Alternaria fungus induces the production of GM-CSF, interleukin-6 and interleukin-8 and calcium signaling in human airway epithelium through protease-activated receptor 2.

RATIONALE Recent studies suggest that host immune responses to environmental fungi may play an important role in the development of allergic diseases, such as human asthma. Epithelium is considered an active participant in allergic inflammation. We previously reported that aspartate protease from Alternaria induces the activation and degranulation of human eosinophils that are mediated through protease-activated receptor 2 (PAR-2). However, our current knowledge on the innate immune responses of epithelium to environmental fungi is very limited. We investigated the responses of epithelium to fungi and the mechanisms of these responses. METHODS Human airway epithelial cell line BEAS-2B and Calu-3 (both from American Type Culture Collection) were incubated with PAR-2 peptides and extracts of various fungi. The cellular responses, including GM-CSF, interleukin (IL)-6, IL-8, eotaxin, eotaxin-2 and RANTES production as well as increases in intracellular calcium concentration ([Ca(2+)](i)), were examined. To characterize the proteases involved in these responses, protease inhibitors such as pepstatin A and alkalo-thermophilic Bacillus inhibitor (ATBI), HIV protease inhibitors and 4-amidinophenylmethanesulfonyl fluoride hydrochloride were used. To investigate the role of PAR-2, PAR-2-agonistic and PAR-2-antagonistic peptides were used. RESULTS PAR-2-activating peptide, but not the control peptide, induced GM-CSF, IL-6 and IL-8 production; these cellular responses were accompanied by a quick and marked increase in [Ca(2+)](i). Among 7 common environmental fungi, only Alternaria induced GM-CSF, IL-6 and IL-8 production and increased [Ca(2+)](i) response. Both cytokine production and increased [Ca(2+)](i) were significantly inhibited by PAR-2 antagonist peptide and by aspartate protease inhibitors (pepstatin A, ritonavir, nelfinavir and ATBI), but not by the PAR-2 control peptide or by other protease inhibitors. CONCLUSIONS Aspartate proteases from Alternaria induce cytokine production and calcium response in airway epithelium that is mediated through PAR-2. This protease-mediated activation of airway epithelium may be implicated in the development and exacerbation of airway allergic disease.