INJURY TO MURINE AIRWAY EPITHELIAL-CELLS BY POLLEN ENZYMES

Background-Pollens are important triggers for asthma but the mechanism of sensitisation to their proteins remains poorly understood. The int rinsic protease activity of some allergens may contribute to sensitisa tion by disrupting the integrity of the airway epithelial barrier. Pol lens release a variety of enzymes, including proteases, upon hydration . The hypothesis that such enzymes might be able to damage airway epit helial cells was therefore tested. Methods-Diffusates from pollens of Lolium perenne (ryegrass), Poa pratensis (Kentucky bluegrass), Acacia longifolia (Sydney golden wattle), or Casuarina distyla (she-oak) were incubated with mouse tracheal epithelial cells in culture and cellula r detachment was quantified using a methylene blue dye binding assay. Results-Diffusates prepared using 100 mg/ml of pollen caused detachmen t of 30-90% of airway epithelial cells in separate experiments. Within each experiment comparable detachment was observed with all diffusate s tested, although total protein in the diffusates varied markedly bet ween species. Viability of the cells recovered after exposure to Acaci a diffusate was higher than after detachment by exposure to Lolium dif fusate. Cellular detachment by all of the diffusates could be almost c ompletely inhibited by addition of 10% serum. Aprotinin, an inhibitor of serine proteases, partially blocked activity in diffusates of Loliu m pollen but not of Acacia pollen. In contrast, alpha(1)-protease inhi bitor and secretory leucocyte protease inhibitor (SLPI) were not able to block the activity of either diffusate at concentrations which inhi bited cellular detachment by trypsin. Conclusions-Proteases released b y pollens are able to cause detachment of ah-way epithelial cells from their substratum in vitro and may not be effectively inhibited by end ogenous antiproteases.