Soluble ICs induce NETs in vitro in human neutrophils that is dependent on endocytosis and is NADPH oxidase independent. (A) Freshly isolated human peripheral blood neutrophils were incubated without or with BSA, BSA/anti-BSA ICs, RBCs, rabbit IgG-opsonized RBCs, plate-bound BSA (Bnd BSA), plate-bound BSA/anti-BSA ICs (Bnd IC), or dextran sulfate for 3 hours at 37°C in the presence or absence of indicated inhibitors. Neutrophils were incubated with PMA for 6 hours as a positive control. For inhibitor studies, cells were pretreated with DMSO (−), diphenylene iodonium (DPI, ROS inhibitor), MβCD (lipid raft inhibitor), cytochalasin D (cytoD; inhibitor of actin polymerization), PP2 (Src inhibitor), piceatannol (Syk inhibitor), LY29004 (PI3K inhibitor), PI103 (PI3K inhibitor), or UO126 (ERK inhibitor) for 30 minutes before incubation with BSA/anti-BSA IC for 3 hours. Sytox Green was added to the supernatant of cells to detect DNA. The fold increase in fluorescence in samples compared with that detected in neutrophils incubated with buffer alone was calculated. IC-treated neutrophils released more DNA than those treated with BSA. Inhibitors of endocytosis blocked NETs. IgG-coated RBCs, bound BSA/anti-BSA ICs, and dextran sulfate failed to induce NETs. N = 3-20 independent experiments. For cells treated with IC alone, *P < .05 compared to cells treated with BSA. For cells treated with PMA and DPI, *P < .05 compared to cells treated with PMA alone. For cells treated with IC and inhibitors, *P < .05, **P < .01 compared to cells treated with IC alone. (B) Human neutrophils were incubated with BSA alone or sICs (left panel), or PMA in the presence and absence of DPI (right panel). Real-time generation of ROS generation was monitored using a luminol-based assay, representative profiles of which are shown. N = 2 independent experiments. (C) Human neutrophils were preincubated with functional blocking antibody to FcγRIIA (αIIA), and/or FcγRIIIB (αIIIB) or their isotype IgG control antibodies for 20 minutes before an additional 15-minute incubation with sICs. NET formation was inhibited in samples containing an FcγRIIIB blocking antibody. N = 3 independent experiments. For cells treated with IC and control IgG, **P < .01 compared to cells treated with BSA and control IgG. For cells treated with IC and blocking antibodies, *P < .05 compared to cells treated with IC and control IgG. (D) Left panel: Murine peripheral blood wild-type neutrophils were incubated with RPMI or ionomycin (positive control) for 2 hours at 37°C. Middle panel: Indicated transgenic murine neutrophils were incubated with BSA or BSA/anti-BSA ICs for 2 hours at 37°C. Fixed and permeabilized cells were stained for CitH3 and DAPI. The percentages of CitH3-positive cells with released chromatin (ie, number of neutrophils with NET structures divided by the total number of CitH3-positive cells × 100) were determined after the indicated treatments. N = 3 independent experiments. Right panel: Representative pictures of FcγRIIA expressing neutrophils treated with BSA or BSA/anti-BSA. Released chromatin (arrowhead) colocalized with DAPI-positive extracellular DNA.