Studies On The Expression Of Endomorphins And Mu-opioid Receptors On Murine Dendritic Cells And Their Functions

Objective: The aim of the present study was to investigate the expression of endomorphins and their high affinity mu-opioid receptors on murine dendritic cells, as well as their functions.Methods: Bone marrow dendritic cells were extracted from normal, 7- to 8-week-old C57 BL/6J mice, purified with anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads and cultured in the presence of GM-CSF. FACS analysis indicated that the purity of dendritic cells was more than 95%. At first, we investigated whether the dendritic cells express the mu-opioid receptors. RT-PCR was used to detect the mu-receptor gene expression on the dendritic cells. Immunofluorescence double staining combined with confocal microscopic observation was used to detect the mu-opioid receptor protein expression with CD11c marker. Quantitative PCR was used to examine the control of mu-opioid receptor expression by different Toll-like receptor ligands. Secondly, we investigated whether the dendritic cells produce and secrete the endomorphins. Immunofluorescence staining was used to detect to expression of endomorphins in the dendritic cells. The enzyme immunoassay (EIA) was used to determine the contents of endomorphins secreted in the supernatant. Finally, on the base of confirmation of the presence of mu-opioid receptor and endomorphins in dendritic cells, we investigated their functional significances. Competitive binding assay was used to measure the forskolin-induced cAMP formation. Western blot was used to determine the phosphorylation of p38MAPK and ERK in the signal pathways. ELISA was used to measure the concentration of various cytokines in the supernatant. 3H-thymidine incorporation was used to detect the proliferation of T cells under the condition of co-culture of T lymphocytes with endomorphin-treated dendritic cells.Results: RT-PCR analysis and double immunofluroscence staining revealed the expression of mu-opioid receptor gene and protein in activated murine dendritic cells. Various ligands of Toll-like receptors could regulate the dynamic expression of mu-opioid receptor in murine dendritic cells. Similarly, we observed that activated murine dendritic cells produce endomorphin in cytoplasm and secrete into the supernatant. Functionally, treatment of DCs with endomorphin-1, one specific agonist of mu-opoid receptor, could inhibit forskolin-induced increase of cAMP levels in activated DCs. The signaling which was triggered by endomorphin-1 could suppress the activation of p38 MAPK and enhance the activation of ERK signaling in LPS-stimulated DCs. Treatment of DCs with endomorphin-1 could increase the production of IL-10 and decrease the production of IL-12 and IL-23. Consistently endomorphins-treated dendritic cells could inhibit the proliferation of T lymphocytes. All the above-mentioned effects of endomorphin-1could be reversed by naloxone (non-specific opioid receptor antagonist) and CTOP (specific mu-opioid receptor antagonist), suggesting that these effects of endomorphin-1 were mediated by mu-opioid receptors.Conclusion: The results demonstrated the inducible expression of functional mu-opioid receptor and endomorphins on activated dendritic cells and the secretion of endomorphins, suggesting that the signaling initiated by endomorphin can alter cellular signaling pathways, modulate the functional properties of dendritic cells and modulate immune response, and impling the involvement of dendritic cells in the crosstalk among the nervous, endocrine and immune systems...