Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR2

Boitano S, Flynn AN, Sherwood CL, Schulz SM, Hoffman J, Gruzinova I, Daines MO. Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR(2). Am J Physiol Lung Cell Mol Physiol 300: L605-L614, 2011. First published February 4, 2011; doi:10.1152/ajplung.00359.2010.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 Ca2+ concentration ([Ca2+](i)), 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-NH2 or 2-furoyl-LIGRLO- NH2, but not the PAR(1)-specific ligand TFLLR-NH2. Desensitization of PAR(2) in 16HBE14o- cells with 2-furoyl-LIGRLO-NH2 or trypsin prevented A. alternata-induced [Ca2+] i changes while desensitization of PAR(1), PAR(3), and PAR(4) with thrombin had no effect on A. alternata-induced Ca2+ responses. Furthermore, the Ca2+ 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 [Ca2+] i in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.