PLD activation in neutrophils stimulated with fMLP or ANCA IgG or by conventional FcγR ligation using either cross-linking antibodies or aggregated IgG.
Phosphatidylbutanol production in 4 × 106 neutrophils primed with 2 ng/mL TNF-α and stimulated with (A) 1 μM fMLP, 250 μg/mL MPO-ANCA, PR3-ANCA, or normal IgG, or with (B) primed, unstimulated cells, or cells stimulated with 1 μg/mL IV.3 (FcRII XL), 1 μg/mL 3G8 (FcRIII XL), or both monoclonal antibodies (FcRII+III XL), followed by cross-linking with 10 μg/mL GAM F(ab′)2, or with (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 1 (■), 5 (░), or 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 triplicates of all samples. Results show mean ± SEM of data pooled from all 3 experiments.
