Dynamic changes of phenotypes and secretory functions during the differentiation of pre-DCs to mature DCs

The dynamic expression of cytokines and phenotypes during the differentiation process of dendritic cell precursors (pre-DCs) to mature dendritic cells (DCs) was investigated, and the effects of inflammatory stimulation with lipopolysaccharide (LPS) on DCs differentiation were understood. The differentiation of bone marrow cells isolated from Balb/c mice was induced to DCs in an 8-day cell culture system with RPMI-1640 complete culture medium containing 10% FBS, 20 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4). On the day 3, 6 and 7 after culture, DCs were divided into non-LPS group and LPS group, given 500 ng/mL LPS for 24 h stimulation and no stimulation respectively. The expression levels of CD11c⁺, MHC-II⁺, CD40⁺, CD80⁺ and CD86⁺ were detected by flow cytometry, and those of IL-2, IL-4, IL-10, IL-12 p70 and IFN-γ in the supernatant by ELISA. On the day 3 and 6 after culture, the expression of IL-2, IL-4, IL-10 and IFN-γ in DCs showed no significant differences between non-LPS group and LPS group, whereas the differences were significant at day 7. The expression levels of cytokines (for IL-2, IL-4, IL-10, IFN-γ and IL-12 p70: 152.86±6.91, 778.33±8.35, 44.55±2.54, 58.26±1.09 and 2423.00±57.21 pg/mL respectively) in LPS group were higher than those in non-LPS group, especially IL-12 p70 increased obviously at day 7. It was concluded that during the differentiation process of pre-DCs to mature DCs, LPS stimulates DCs producing large amounts of IL-12 p70 and Th1-type cytokines as compared with Th2-type cytokines, and day 7 may be a key time-point for DCs polarization.