The Effect Of Endomorphin-1on The Human Dendritic Cells And Its Mechanism

BackgroundRheumatoid arthritis (RA) is one of the most common inflammatory joint diseases.The clinical manifestations of RA are broadly, the continuity and progress of peripheraljoint synovial inflammation, pannus formation, and the resulting damage of bone andcartilage are the main features of RA. The pathogenesis of RA is still unclear, theetiology of RA is most likely associated with multifactorial factors. RA can be seen asthe auto-immune disease mediated by T cell activation which induced by unknownantigen.Dendritic cells (DCs) are the most effective and professional antigen presentingcells (APC) that initiate primary immune responses. They are located at surveillancesites where they capture and process antigens. And they are matured gradually in theprocess of transition to peripheral lymphoid tissue. Mature DCs can present antigenicpeptides to T cells, then DCs can express lymphocyte costimulatory molecules, initiateand regulate T cells and B cells immune response, secrete cytokine, then produce thecorresponding biological effect. So, DCs play a critical role in RA.Endomorphin (EM) is an endogenous ligand of mu-opioid receptor, and has a widerange of biological effects, such as analgesia, cardiovascular effects and immuneresponses. It is reported EM-1inhibited the secretions of IL-6, IL-8in osteoarthritis andrheumatoid arthritis patient’s synovial fluid, prompting EM-1has the anti-inflammatoryeffect. There has rarely reported that EM-1can affect the differentiation, maturation andimmune function of DCs, and participate in the formation and development of RA, so itis worthy of our consideration.Objectives：1. To observed the effects of EM-1on maturation and function of the human monocyte-derived dendritic cells;2. To explore the roles of TLR2and TLR4; on EM-1’s effect on immune function of dendritic cells;3. To reveal the mechanisms of EM-1on immunomodulatory of dendritic cells and provide the theoretical and experimental evidence for EM-1treatment on rheumatoid arthritis.Methods:Healthy human peripheral blood was collected and mononuclear cells were isolated.After monocyte-enriched cells were cultured with GM-CSF and IL-4, at the sixth days,the imDCs were stimulated with EM-1or (and) LPS, the cells were divided into fourgroups: Blank group (without any factors), EM-1group (treated with EM-1at1×10-6mmol/l), lipopolysaccharide group (treated with LPS at100ng/ml), LPS+EM-1group (treated with EM-1at1×10-6mmol/l and LPS at100ng/ml), cultured for48hours.1. Cells were labeled with CD11c fluorescent antibody and observed under inverted microscope, the purity of DCs populations was identified by flow cytometry.2. The cells were stimulated with different treatments and harvested, the expressions of the surface markers (CD83, CD80, CD86, HLA-DR, CCR7and MOR) were analyzed by flow cytometry; the levels of interleukin-10and interleukin-12in the supernatants were detected by ELISA．3. The cells proliferation of allogeneic T cells had co-cultured with DCs, and the proliferation of T cells were analyzed by CFSE method. At the same time, the levels of IFN-γ and IL-12in the supernatants of mixed lymphocyte proliferation experiment were detected by ELISA.4. DCs were collected from different groups, and the expressions of TLR2,4on DCs were analyzed by Flow cytometry. The expressions of TLR2and TLR4at mRNA level in DCs were measured by RT-PCR.Results:1. The results showed that compared with the control group, the expressions of CD80, CD86, CCR-7and HLA-DR on dendritic cells were increased after treatment with EM-1, in contrast to LPS group, the expressions of HLA-DR and MOR were increased in EM-1+LPS group, as well as the increase of CD83, CD80, CD86and CCR-7,the secretion of IL-10and IL-12were inhibited when the dendritic cells were treated with EM-1;2. In the mixed lymphocyte reactions, EM-1inhibited the stimulatory capacity of dendritic cells on T lymphocytes. At the same time, EM-1stimulation inhibited the secretions of IL-12and IFN-γ in the mixed lymphocyte reactions;3. Flow cytometry and RT-PCR results showed that the expressions of TLR2and TLR4in DCs was down-regulated when DCs were treated with EM-1.Conclusions:1. EM-1enhanced the expressions of HLA-DR, CD83, CD80, CCR-7and CD86on DCs, reduced the secretion of IL-12and IL-10, suggesting EM-1may be involved in the DC mediated inflammatory response;2. EM-1inhibited the secretions of IL-12and IFN-γ, while suppressed the proliferation of T lymphocytes. EM-1had anti-inflammatory effect, which may be relate to the immune function of DCs;3. The effects of EM-1on the immune function of DCs may be associated with the expressions of TLR2and TLR4on the DCs.