Injury to murine airway epithelial cells by pollen enzymes

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Abstract
BACKGROUND Pollens are important triggers for asthma but the mechanism of sensitisation to their proteins remains poorly understood. The intrinsic protease activity of some allergens may contribute to sensitisation by disrupting the integrity of the airway epithelial barrier. Pollens release a variety of enzymes, including proteases, upon hydration. The hypothesis that such enzymes might be able to damage airway epithelial cells was therefore tested. METHODS Diffusates from pollens ofLolium 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 cellular detachment was quantified using a methylene blue dye binding assay. RESULTS Diffusates prepared using 100 mg/ml of pollen caused detachment of 30–90% of airway epithelial cells in separate experiments. Within each experiment comparable detachment was observed with all diffusates tested, although total protein in the diffusates varied markedly between species. Viability of the cells recovered after exposure to Acacia diffusate was higher than after detachment by exposure to Loliumdiffusate. Cellular detachment by all of the diffusates could be almost completely inhibited by addition of 10% serum. Aprotinin, an inhibitor of serine proteases, partially blocked activity in diffusates ofLolium pollen but not of Acacia pollen. In contrast, α1-protease inhibitor and secretory leucocyte protease inhibitor (SLPI) were not able to block the activity of either diffusate at concentrations which inhibited cellular detachment by trypsin. CONCLUSIONS Proteases released by pollens are able to cause detachment of airway epithelial cells from their substratum in vitro and may not be effectively inhibited by endogenous antiproteases.