Mechanotransduction and Eosinophil Function

描述(由申请人提供)： 嗜酸性粒细胞是哮喘的主要炎症细胞，它产生大量的5-脂氧合酶衍生的二十烷类化合物，白三烯(LT)C4。LTC4及其衍生物LTD4和LTE4是强有力的支气管收缩因子和哮喘气道炎症的有力介质。在哮喘患者中，随着肺充气，气道扩张和伸展，侵入气道壁的嗜酸性粒细胞和附着在呼吸道上皮细胞的管腔内嗜酸性粒细胞经历周期性机械拉伸。为了探索这种动态机械环境是否可能影响哮喘的呼吸道炎症，我们研究了循环应变对体外贴壁的人嗜酸性粒细胞合成白三烯的影响。我们观察到，与在静态条件下培养相比，贴壁的嗜酸性粒细胞受到循环应变，对激动剂刺激的LTC4合成表现出明显的抑制。初步数据表明，这种抑制作用需要完整的肌动蛋白细胞骨架，并依赖于响应循环应变而产生的活性氧物种，导致磷脂酶介导的花生四烯酸释放和5-脂氧合酶活性降低。因此，我们假设，黏附的嗜酸性粒细胞的周期性机械拉伸触发了依赖于Rho和细胞骨架的信号，导致了活性氧的产生。这些现象导致嗜酸性粒细胞胞浆磷脂酶A2和5-脂氧合酶失活，从而抑制了半胱氨基白三烯的合成。通过阐明循环应变抑制嗜酸性粒细胞白三烯合成的机制，拟议的研究将提供关于哮喘呼吸道炎症的主要潜在决定因素的新信息，该决定因素以前尚未被认识到。 为了更好地了解机械应力对呼吸道细胞的影响，我们还开发了一种新的组织工程化的三维气道壁模型，在该模型中，上皮细胞在胶原凝胶中的成纤维细胞上方的气液界面上分化。模型中的凝胶和细胞可以被机械压缩。我们将在气道模型中加入嗜酸性粒细胞，以确定在上皮细胞和成纤维细胞存在的情况下，压缩机械应力对嗜酸性粒细胞白三烯合成的影响。

DESCRIPTION (provided by applicant): Eosinophils, the predominant inflammatory cells in asthma, produce large amounts of the 5-lipoxygenase-derived eicosanoid, leukotriene (LT)C4. LTC4 and its derivatives, LTD4 and LTE4, are powerful bronchoconstrictors and potent mediators of asthmatic airway inflammation. In asthma, eosinophils infiltrating the airway wall and intraluminal eosinophils adherent to the airway epithelium undergo cyclic mechanical stretch as the airways distend and elongate with lung inflation during ventilation. To explore whether this dynamic mechanical environment might influence airway inflammation in asthma, we have investigated the effect of cyclic strain on leukotriene synthesis by adherent human eosinophils in vitro. We have observed that adherent eosinophils subjected to cyclic strain, as compared to culture under static conditions, exhibit marked inhibition of LTC4 synthesis in response to agonist stimulation. Preliminary data indicate that this inhibitory effect requires an intact actin cytoskeleton and depends on reactive oxygen species generated in response to cyclic strain, leading to reduced phospholipase-mediated release of arachidonic acid and 5-lipoxgenase enzyme activity. Thus, we hypothesize that cyclic mechanical stretch of adherent eosinophils triggers Rho- and cytoskeleton-dependent signaling, leading to generation of reactive oxygen species. These phenomena result in inactivation of cytosolic phospholipase A2 and 5-lipoxygenase in eosinophils, causing inhibition of cysteinyl leukotriene synthesis. By elucidating the mechanisms by which cyclic strain inhibits leukotriene synthesis in eosinophils, the proposed investigation will provide new information about a major potential determinant of asthmatic airway inflammation that has not previously been recognized. To better understand the effects of mechanical stress on cells in the airway, we have also developed a novel tissue-engineered, 3-dimensional model of the airway wall in which epithelial cells are differentiated at an air-liquid interface overlying fibroblasts in a collagen gel. The gel and cells in the model can be mechanically compressed. We will add eosinophils to the airway model to determine the effects of compressive mechanical stress in the presence of epithelial ceils and fibroblasts on eosinophil leukotriene synthesis.