Flow-cytometry analyses of properdin-deficient and wild-type bone marrow and blood neutrophils. (A) Frequencies of Ly6GlowCD11b+ and Ly6GhighCD11b+ cells were less in the BM of properdin-deficient (KO) mice with arthritis than in those of the wild-type (WT) group. The data are representative of three separate experiments involving four mice/group. (B) RANKL was expressed on Ly6GhighCD11b+ cells isolated from wild-type arthritic mice but not on those from properdin-deficient mice with CAIA. The values of the mean fluorescence intensity (MFI) are presented in each histogram. (C) Ly6GhighCD11b+ BM cells from properdin-deficient and wild-type mice expressed C5aR as presented in one individual experiment. (D) Histograms show upregulated RANKL expression on arthritic wild-type neutrophils isolated from blood at Day 10 of CAIA. The data on the graph represent the mean ± SD of the percentage of Ly6G+ RANKL+ cells in three experiments involving four mice/group; **P < 0.01; ***P < 0.001; Student t test. (E) Properdin-deficient arthritic neutrophils isolated from blood have higher C5aR expression than do wild-type cells, as shown on the histograms. The data on the graph are expressed as the mean ± SD of Ly6G+C5aR+ cells from three experiments involving four mice/group. *P < 0.05; ***P < 0.001; Student t test. (F) C5L2 was expressed on human blood CD16+ cells but not on mouse blood Ly6G+ WT cells when compared with the isotype controls of mouse IgG2a, for human C5L2 antibody and of rat IgG2b, a control for mouse C5L2 antibody. The data are representative of three separate experiments involving four mice/group.