p85 PI3K activity in neutrophils stimulated with fMLP or ANCA IgG or by conventional FcγR ligation using either cross-linking antibodies or aggregated IgG.
p85 PI3K activation measured by in vitro kinase assays in anti-p85α immunoprecipitates from aliquots of 4 × 106 neutrophils primed with 2 ng/mL TNF-α and stimulated with either (A) 1 μM fMLP, 250 μg/mL MPO-ANCA, PR3-ANCA, or normal IgG or (B) 1 μg/mL IV.3 (FcRII XL), 1 μg/mL 3G8 (FcRIII XL), or 1 μg/mL both anti-FcγR monoclonal antibodies (FcRII+III XL) followed by 10 μg/mL GAM F(ab′)2 cross-linking antibody or (C) primed, unstimulated cells or cells stimulated with either 1 μM fMLP, 250 μg/mL normal IgG, or 250 μg/mL heat-aggregated IgG for 30 seconds (■), 1 minute (░), and 15 minutes (▨). All experiments were repeated 3 times using neutrophils from different donors, 3 different MPO-ANCA and PR3-ANCA IgG preparations, and 2 different normal IgG samples (native and heat-aggregated) and duplicates of all samples. Results show mean ± SEM of data pooled from all 3 experiments.
