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.
