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The mechanisms by which endotoxins mediate neutrophil transepithelial migration and lung inflammation are unclear. It was hypothesized that both the presence and orientation of epithelial cells are critical to endotoxin-induced neutrophil migration. Neutrophil migration was compared through naked filters and filters with A549 lung epithelial monolayers grown on the upper and lower surface of permeable filters to simulate the apical and basal directional movement of neutrophils, respectively. The endotoxin, Pseudomonas aeruginosa lipopolysaccharide, was placed below the filter, acting as either a basal or an apical stimulus. Endotoxin without serum failed to stimulate neutrophil migration. In the presence of 1% human serum, endotoxin-induced neutrophil migration through naked filters was dose dependent. Endotoxin-induced neutrophil migration across A549 monolayers was minimal when the monolayers were cultured on the upper surface of the filters (basal stimulus). In contrast, neutrophil transepithelial migration was much greater and dependent on both dose and time when the monolayer was cultured on the lower surface of the filter (basal to apical neutrophil directional movement). Furthermore, enhanced neutrophil transepithelial migration was greater with an apical than with a basal stimulus. Endotoxin-induced neutrophil transepithelial migration was markedly inhibited (&gt;95%) by actinomycin D pretreatment of the monolayers, suggesting the necessity for intact protein synthesis capacity of the A549 cells. Thus, both the presence and orientation of airway epithelium are key in supporting endotoxin-mediated lung neutrophilic responses.

Orientation and presence of epithelium are key to endotoxin-induced neutrophil migration