MHC-II and costimulatory molecules are specifically upregulated on neutrophils that have phagocytized RBCs. (A) Schematic overview showing magnetic selection of the neutrophils that have phagocytosed RBC-ops. RBCs were biotinylated and coupled to magnetic streptavidin beads. RBCs were opsonized and incubated with neutrophils for 1 hour. Next, RBCs were lysed and phagocytosing neutrophils were obtained by magnetic selection. (B) Cytospins of the nonphagocytosing and phagocytosing neutrophils after magnetic separation. These images were blindly chosen and are representative for 5 different experiments. May-Grünwald Giemsa stain; original magnification ×500. (C-G) Surface expression of HLA-DR, CD40, and CD80 on nonphagocytosing and phagocytosing neutrophils. Data represent the MFI values of HLA-DR, CD40, and CD80 (mean ± SEM; n = 4-10). (H) Relative levels of mRNA for HLA-DR in neutrophils ± RBC-ops measured by quantitative PCR. Data are normalized for GUS expression (mean ± SEM; n = 4). Asterisks above the 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 < .01; *P < .05).