Fig. 3. LL-37 induction of neutrophil chemotaxis is blocked by Pgp3.

(A) Mouse bone marrow-derived neutrophils were used for measuring chemotaxis. The neutrophils were placed in upper chamber in 200μl chemotaxis medium at the cell density of 2 million cells per ml while the chemoattractant LL-37 in different concentrations as listed at the bottom of the figure was added to the lower chambers filled with 800μl of the same chemotaxis medium. The upper and lower chambers were separated by 3μM pore-size of inserts. After two hours of incubation at 37°C, the cells in both upper and lower chambers were counted, based on which, the % of neutrophils migrated from the upper to lower chambers was calculated from 3 independent experiments and displayed along the Y-axis. Note that LL-37 at 12μM exhibited strongest chemoattraction ability, which was used for testing whether Pgp3 could affect LL-37-induced chemoattraction in (B). The experiments in (B) were carried out as described above except that different chemoattractants were prepared, including LL-37 at 12μM alone, LL-37 pre-incubated with varying concentrations of Pgp3, MIP2 at 4ng/ml alone, MIP2 pre-incubated with 36μM Pgp3 and Pgp3 alone at 36μM. The pre-incubation was carried out by mixing LL-37 or MIP2 with Pgp3 at the concentrations as indicated at the bottom of the figure for 30min at 37°C in chemotaxis medium and 800μl of the mixtures was added to the corresponding lower chambers. Note that the % of neutrophils migrated into the lower chambers that contain the LL-37/Pgp3 mixtures were significantly lower than that from the well with LL-37 alone (**p<0.01, Wilcoxon ran sum).