GSK-3 is constitutively active in and inhibits spontaneous maturation of immature MoDCs. Immature MoDCs were washed on days 5 to 7 of culture and resuspended in culture medium at a concentration of 2 to 3 × 105 cells/mL. Cells were either continued in GM-CSF and IL-4 (iDC) or activated with E coli (1:1000). LiCl (10 mM) or SB415286 (10 μM) was added 20 minutes prior to activation or together with GM-CSF and IL-4. (A) Western blot analysis of DC lysates harvested 2 hours following activation and/or addition of LiCl. β-Actin was used to control for protein loading. One representative of 4 experiments is shown. (B) Flow cytometry analysis and IL-6 ELISAs of the supernatants were performed 36 hours after exposure to LiCl. Mean fluorescence values of CD80, CD86, HLA-A, -B, and -C, and HLA-DR and IL-6 levels in the supernatants are shown for MoDCs relative to MoDCs cultured in the absence of LiCl. Shown are the means ± SD; n = 4 (FACS) and n = 14 (for IL-6). *P < .05 compared with MoDCs not exposed to LiCl. (C) Mean fluorescence values of CD80, CD83, CD86, CD14, and IL-6 levels in the supernatants are shown for MoDCs relative to MoDCs cultured in the absence of SB415286. Shown are the means ± SD; n = 3-6 (FACS) and n = 14 (for IL-6). *P < .05 compared with MoDCs not exposed to LiCl.
