IL-8 increases the susceptibility of polarized airway epithelia to AdV entry, apical CAR^Ex8 protein expression, and neutrophil adhesion at the apical surface.

Abstract

<p>The apical surfaces of polarized A-D) Calu-3 cells or F-I) primary human airway epithelia were exposed to IL-8 for 4 h. A) Mock (0) or IL-8-exposed Calu-3 epithelia were transduced with AdV5 from the apical surface and analyzed 24 h later for the fold change in viral genomes (Vg) relative to GAPDH by qPCR. B) TER before or after IL-8 (30 ng/ml) exposure. C) Western blots for CAR^Ex8, total CAR, actin, and E-cadherin protein expression in lysates or D) CAR^EX8 and actin after apical surface-specific biotinylation. E) CAR^Ex8 and actin protein expression in lysates from Calu-3 cells exposed to IL-8 for different lengths of time. The apical surface of polarized primary airway epithelial cells were exposed to IL-8 and F) CAR^Ex8, actin, and E-cadherin protein expression in lysates or G) after apical surface-specific biotinylation. H) Polarized primary human airway epithelia were either mock or IL-8 treated for 4 h. Cells were then either untreated or treated with purified AdV5 FK, as indicated, followed by an adhesion assay with primary neutrophils stained with calcein green. Bound neutrophils were imaged using fluorescence microscopy (10X lens, white bar = 150 μm) and I) quantified using Metamorph software. Error bars represent the SEM from three independent experiments: *p < 0.05, A and B by one-way ANOVA or I, IL-8 treatment versus untreated or FK treated.</p