Mesenchymal cell fibrogenesis triggered by epithelial cells in asthma

（申请人的摘要）哮喘患者气道的结构变化， 包括上皮下纤维化，被认为在 哮喘特有的生理变化。间充质细胞，包括 肺成纤维细胞或其中的一个亚类称为肌成纤维细胞，似乎是 胶原蛋白III、纤连蛋白和腱生蛋白的重要来源， 哮喘性脊髓炎纤维化事件似乎是协调的， 支气管上皮细胞的变化。对此的总体假设是 该项目指出，哮喘患者的上皮细胞分泌的物质， 作用于间充质细胞（肺成纤维细胞和气道平滑肌细胞）， 产生哮喘气道特征的结构变化。我们有 开发了一种在细胞界面的实验方法 生物学/生物化学和临床哮喘，其中特定元素存在于 产生这些病理效应的生物体。这种方法 有3个关键要素：1）2周、4次支气管镜检查方案（“从 炎症”或RFI协议），其中人类哮喘患者经历气道 取样（支气管肺泡灌洗，支气管刷检以获得上皮细胞 细胞，支气管内钳活检）和节段性抗原激发（SAC）， 在激发前（基线时）， 炎症反应（激发后24小时），以及在其修复期间， 可表征消退期（激发后1周和2周）; 2） 上皮细胞的体内、短期、空气-流体界面培养， 其中哮喘细胞保留了重要的方面 他们的哮喘表型;和3）上皮细胞衍生的生物测定 其中测试上皮细胞上清液的纤维化因子， 能够刺激III型胶原蛋白和其他细胞外基质的产生， - 来自间充质细胞的基质（ECM）组分，包括肺成纤维细胞和 气道平滑肌细胞，从而使我们能够表征和识别 重要的纤维化介质。使用这些工具，我们将：1）确定 上皮细胞分泌纤维化生长因子的动力学 刺激肺成纤维细胞和气道平滑肌细胞产生ECM 在体内免疫触发（SAC）后，并确定这些事件是否 仅限于哮喘患者，不存在于非哮喘性鼻炎患者中， 非过敏、非哮喘对照; 2）鉴别活性成分 由哮喘上皮细胞分泌; 3）确定急性气道 嗜酸性粒细胞反应是哮喘上皮细胞分泌 纤维化因素以及是否是机械的，以及免疫损伤， 可以触发它们的合成;以及4）识别信号的元素是否 涉及NF-κ B、MAP激酶和/或Smad途径的转导途径是 参与肺成纤维细胞和气道平滑肌细胞的ECM分泌 由哮喘上皮细胞的活性因子触发。这些 研究将提供关于哮喘气道重塑的新信息。

(Applicant's Abstract) Structural changes in the airways of asthmatics, including subepithelial fibrosis, are thought to play an important role in the physiologic changes characteristic of asthma. Mesenchymal cells, including lung fibroblasts or a subcategory of them called myofibroblasts, appear to be important sources of collagen III, fibronectin, and tenascin, components of the asthmatic subepithelium. Fibrogenic events appear to be coordinated by bronchial epithelial cells in asthmatics. The overall hypothesis for this project states that epithelial cells from asthmatics secrete substance(s) that act on mesenchymal cells (lung fibroblasts and airway smooth muscle cells) to produce the structural changes characteristic of asthmatic airways. We have developed an experimental approach at the interface of cell biology/biochemistry and clinical asthma in which specific elements present in vivo that produce these pathological effects can be identified. This approach has 3 key elements: 1) a 2 week, 4 bronchoscopy protocol ("Recovery from Inflammation," or RFI protocol) in which human asthmatics undergo airway sampling (broncholaveolar lavage, bronchial brush biopsy to obtain epithelial cells, endobronchial forceps biopsy) and segmental antigen challenge (SAC) so that inflammatory events before challenge (at baseline), at the height of the inflammatory response (24 hrs after challenge), and during its repair and resolution phases (1 wk and 2 wks after challenge) can be characterized; 2) ex vivo, short term, air-fluid interface culture of epithelial cells from asthmatics and controls in which the asthmatic cells retain important aspects of their asthma phenotype; and 3) a bioassay for epithelial cell-derived fibrogenic factors in which epithelial cell supernatants are tested for their ability to stimulate production of collagen type III and other extracellular matrix (ECM) components from mesenchymal cells, including lung fibroblasts and airway smooth muscle cells, thus allowing us to characterize and identify important fibrogenic mediators. Using these tools we will: 1) Determine the kinetics of the secretion of fibrogenic growth factors from epithelial cells which stimulate lung fibroblasts and airway smooth muscle cells to produce ECM after an in vivo immunologic trigger (SAC), and determine whether these events are restricted to asthmatics and not present in nonasthmatic rhinitics and nonallergic, nonasthmatic controls; 2) Identify the active principle(s) secreted by asthmatic epithelial cells; 3) Determine whether an acute airway eosinophilic response is necessary for asthmatic epithelial cells to secrete fibrogenic factors and whether mechanical, as well as an immunologic injury, can trigger their synthesis; and 4) Identify whether elements of signal transduction pathways involving NF-kB, MAP kinases, and/or Smad pathways are involved in ECM secretion by lung fibroblasts and airway smooth muscle cells triggered with active factors derived from asthmatic epithelial cells. These studies will provide novel information concerning airway remodeling in asthma.