Frequencies of neutrophils and CD4+ T cells in spleen of properdin-deficient and wild-type mice. (A) Ly6GlowCD11b+ and Ly6GhighCD11b+ cells were determined in spleen of properdin-deficient and wild-type mice. The representative experiment shows increased frequencies of splenic Ly6GhighCD11b+ cells in arthritic mice. (B) Elevated expression of co-stimulatory molecule CD86 on wild-type arthritic Ly6GhighCD11b+ cells. The histograms are representative of three separate experiments involving five mice/group. (C) Flow-cytometry analyses of splenocyte population demonstrated increased percentage of CD4+ T cells in wild-type arthritic mice compared with the properdin-deficient CAIA group. The mean ± SD of the CD4+ T cells in spleen are presented on the graph, ***P < 0.001; Student t test. (D) Activation marker CD69 was barely expressed on the arthritic wild-type and properdin-deficient CD4+ T splenic populations. The histogram is representative of three separate experiments involving five mice/group. The ability of splenic CD4+ T cells to secrete (E) IFN-γ, (F) IL-6, and (G) IL-17 was determined with ELISA. Cells (1 × 106 cells/ml) were isolated from PBS or CAIA groups at Day 10 of disease and were stimulated in 48-well plates with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (5 μg/ml) antibodies (αCD3/28) or with ConA (2 μg/ml; where indicated), and in the presence of murine recombinant IL-2 (10 ng/ml). A control group of nonstimulated cells (unstim) was included in the experiments. After 48 hours of culture, supernatants were collected for cytokine determination. Data represent the mean ± SD. *P < 0.05; **P < 0.01; and ***P < 0.001; Student t test.