PI3K signal activation was crucial for TIMP-3–mediated suppression of IL-12 production but not for TIMP-3–mediated repression of CD86 induction. Various concentrations of wortmannin or DMSO were used to treat imDC for 1 hour before their further stimulation with LPS or with LPS plus TIMP-3. (A) Two days later, production of IL-12, IL-10, and TNF-α in the cell-free supernatants was assayed by ELISA. (B-C) Reversal of TIMP-3–mediated IL-12 suppression by PI3K inhibitor wortmannin in DCs. Panel C was calculated based on the data collected in panel B. (D) The time-course tracking of TIMP–3 induced IL-12 down-regulation and its reversal by PI3K inhibitor wortmannin. The culture supernatants were collected at different time points for IL-12 assay by ELISA. (E) Whole-cell lysates from imDCs with LPS (1 μg/mL) with or without TIMP-3 (50 ng/mL) treatment for 30 minutes were subjected to Western blot analysis of phosphorylated Akt. (F) The same experiment was performed as in panel A, and the expression of CD86 was analyzed by flow cytometry. (G) Expression of MARCH1 mRNA in DCs matured with LPS (1 μg/mL) with or without TIMP-3 (50 ng/mL) for indicated times was analyzed by quantitative RT-PCR. Data are the mean ± SD from 3 independent experiments. *P < .05.