Mesenchymal cell fibrogenesis triggered by epithelial cells in asthma

(申请人摘要)哮喘患者呼吸道的结构变化， 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 肺成纤维细胞或其亚类称为肌成纤维细胞，似乎是 III型胶原、纤维连接蛋白和肌腱蛋白的重要来源 哮喘的上皮下层。致纤维化的事件似乎是由 哮喘患者的支气管上皮细胞。对此的总体假设是 项目称哮喘患者上皮细胞分泌物质(S) 作用于间充质细胞(肺成纤维细胞和呼吸道平滑肌细胞)以 产生哮喘呼吸道特有的结构变化。我们有 在细胞界面上开发了一种实验方法 特定元素存在的生物/生化与临床哮喘 产生这些病理效应的活体可以被识别出来。这种方法 有3个关键要素：1)2周、4次支气管镜检查方案 炎症，或RFI方案)，其中人类哮喘患者经历呼吸道 取材(支气管肺泡灌洗、支气管刷活检取上皮 细胞、支气管钳活检)和节段性抗原挑战(SAC) 挑战前的炎症事件(在基线上)，在 炎症反应(挑战后24小时)，以及在其修复和 可以确定解决阶段(挑战后1周和2周)的特征；2)例如 活体、短期、气液界面培养兔肾上皮细胞 哮喘患者和对照组中哮喘细胞保留的重要方面 他们的哮喘表型；3)上皮细胞来源的生物测定 检测上皮细胞上清液中的促纤维化因子 刺激III型胶原和其他细胞外胶原生成的能力 间质细胞的基质(ECM)成分，包括肺成纤维细胞和 呼吸道平滑肌细胞，从而使我们能够表征和识别 重要的促纤维化介质。使用这些工具，我们将：1)确定 上皮细胞分泌成纤维生长因子的动力学研究 刺激肺成纤维细胞和呼吸道平滑肌细胞产生细胞外基质 在体内免疫触发(SAC)之后，并确定这些事件 仅限于哮喘患者，而不存在于非哮喘鼻炎患者和 非过敏、非哮喘对照；2)确定有效成分(S) 由哮喘上皮细胞分泌；3)确定急性呼吸道是否 嗜酸性粒细胞反应是哮喘上皮细胞分泌的必要条件 无论是机械性的还是免疫性的损伤， 可以触发它们的合成；以及4)识别信号的元素 涉及核因子-kB、MAP激酶和/或Smad通路的信号转导通路包括 肺成纤维细胞和气道平滑肌细胞分泌细胞外基质的研究 由哮喘上皮细胞中的活性因子触发。这些 研究将为哮喘的气道重塑提供新的信息。

(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.