| Dendritic cells (DCs), which are potent professional antigen presenting cells (APC), play the unique role in immune system because of their abilities to activate naive T cells and initiate a primary immune response. DCs may function as immune-stimulating cells as well as tolerance-inducing cells. DCs change immunophenotypically and functionally during their maturation. Immature DCs display tolerance-inducing activities, though they tum to exhibit their immunogenicity after maturation. Recent studies have outlined that exploring the immune-regulatory capacities of dendritic cells holds great promise for the treatments and the prevention of cancer, infection, autoimmune disease, transplant rejection. So the factors and mechanisms related to the development and maturation of DCs have been the research hotspots in the field of immunology.The neuropeptide, a-melanocyte stimulating hormone (a-MSH) is well known for its anti-inflammatory and immunomodulating capabilities, and the biological activities of a -MSH are mediated by MC1R-MC5R. Human monocyte-derived dendritic cells (MoDC) express MC-IR and the expression of the costimulatory molecules CD86 and CD40 is downregulated on DC in the presence of a -MSH. Thus, a -MSH may exert its immunomodulating actions by altering the maturation and function of DCs. It is known that tumor necrosis factor-a (TNF-a) and triggering receptor expressed on myeloid cells-1 (TREM-1) exert the enhancing actions on the activation and maturation of DCs. Based on the above researches, whether a-MSH has the regulatory functions on TNF-a or activated TREM-1 inducing the maturation of MoDC and the related mechanisms are investigated.Part I Regulatory actions of a -Melanocyte Stimulating Hormone on the Maturation of Human Monocyte-derived Dendritic CellsConventional method was used to obtain monocytes in leukocyte-enriched buffy coat from healthy donors, and MoDCs were induced in the presence of GM-CSF plus IL-4. MoDCs were divided into 4 groups: MoDCs induced only in the presence of GM-CSF plus IL-4 (MoDC); mature MoDCs induced by TNF-a or agonist anti-TREM-1 mAb (TNF-a -treated MoDC or anti-TREM-1-treated MoDC); MoDCs treated by a-MSH (a-MSH-treated MoDC); MoDCs cultured with a-MSH each time 2h before the stimulation of TNF-a or anti-TREM-1 mAb (a -MSH+TNF-a -treated MoDC or a -MSH+ anti-TREM-1-treated MoDC). After 8 days of culture above mentioned, DCs were harvested to perform the experiments listed as follows: (1) observation of the morphological changes; (2) FACS analysis of the phenotype and endocytosis ability; (3) examination of the ability stimulating the allogenic T cell proliferation by MLR; (4) test of the antigen presenting capability; (5) detection of IL-12 secretion in the supernatant by ELJSA.The results showed that compared with TNF-a -treated MoDC which had the characterization of mature DC, the membrane protuberances of a-MSH+TNF-a-treated MoDC were less and blun observed under optical microscope. FACS analysis showed that the expression of MHC-II, CD86,CD83 and CDla on a -MSH + TNF-a -treated MoDC was notably downregulated.The uptake of antigen (FITC-OVA) was enhanced by a-MSH + TNF-a-treated MoDC compared with those of TNF-a -treated MoDC. The abilities stimulating the proliferation of allogenic T cells and presenting antigen were also impaired. Mature TNF-a -treated MoDC secreted more IL-12 than that of a -MSH+TNF-a -treated MoDC. All these results indicate that MoDC maturation induced by TNF-a was inhibited in the presence of a -MSH.Compared with anti-TREM-1-treated MoDC which had the characterization of mature DCs, a-MSH +anti-TREMl-treated MoDC observed under opticalmicroscope had less and blunt dendrites. FACS analysis showed that the expression of MHC-II, CD86, CD83 and CDla on a -MSH + anti-TREM-1 -treated MoDC was downregulated markedly. Both day 3 MoDC and day 7 MoDC expressed the low levels of TREM-1 protein. TREM-1 expression on Day 7 MoDC was induced to a higher level following stimulation with LPS for 24h and decreased by adding a-MSH.
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