Author(s): Winton HL, Wan H, Cannell MB, Gruenert DC, Thompson PJ,
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Abstract BACKGROUND: Allergenic and non-allergenic proteinases from house dust mites (HDM) cause loss of adhesion between airway epithelial cells that may result in a loss of functional cohesion between the cells and thus assist in allergen presentation. Improved cellular assay systems are needed to ascertain the mechanisms involved. OBJECTIVES: To survey a series of epithelial cell lines (Calu-3, 16HBE14o-, NCI-H292 and A549 from human airways, and MDCK from dog kidney) and establish their utility for studies of the effects of HDM proteinases from D. pteronyssinus on epithelial permeability. To develop an improved method for measuring changes in epithelial permeability induced by HDM proteinases and other provocants. METHODS: The permeability of epithelial monolayer cultures to mannitol was calculated from measurements of clearance using a technique that permits mathematical estimation and reduction of non-cellular diffusional constraints. Permeability was studied under control conditions and after perturbation of monolayers with HDM proteinases (separated into serine- and cysteine-proteinase classes) or chelation of extracellular Ca2+. Fluorescent antibody staining was used to investigate whether the cells expressed tight junctions (staining of ZO-1), desmosomes (staining of desmoplakin) and zonulae adherentes (staining of E-cadherin). RESULTS: The Calu-3 line was identified as an airway cell line that expressed functional tight junctions, desmosomes and zonulae adherentes. Calu-3 monolayers exhibited a low clearance and permeability to mannitol, similar to that seen in the extensively characterized MDCK cell line. Clearance and permeability were significantly increased by treatment with either HDM proteinase fraction or by calcium chelation. 16HBE14o- cells also had a low permeability to mannitol under control conditions and expressed a similar repertoire of functional proteins from major intercellular junctions. In contrast, NCI-H292 and A549 cell lines were functionally deficient in tight junctions, although they did express desmosomes and zonulae adherentes to a greater extent. Epithelial permeability was found to be a more appropriate and sensitive index of epithelial perturbation than was tracer clearance. CONCLUSION: These results suggest that the Calu-3 and 16HBE14o- cell lines are useful tools in studying the mechanism of HDM proteinases on airway epithelial cell function. HDM proteinases of both cysteine and serine mechanistic classes were found to perturb epithelial adhesion and function.
This article was published in Clin Exp Allergy
and referenced in Journal of Bioequivalence & Bioavailability