Figure 1
Ability of whole ANCA and ANCA F(ab′)2 to induce azurophilic granule release in primed neutrophils. Neutrophils (2.5 × 105) were primed with TNF-α (2 ng/mL) in the presence of cytochalasin B (10 µmol/L) for 15 min. Neutrophils were then treated with ANCA IgG, normal control IgG (200 µg/mL) or the positive control fMLF (1 µmol/L) for 15 min (A). Neutrophils were also treated in the absence of cytochalasin B (B) and treated as above but allowed to adhere onto a fibrinogen-coated (50 µg/mL) plate for 2 h. After treatment, supernatants were collected and analyzed for MPO using a specific substrate. Data have been normalized as a percentage of the unstimulated (unstim) control cells. For neutrophils primed with TNF-α in the presence of cytochalasin B, the data are also expressed as a percentage of total MPO from lysates (C). Primed neutrophils (2.5 × 105), in the presence of cytochalasin B, were also treated separately with ANCA IgG (200 µg/mL), F(ab′)2 fragments, Fc fragments or preparations of both (300 µg/mL) from the same antibody preparation for 15 min (D). Data were normalized as a percentage of the unstimulated control cells. Data are a mean of five different neutrophil donor responses using a panel of two normal IgG preparations and three ANCA preparations (one MPO-ANCA, two PR3-ANCA). ***P < 0.001, **P < 0.01, *P < 0.05 by one-way ANOVA with posttest. Tested data samples were compared with normal IgG-treated (A, B and C) or ANCA IgG-treated (D) samples.