Figure 5

The effect of DGK inhibition on ANCA-induced intracellular calcium release. Neutrophils (2 × 10⁶) were preincubated with INDO-1 (1 µmol/L) dye for 30 min before priming. Paired samples of cells were also treated with R59022 (18 µmol/L) for 10 min before priming. The ratio of the fluorescent intensity was observed for 100 s before treating neutrophils with ANCA (200 µg/mL) and recording changes in fluorescent intensity for 500 s. Paired readings were recorded for neutrophils treated in the presence of PA (12.5 µmol/L). Mean data (A) are shown as the increase in calcium with the highest value of calcium subtracted from the baseline levels. A representative time course assay is shown with fluorescent intensity ratio converted to calcium concentrations (B). To assess the relative contributions of the intracellular Ca²⁺ store and the influx of extracellular Ca²⁺ in response to ANCA IgG stimulation, neutrophils were stimulated in Ca²⁺ free medium containing EGTA, and then after a response plateau, Ca²⁺ was added to back to the cell suspension. A paired neutrophil sample also received R59022 (18 µmol/L) before priming. Mean data for both the release of intracellular Ca²⁺ and influx of extracellular Ca²⁺ are shown (C). A representative time course assay is also shown (D). The ability of ionomycin to restore exocytosis in R59022-inhibited cells was also assessed (E). Primed neutrophils (2.5 × 10⁵) were treated with ANCA IgG (200 µg/mL) or control normal IgG (200 µg/mL) for 15 min in the presence or absence of ionomycin (5 nmol/L). Paired samples of cells were treated with R59022 (18 µmol/L) for 10 min before priming. Supernatants were collected and analyzed for the activity of MPO using a specific substrate. A representative time course assay is shown for the ability of ionomycin (5 nmol/L) to restore calcium levels in R59022-inhibited cells (F). Data have been normalized as a percentage of the unstimulated (unstim), uninhibited control cells. All graphs show the mean of four different neutrophil donor responses using a panel of two normal IgG preparations and two ANCA preparations (one MPOANCA, one PR3-ANCA). *P < 0.05, **P < 0.01, ***P < 0.001 by a paired Student t test.