Dept. of Physiology, Arizona Respiratory Center, Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ, USA.
Am J Physiol Lung Cell Mol Physiol. 2011 Apr;300(4):L605-14. doi: 10.1152/ajplung.00359.2010. Epub 2011 Feb 4.
Allergens are diverse proteins from mammals, birds, arthropods, plants, and fungi. Allergens associated with asthma (asthmagens) share a common protease activity that may directly impact respiratory epithelial biology and lead to symptoms of asthma. Alternaria alternata is a strong asthmagen in semiarid regions. We examined the impact of proteases from A. alternata on lung inflammation in vivo and on cleaving protease-activated receptor-2 (PAR(2)) in vitro. A. alternata filtrate applied to the airway in nonsensitized Balb/c mice induced a protease-dependent lung inflammation. Moreover, A. alternata filtrate applied to human bronchial epithelial cells (16HBE14o-) induced changes in intracellular Ca(2+) concentration (Ca(2+)), consistent with PAR(2) activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR(2) specific ligands SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2), but not the PAR(1)-specific ligand TFLLR-NH(2). Desensitization of PAR(2) in 16HBE14o- cells with 2-furoyl-LIGRLO-NH(2) or trypsin prevented A. alternata-induced Ca(2+) changes while desensitization of PAR(1), PAR(3), and PAR(4) with thrombin had no effect on A. alternata-induced Ca(2+) responses. Furthermore, the Ca(2+) response to A. alternata filtrates was dependent on PAR(2) expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR(2) to induce rapid increases in human airway epithelial Ca(2+) in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.
变应原是来自哺乳动物、鸟类、节肢动物、植物和真菌的多种蛋白质。与哮喘相关的变应原（asthmagens）具有共同的蛋白酶活性，可能直接影响呼吸道上皮生物学并导致哮喘症状。链格孢Alternaria alternata 是半干旱地区的一种强烈的哮喘变应原。我们研究了链格孢Alternaria alternata 蛋白酶对体内肺炎症和体外切割蛋白酶激活受体-2（PAR(2)）的影响。在未致敏的 Balb/c 小鼠的气道中应用链格孢Alternaria alternata 滤液可诱导依赖蛋白酶的肺炎症。此外，链格孢Alternaria alternata 滤液应用于人支气管上皮细胞（16HBE14o-）可诱导细胞内 Ca(2+)浓度 (Ca(2+)) 变化，与 PAR(2) 激活一致。这些作用被滤液的热失活或丝氨酸蛋白酶抑制所阻断，并且被 PAR(2) 特异性配体 SLIGRL-NH(2) 或 2-糠酰基-LIGRLO-NH(2) 模拟，但不是 PAR(1)-特异性配体 TFLLR-NH(2)。用 2-糠酰基-LIGRLO-NH(2)或胰蛋白酶对 16HBE14o-细胞中的 PAR(2)进行脱敏可防止链格孢Alternaria alternata 诱导的 Ca(2+)变化，而用凝血酶对 PAR(1)、PAR(3)和 PAR(4)进行脱敏对链格孢Alternaria alternata 诱导的 Ca(2+)反应没有影响。此外，HeLa 细胞稳定转染模型中 PAR(2)表达对滤液的 Ca(2+)反应有依赖性。这些数据表明，链格孢Alternaria 蛋白酶通过 PAR(2)诱导体外人气道上皮细胞 Ca(2+)的快速增加和体内细胞募集。这些反应可能是过敏性哮喘发展的关键早期步骤。
