FcgRIIA and TLR7/8 activation regulates phagocytosis of RNP-ICs. (A) Neutrophils were incubated with antibodies against FcgRs before stimulation with RNP-ICs. Phagocytosis was quantified by flow cytometry and compared with isotype antibody added (percentage of control). The experiment was repeated three times; combined results are shown and compared using paired Student’s t test (P = 0.013; P < 0.0001; P = 0.0009 for FcgRI, FcgRIIA, and FcgRIIIB, respectively). (B) TLR7/8 activation was inhibited by RNase or TLR7-9 iODN treatment before incubation of RNP-ICs with neutrophils and phagocytosis analyzed by flow cytometry. The experiment was repeated three times (ODN) or six times (RNase); combined results are compared using paired Student’s t test (P = 0.015; P = 0.0006; P = 0.014 for SLE IgG, huRNase, and TLR7-9 iODN, respectively). (C) Neutrophils were incubated with human (hu)RNase or HAGG and analyzed for IgG-Fc binding by flow cytometry. The experiment was repeated three times; combined results are shown. (D) Neutrophils were stimulated with R848 before incubation with RNase-treated RNP-ICs, HAGG, beads or zymosan. The results are expressed as phagocytosis as compared with no R848 added (% of control). The experiment was repeated six (zymosan), eight (RNP-IC+RNase), nine (HAGG), or ten (beads) times; combined results are shown and compared using paired t test (P = 0.0005, P = 0.0001, P < 0.0001, and P = 0.017 for RNP-IC+RNase, HAGG, beads, and zymosan, respectively). (E) Neutrophils, treated with or without R848 followed by cytochalasin B (CytoB; 5 µM), were analyzed for binding and uptake of RNP-ICs by flow cytometry. The experiment was repeated six times; combined results are shown and compared using paired Student’s t test (P < 0.0001 for IC vs. IC+CytoB; P = 0.0066 for IC vs. IC+R848; P = 0.0078 for IC+CytoB vs. IC+R848+CytoB; and P = 0.0158 for IC+R848 vs. IC+R848+CytoB). *, P < 0.05; **, P < 0.01; ***, P < 0.001.