| Dendritic cells (DCs) are currently known to be the most potent professional antigen-presenting cells, which have the unique function of stimulating naive T cells. The antigen specific adaptive immune response and Thl/Th2/Trg balance ultimately depend on the maturation status of DC. It is only the mature DC that can express the full complement of co-stimulatory molecules, adhesion molecules and antigen-presenting MHC molecules and cytokines required for effective T cell activation .There are a number of factors, such as LPS and CpG-ODN, that effect on the maturation of DCs ,which alter the development of adaptive immune respond and the Th balance.The bacterial or viral DNAs and and synthetic CpG-oligodexynucleotides (CpG-ODN)., TLR9 ligands, as well as LPS (TLR4 ligands) can induce DCs maturation and trigger adaptive immune responses. Due to cross-tolerance between LPS and other inflammation factor, we speculated that CpG-ODN plays a similar role in immune tolerance .we investigated the effect of CpG-ODN on differentiation and maturation of DCs and compared the intracellular signals in sustained CpG-ODN stimulating DCs versus single CpG-ODN stimulating DCs.Conventional method was employed to culture murine bone marrow derived dendritic cells (BMDCs) induced by GM-CSF. In the process the group with sustained CpG-ODN stimulation, which mimic the sustained infection state in vivo, was aimed to study the effect of long-termed CpG-ODN existence on the maturation of DCs. Meanwhile, the group without CpG-ODN was used as negative control and the group stimulated by CpG-ODN in the last 36 hours was called 'single CpG-ODN stimulation control'. After 7 days of culturing, DCs were harvested to perform the experiments listed as follows: (1)comparing the morphological difference; (2)measuring the phenotype and endocytosis ablity with flow cytometry analysis; (3)detecting cytokines secreted in the supernatant by ELISA; (4) examining the capability of a proliferation esentation by MLR; (5) apoptosis detection by rhodamine123; (6) measuring cell cycle with flow cytometry analysis; ?studying expression level of some signal transduction molecules by Western-blot.The results are showed as follows:Part I. Effect of sustained CpG-ODN on the phenotypes and functions of DCs.Researches have confirmed that DCs undergo a process of maturation when they primed by CpG-ODN. Mature DCs are known as upregulated expression of MHC II, CD86,CD40 etc., and increase in the capability of stimulating allogenic T cell proliferation. However, our work indicated that the maturation and function of BMDCs were remarkably slacked when co-culturing BMDCs with 2jag/ml CpG-ODN from the early stage of differentiation from bone marrow progenitors until day 7. The cell numbers in sustained CpG-ODN stimulated BMDC were reduced ((75 + 8. 066)X104 /ml vs (226?5. 664)X 104 /ml in single and (157 + 8. 981) X 104 /ml in no CpG-ODN control). Because no apoptosis was found with Rhodaminel23 dyeing in all groups, the cell cycle was detected by FACS. The results showed that the cells in G2 phase were increased in sustainedCpG-ODN group. Under optical microscope, the rumples and protuberances on the membrane of BMDC were less and blunt. FACS analysis showed that the expression of MHC II. CD86. and CD40 on sustained CpG-ODN stimulated BMDCs is notably downregulated but the uptake of antigen (FITC-OVA) was enhanced compared to those of single CpG-ODN stimulation. The ability of stimulating proliferation of allogenic and homogenic T cells was also impaired. Besides, cytokines released in the supernatant were examined and the results showed that mature BMDC secreting more IL-12p70 than that of BMDCs under sustained CpG-ODN stimulation. All these results made us believe that BMDCs stimulated by sustained CpG-ODN are immature.Part II. Mechanisms of immature BMDCs suppressed by sustained CpG-ODN stimulaton.Most studies have focused on MyD88-mediated pathway. Triggered after TLR ligation, TLR results in the recruitment of MyD88, which interacts with IRAK; this leads... |
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