Effect Of Jagged-1 On The Differentiation And Maturation Of Murine Myeloid-derived Dendritic Cells

Objective: To investigate the in vitro effect of soluble Jagged-1/Fc(Jagged-1) on the differentiation and maturation of murine myeloid-derived dendritic cells(DCs) induced by rmGM-CSF plus rmIL-4. Methods: The method for generating large quantities of high pure matured dendritic cells under rmGM-CSF plus rmIL-4 conditions from mouse bone marrow was established. The influences of Jagged-1. DAPT, LPS, Zymosan A on the morphology of DCs was checked, and the expression of NICD,Hes-1,Deltex-1 that downstream target of Jagged-1-Notch signaling were turned to westernblot. Double fluorescin-conjugated monoclonal antibody labeling technique and flow cytometry were used to evaluate the expression of differentiation mark CD11c and maturation mark MHC-Ⅱ, CD86, CD80, CD40 on the DCs induced by Jagged-1; the expression of interleukin 10 (IL-10), interleukin 4 (IL-4). transforming growth factorβ(TGF-β), interferon-gamma (IFN-γ), tumor necrosis factor a (TNF-α), interleukin 6 (IL-6), interleukin 2 (IL-2), interleukin 12 (IL-12) in the supernatant of DCs and lymphocytes that treated with Jagged-1, DAPT, LPS. Zymosan A were detected by LuminexTM system and enzyme-labeled immunosorbent assay (ELISA). Results: Two days after rmGM-CSF plus rmIL-4 treament, the product DCs, which grew as characteristic semi-anchoring clusters that keep growing till the day 5. Then the DCs cluster dismissed and enlarged the cell itself with dendritic morphology that differ from each treatment. A significant up-regulation of CD11c, MHC classⅡ. CD80. CD40 and CD86 surface expression on Jagged-treated DCs was detected. DCs exposed to LPS and Zymosan A showed similar expression levels as DCs treated with Jagged-1. One hour after Jagged-1 treament only little NICD(Notch Intracellular Domain) was deceted. but reached its peak at 6 h till 24 h with vary significantly compared to DAPT, LPS, Zymosan A treatments. Hes-1,Deltex-1 showed a delayed expression pattern that reach the peak at 24 h but last to 48 h. Cells that incubated with DAPT, LPS, Zymosan A had little expression all the time, especially Zymosan A that had undecteable level sometime. Compared with LPS or Zymosan A, the IL-4 expression of Jagged-1-treated DCs increased significantly and the expression of TNF-αdecreased markedly butγ-secretase inhibitor DAPT reversed the inhibitied expression trend of TNF-α,IL-12 and reversed it to the control level. Jagged-1 altered the amount of IL-l0,IL-6,IL-2,IL-12 and IFN-γexpression moderately and had no effect on TGF-βexpression. Lymphocytes that treated with Jagged-1 downregulated all the cytokins except TGF-βand IL-6, but the expression of IFN-γ, TNF-αand IL-4 depressed significantly. Compared to Jagged-1, LPS and Zymosan A upregulated the expression of IFN-γ,TNF-α,IL-10,IL-12 markedly but with less TGF-β. Moreover, Jagged-1 weaken the the activation capability of DCs for allogeneous lymphocytes. Conclusion: The results suggest that Jagged-1 can promote the differentiation and maturation of DCs from bone marrow cells through its downtargets-Hes-1 and Deltex-1. Jagged-1-induced DCs promoted the expression of Th2 cytokines IL-4 and IL-10 but inhibited the expression of Th1 cytokines IFN-γ, IL-12, TNF-a and prime na(?)ve T cells to the Th2 cells that prone to generate transplantation tolerance.