Distinct glucocorticoid responsiveness of BMDCs. (A) Flow cytometric assessment of expression levels of CD86 and MHC II on BMDCs from control (CON), DEX (10nM, 24 hours), LPS (1 ng/mL, 24 hours), and LPS+DEX treated cultures shows that LPS induced both CD86 and MHC II, which was blocked by DEX. Experiments were repeated at least 4 times. (B) LPS (1 ng/mL, 24 hours) significantly induced TNF-α and IL-6 production by BMDCs. DEX (10nM, 24 hours) efficiently blocked the LPS-induced cytokine production. Averages (± SD) from 4 experiments are shown (*significantly greater than other treatment groups, P < .05; 1-way ANOVA followed by Tukey posthoc test). (C) T cell proliferation (CFSE) in CD4⁺ T cells and LPS-activated BMDC coculture was repressed when BMDCs were pretreated with DEX (10nM, 24 hours). Experiments were repeated at least 4 times. (D) Immunocytochemistry analysis revealed the expression of GR in BMDCs in vehicle, DEX (10nM, 24 hours), LPS (1 ng/mL, 24 hours), or LPS+DEX treated cells. The inset shows the detailed DCs morphology. Scale bar, 10 μm. (E) FITC-dextran uptake activity was blocked by DEX in immature DCs. BMDCs were pretreated with vehicle or DEX (10nM) for 24 hours. Averages (± SD) of 4 experiments are shown (*significantly less than other treatment groups, P < .05; 1-way ANOVA followed by Tukey posthoc test). (F-G) Mature BMDCs were more sensitive to glucocorticoid killing than immature DCs. Immature and mature BMDCs were cultured and purified on a Moflo cell sorter as described in “Methods.” Sorted cells were treated with vehicle, DEX (100nM) ± RU486 (100nM) for 24 hours. Both annexinV⁺ (F) and dead cell (DAPI⁺; G) staining indicated that mature BMDCs were more sensitive to glucocorticoid killing. Averages (± SD) of 5 experiments are shown (*significantly greater than other treatment groups (annexin staining) or significantly greater than CON and RU (DAPI staining), P < .01; 1-way ANOVA followed by Tukey posthoc test).