Allergens as proteases: an Aspergillus fumigatus proteinase directly induces human epithelial cell detachment

Allergic bronchopulmonary aspergillosis (ABPA) is characterized by pulmonary and systemic allergic and inflammatory processes triggered by fungal antigens. Airway damage is a feature of this disorder, and although Aspergillus-derived proteinases have been described, the capacity of Aspergillus, however, to directly induce damage to human epithelium has not previously been studied. We therefore cultured Aspergillus fumigatus from two patients with ABPA, extracted mycelial products by sonication and filtration, and then evaluated their capacity to induce epithelial cell (EC) desquamation from basement membrane using an in vitro model that uses intact human amniotic EC and native basement membrane. A. fumigatus extracts induced detachment of EC in a dose-dependent fashion, producing up to 34% +/- 6% detachment (p less than 0.05 compared to medium alone). Enzyme analysis of A. Fumigatus extract using synthetic substrates revealed the presence of a number of different enzymes; therefore, studies with specific proteinase inhibitors were undertaken to identify the proteinase(s) responsible for detachment. A. Fumigatus-induced desquamation was partially inhibited by phenylmethylsulfonylfluoride and substantially inhibited by glutathione and N-acetylcysteine, but not by alpha 1-antitrypsin, 1,10 phenanthroline, ethylenediaminetetraacetic acid, aprotinin, or soybean trypsin inhibitor at concentrations that inhibit other serine- or metalloproteinases. Gel filtration of the extract with a Sepharose 6B column revealed that the major epithelium-detaching activity appeared in the 20 to 35 kd fraction. Comparison with proteinase standards suggested a role for a chymotrypsin-like proteinase. Thus, A. fumigatus releases a proteinase that is directly able to induce EC detachment.(ABSTRACT TRUNCATED AT 250 WORDS)