Figure 2
FPR2 mediates scolopendrasin IX-stimulated neutrophil activation. (A) Fura-2 loaded vector-, FPR1-, or FPR2-expressing RBL-2H3 cells were stimulated with 1 μM fMLF, 1 μM MMK-1, or 50 μg/ml scolopendrasin IX. Cytosolic calcium levels were determined fluorometrically using a spectrofluorophotometer. The peak levels of cytosolic calcium were recorded. (B) Neutrophils were pre-incubated with vehicle or WRW4 (40 μM) for 30 min, and then the cells were applied to the upper well of a multi-well chamber containing several concentrations (0 μg/ml, 0.1 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, and 50 μg/ml) of scolopendrasin IX or WKYMVm (1 μM) for 90 min. The number of migrated cells was determined through counting under a light microscope. (C) Vector- or FPR2-expressing RBL-2H3 cells were applied to the upper well of a multi-well chamber containing several concentrations (0 μg/ml, 0.1 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, and 50 μg/ml) of scolopendrasin IX or MMK-1 (1 μM) for 4 h. (D) Vector- or FPR2-expressing RBL-2H3 cells were re-suspended in Tyrode’s buffer and incubated with several concentrations (0 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, and 50 μg/ml) of scolopendrasin IX or 1 μM of WKYMVm for 30 min. The peptide-induced secretion of β-hexosaminidase was determined. Data are presented as mean ± SD (n = 10 for B–D). Data are representative of two independent experiments (A). Data in panels are representative of two independent experiments (B–D). *p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle-treated control; ###p < 0.001 compared with control without WRW4 treatment.
