Mechanotransduction and Eosinophil Function

描述（由申请人提供）： 嗜酸性粒细胞是哮喘中的主要炎症细胞，产生大量的5-脂氧合酶衍生的类花生酸，白三烯（LT）C4。LTC 4及其衍生物LTD 4和LTE 4是强有力的支气管收缩剂和哮喘气道炎症的有效介质。在哮喘中，浸润气道壁的嗜酸性粒细胞和粘附于气道上皮的管腔内嗜酸性粒细胞经历周期性机械拉伸，因为气道在通气期间随着肺充气而扩张和伸长。为了探讨这种动态力学环境是否会影响哮喘气道炎症，我们研究了周期性应变对体外粘附的人嗜酸性粒细胞合成白三烯的影响。我们已经观察到，粘附嗜酸性粒细胞进行循环应变，相比静态条件下的培养，表现出显着的抑制LTC 4合成的激动剂刺激。初步的数据表明，这种抑制作用需要一个完整的肌动蛋白细胞骨架，并依赖于反应产生的活性氧物种的循环应变，导致减少磷脂酶介导的释放花生四烯酸和5-脂氧合酶的酶活性。因此，我们假设粘附的嗜酸性粒细胞的周期性机械拉伸触发Rho和细胞因子依赖性信号传导，导致活性氧的产生。这些现象导致嗜酸性粒细胞中胞质磷脂酶A2和5-脂氧合酶失活，从而抑制半胱氨酰白三烯合成。通过阐明循环应变抑制嗜酸性粒细胞中白三烯合成的机制，拟议的研究将提供有关哮喘气道炎症的主要潜在决定因素的新信息，这是以前没有认识到的。 为了更好地了解机械应力对气道细胞的影响，我们还开发了一种新型的气道壁组织工程三维模型，其中上皮细胞在胶原蛋白凝胶中覆盖成纤维细胞的气液界面处分化。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.