4增加。进一步的初步调查 研究表明，与纤维连接蛋白结扎会导致血管活性增加 胞浆磷脂酶A2(CPLA2)和sPLA2活性增加 对应 这些异构体的移位。利用特定的单抗 抗β2亚基或抗VLA-4抗体(MAb)及选择性 使用 针对PLA2亚型的单抗，建议进行以下研究 考查 分子粘附力增强刺激的假说 收缩 通过增加LTC4的分泌而对人类呼吸道产生影响 起因于 黏附诱导移位后cPLA2活性增加 核膜和sPLA2转化为质膜/灌流液。这些 调查应建立一种机制，说明 选择性激活人嗜酸性粒细胞并建立其直接 这一机制与人体增强收缩的相关性 气道口 处于高反应状态的平滑肌。从这些数据派生的数据 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.