Neutrophils upregulate MHC-II and costimulatory molecules following RBC phagocytosis. (A-B) RBC phagocytosis by neutrophils was measured by incubating neutrophils ± DiD-labeled unopsonized RBCs (RBC) or anti-GPA RBC-ops (RBC-ops) for 45 minutes. Subsequently, RBCs were lysed and the percentage of RBC-phagocytosing neutrophils was measured by flow cytometry. (A) Approximately 45% of neutrophils phagocytose RBC-ops (mean ± standard error of the mean [SEM]; n = 5). (B) Phagocytosis of RBC-ops by neutrophils was confirmed by cytospin. These images where blindly chosen and are representative for 5 different experiments. May-Grünwald Giemsa stain; original magnification ×500. (C-E) Surface expression of HLA-DR (MHC-II), CD40, and CD80 on freshly isolated neutrophils (T = 0) and neutrophils cultured for 20 hours (T = 20) with or without RBCs or RBC-ops. (F-H) Surface expression of HLA-DR, CD40, and CD80 on freshly isolated neutrophils (T = 0) and neutrophils stimulated with RBC-ops, IFNγ, TNFα, or GM-CSF and cultured for 20 hours (T = 20). The data represent the MFI values of HLA-DR, CD40, and CD80 (mean ± SEM; n = 5-7). Asterisks above straight-line bars represent significant differences compared with T = 0 control; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (****P < .0001; ***P < .001; **P < .01; *P < .05). IFN, interferon; ns, nonsignificant.