Figure 3.

Mechanisms of pepsin-induced neutrophil migration and barrier integrity disruption. Pepstatin A (20 µg/ml), an inhibitor of pepsin, blocked neutrophil (PMN) migration induced by exposure to pepsin (50 µg/ml) at pH 3 (A) and prevented pepsin-induced barrier integrity disruption (HRP flux) at doses of pepsin ranging from 12.5–50 µg/ml pepsin at pH 3 (B). Pepstatin A failed to prevent neutrophil migration induced by PAO1 infection or following the application of a chemoattractant gradient of fMLP (100 nM) (C). An inhibitor of 12-lipooxygenase (CDC) failed to interfere with neutrophil migration induced by pepsin at pH 3 while PMN migration in response to PAO1 infection at pH 7.4 was significantly reduced (D). Exposure of pepsin (50 µg/ml) to NaOH bringing the solution pH up to between 8.5 and 10 followed by return to pH 3 by adding HCl and then applying to H292 lung epithelial monolayers abolished both the PMN transmigratory response and the increased HRP flux associated with control pepsin (50 µg/ml) at pH 3 treated in parallel with HCl only (E). Panels A–E are representative internally controlled experiments conducted on at least three separate occasions yielding similar results. Each data point depicts the mean +/− SD of triplicate wells assayed (n = 3). Differences were considered significant at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment.