The effect of NEI, neutrophil elastase, and PG-1 added in vitro on the antimicrobial activity of porcine wound fluid.

(A) To determine the effect of NEI on the in vitro antimicrobial activity, wound fluids were either treated with solvent in vivo (24-hour treatment) and in vitro (2-hour treatment) (“solvent/solvent”); solvent in vivo and 0.25 mM NEI in vitro (“solvent/NEI”); 0.25 mM NEI in vivo and solvent in vitro (“NEI/solvent”); or 0.25 mM NEI in vivo and in vitro (“NEI/NEI”). Antimicrobial activity was measured by RDA. NEI added in vitro to the wound fluids had no effect on antimicrobial activity against S epidermidis and E coli under the assay's most sensitive conditions (10 mM NaP) (n = 3). (B) Neutrophil elastase (E) added in vitro at 100 μg/mL final concentration to wound fluid that had been treated with NEI in vivo fully restored the antimicrobial activity under RDA against E coli compared with solvent-only control (S) and NEI-treated wound fluids (n = 3). (C) The average concentrations of protegrin in wound fluid treated in vivo with solvent (hatched bar) NEI (solid bar) were determined by semiquantitative Western analysis (n = 3). The in vitro antimicrobial activity of the same wound fluid that had been treated in vivo with NEI could be restored to that of the solvent-only control with the addition of an average of 36 μg/mL PG-1 (RDA against E coli, shaded bar) (n = 3). Note that the difference in measured protegrin concentration between the NEI- and solvent-treated fluids is equivalent to the restorative concentration of protegrin. Error bars represent SEM.