Figure 1.

Neutrophil migration, barrier integrity disruption, and cytotoxicity in response to varying pepsin concentrations and pH. The impact of pepsin on H292 monolayers following a 1-hour exposure was examined at a concentration range of 0.2 to 100 µg/ml, suspended in HBSS at pH 3, 5, and 7.4. Pepsin (3 to 100 µg/ml) suspended in pH 3 only induced significant neutrophil (PMN) migration (A) and increased HRP flux (B). Pepsin (≤1 µg/ml) suspended in pH 3 failed to cause a significant PMN migratory response or increase HRP flux. (C) A complete reduction in MTT conversion was observed at all concentration of pepsin (3 to 100 µg/ml) suspended in pH 3 (D) and complete reduction in MTT conversion was observed by 1 hour of treatment in pH 3 buffer without pepsin. (E) All positive and negative assay controls were conducted in pH 7.4 HBSS. For neutrophil migration, HBSS alone and infection with non-pathogen E. coli (MC1000) served as negative controls and a gradient of 100 nM fMLP and infection with pathogenic P. aeruginosa (PAO1) served as positive controls. For HRP flux and MTT conversion, HBSS alone served as a negative control and treatment with 0.1% detergent triton X-100 (Tx-100) served as a positive control. 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 quadruplicate wells assayed (n = 4). Differences between pepsin (pH 3) and HBSS (pH 7.4) negative control were considered significant at p ≤ 0.05 and noted by the symbol (*). P values involving multiple comparison were calculated by one way ANOVA with Dunnett’s test to compare the impact of individual pepsin concentrations (at pH 3) with HBSS control (at pH 7.4) within an internally controlled experiment.