TLR7/8-activated neutrophils shed FcgRIIA from monocytes and pDCs. (A and B) PBMCs were coincubated with neutrophils (PMNs) in the presence of R848 and a pan-protease inhibitor. Levels of FcgRIIA on (A) monocytes (CD14⁺) and (B) pDCs (CD304⁺) were determined by flow cytometry and expressed as FcgRIIA (percentage of control) as compared with PBMCs incubated in medium in absence of neutrophils. In A, the experiment was repeated 11 times with the exception of the proteinase inhibitor (n = 5); combined results are shown and compared using paired t test (PMN+PBMC, P < 0.0001; PMN+PBMC vs. PMN+PBMC+R848, P < 0.0001; PMN+PBMC+R848 vs. PMN+PBMC+R848+Prot.inh., P = 0.002). In B, the experiment was repeated seven times; combined results are shown and compared using paired Student’s t test (PMN+PBMC, P = 0.0261; PMN+PBMC vs. PMN+PBMC+R848, P = 0.0002; PMN+PBMC+R848 vs. PMN+PBMC+R848+Prot.inh., P = 0.0128). (C) Monocytes were analyzed for the expression of FcgRI (CD64), as well as FcgRIIA using the monoclonal antibodies IV.3 and FUN2. The experiment was repeated four times; combined results are shown and compared using paired Student’s t test (P = 0.008). (D) Neutrophil supernatant, derived from nonstimulated (no, n = 5) or R848-stimulated (R848, n = 9) neutrophils, were added to monocytes in presence of indicated inhibitors (CMK; furin inhibitor, n = 5, and Pan; pan-protease inhibitor, n = 5) and monocyte FcgRIIA levels analyzed by flow cytometry. Combined results are shown and compared using paired Student’s t test (R848, P < 0.0001; R848+Pan, P = 0.003). (E) Neutrophil supernatant were added to monocytes and expression of FcgRIIA (IV.3 and FUN2) as well as FcgRI (CD64) analyzed by flow cytometry. The experiment was repeated four times; combined results are shown and compared using paired Student’s t test (P = 0.0043). *, P < 0.05; **, P < 0.01; ***, P < 0.001.