Figure 3. Neutrophil cPLA2α is required for efficient neutrophil trans-epithelial migration in response to epithelial infection with PAO1.

A) Healthy primary human neutrophils were pretreated for 60 min. with multiple doses of cPLA2α inhibitor or vehicle control (DMSO 1:500) and applied to the basolateral aspect of PAO1-infected H292 epithelial monolayers. B) Neutrophils were pretreated for 30 min. with cPLA2α inhibitor (6μM) or vehicle control (DMSO 1:1000) and migrated in response to PAO1 infection, non-pathogenic MC1000 or mock infection (HBSS), or towards an exogenous IL-8 gradient placed in the apical compartment of the Transwell. C) H292 monolayers were pretreated with cPLA2α inhibitor (6μM) or vehicle control (DMSO 1:1000) prior to infection with PAO1, MC1000 or HBSS. Untreated neutrophils were applied to the basolateral surface and allowed to migrate towards infection, or towards exogenous IL-8. For all experiments, following a 2h migration, relative neutrophil migration was assessed by total myeloperoxidase activity in the apical compartment and corrected for using standard curves generated for each drug treatment of neutrophil. Data are shown as mean corrected value +/− standard deviation. Experiments were performed on at least 3 occasions with n≥3 technical replicates. P values were calculated by ANOVA with Dunnett’s test (A) or paired Student’s t-test (B, C). P values ≤0.05 was consider significant.