An in vitro system to model pulmonary epithelial barrier dysfunction mediated by immune effector cells

Abstract

An assay of epithelial barrier function was developed to monitor immune-mediated changes in lung permeability that may be occurring during pulmonary allograft rejection and inflammatory lung diseases. Lung tissue was obtained from minipigs, digested with collagenase (1 mg/ml) overnight, and propagated in RPMI 1640 tissue culture medium. Cells with an epithelioid morphology were purified by differential detachment using trypsin-ethylenediaminetetraacetic acid and were characterized as epithelial by positive staining with an anti-cytokeratin monoclonal antibody. Monolayers of these epithelial cells were cultured on porous tissue culture inserts, and transmonolayer resistance values were measured. Transmonolayer resistance values reached a mean of 5487 +/- 2882 omega (mean +/- 95% confidence interval; n = 9) after 5 days in culture. These values indicated the presence of functional intercellular tight junctions between the cells. Addition of cytotoxic immune effector cells to the cultured monolayers caused a rapid reduction in the transmonolayer resistance values, whereas unstimulated splenocytes failed to produce this effect. Comparison of these results with those obtained in parallel experiments performed with standard isotopic cytotoxicity assays indicated the sensitivity of the transmonolayer resistance technique. The assay described in this report will enable in vitro modeling of epithelial permeability damage mediated by both activated lymphoid cells and their soluble products.