Figure 2
The effect of the DGK inhibitor R59022 on exocytosis from ANCA IgG-stimulated primed neutrophils. TNF-α primed neutrophils in the presence of cytochalasin B (2.5 × 105) were treated with ANCA IgG, normal control IgG (200 µg/mL) or the positive control fMLF (1 µmol/L) for 15 min. Paired samples of cells were also treated with R59022 (18 µmol/L) or VU0155069 (1 µmol/L) for 10 min before priming. Supernatants were collected and analyzed for MPO using a specific substrate (A). Primed neutrophils in the presence of cytochalasin B (2.5 × 105) were treated with ANCA IgG as above in the presence or absence of R59022; CD63 surface expression was then analyzed by flow cytometry (B). Neutrophils were also primed with TNF-α for 15 min in the presence of fMLF (1 µmol/L) as a substitute for cytochalasin B, treated in suspension as above and measured for MPO release (C). Primed neutrophils in the presence of cytochalasin B (2.5 × 105) were treated with ANCA IgG as above in the presence or absence of PA or DAG (both at 12.5 µmol/L). Paired samples of cells were also treated with R59022 (18 µmol/L) for 10 min before priming. Supernatants were collected and analyzed for the activity of MPO (D). Data are the mean of six different neutrophil donor responses using a panel of two normal IgG preparations and three ANCA preparations (one MPO-ANCA, two PR3-ANCA). All data represent values that have been normalized as a percentage of the unstimulated (unstim) uninhibited control cells with optical density (OD) (A, C and D) or geometric mean (B) as the primary measurement. ***P < 0.001, **P < 0.01, *P < 0.05 by one-way ANOVA with posttest.