Effects of estrogen on Th17 cells in joints of mice with established collagen-induced arthritis. DBA/1 mice were ovariectomized, subjected to collagen-induced arthritis (CIA) and treated with 17β-estradiol (E2; 0.83 μg/day) or placebo. (A) Joint cells from mice with CIA terminated day 49 were stimulated in triplicates with phorbol 12-myristate 13-acetate and ionomycin for 40 to 42 hours in interleukin (IL)-17 ELISPOT assay. The number of spot-forming cells (SFCs) was counted in a blinded manner these are shown as SFCs per total number of cells in wells. Total numbers of (B) T helper 17 cells (Th17; IL-17+CD4+) and (C) neutrophils (CD11b+F4/80−Gr-1hi) in joints from mice with CIA terminated day 35 were determined by flow cytometry. The absolute number of cells was derived by multiplying the frequency of Th17 cells or neutrophils by the total number of cells acquired from an automated cell counter in a corresponding sample. All data shown are arithmetic mean ± SEM derived from single experiments with n = 8 or 9 mice/group. *P <0.05 (Student’s t-test).