PGE2 Regulates The Function Of Bone Marrow-derived Dendritic Cells Through EP4-cAMP Signaling Pathway And The Effect Of CP-25

Prostaglandin E2 (PGE2) is an important metabolite of arachidonic acid substances. It has a strong immune activity, and regulates the function and survival of immune cell. PGE2 receptors are divided into four subtypes:EP1, EP2, EP3, and EP4. PGE2 binding to various EP receptors leads to inflammation. Dendritic cells (DCs) are a population of highly specialized antigen-presenting cells (APCs). DCs can regulate innate and adaptive immunity. According to the different state, DCs have a dual role:immunogenicity and tolerability. The mature dendritic cells (mDCs) promote immune function, while the immature dendritic cells (iDCs) show the characteristics of immune tolerance. The effect which involved in the inflammatory and immune responses is that PGE2-EP4-Gs protein (stimulatory G protein) signaling pathway regulates cyclic adenosine monophosphate (cAMP) levels. The combination of β-arrestin2 and G protein-coupled receptor kinase 2 (GRK2) cause the endocytosis of cell surface receptors and receptor desensitization and negatively regulate G protein signaling pathway. PGE2 can promote maturation of DCs by reducing the ability to uptake antigens, and enhancing antigen-presenting capacity of mDCs. However, whether PGE2 regulation of DCs through EP4-cAMP pathway, as well as whether β-arrestin and GRK involved in the regulation have not been reported.Our previous report is that Pae has anti-inflammatory and immunomodulatory effects. CP-25, a new Pae monomer derivative, is derived from Pae structural modification. The report in vivo experiments is that CP-25 significantly relieves and treats inflammation in CIA mice,which its related to the regulation of the T, B lymphocytes functions. Whether CP-25 can regulate the function of DCs, whether the regulatory role of CP-25 is related to EP4-cAMP pathway, the expressions of P-arrestin and GRK. It is unclear. The present work is to investigate that PGE2 regulates the function of DCs and the effect of CP-25.Aim:To investigate the effect of PGE2 on the function of bone marrow-derived dendritic cells and its mechanisms. To investigate the regulatory effect of CP-25 on the function of bone marrow-derived dendritic cell under the stimulation of PGE2 and its mechanism, which reveal the mechanism of PGE2 involved in the regulation of DCs function through receptors and the effect of CP-25, and provide an improtant experimental evidence.Methods:Bone marrow-derived DCs was research subjects. The changes of the expression of EP4, cAMP levels, the expressions of β-arrestin2 and GRK2 in bone marrow-derived DCs stimulated by PGE2 under the different concentrations were analyzed. The effects of CP-25 on the above changes were also observed. The expressions of CD40, CD 80, CD83, MHC-Ⅱ on DCs and the ability of antigen uptake were analyzed by flow cytometry. The expression of EP4 on DCs was analyzed by flow cytometry, which use the mean fluorescence intensity as the level of expression. T cells proliferation was determined by the 3-(4,5-2 dimethylthiazal-2yl) 2, 5-diphenyltetrazoliumbromide (MTT) assay. The cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The expressions of GRK2 and β-arrestin2 were determined by western blot. Results: 1. PGE2 promotes the maturation of DCs and increased the expression of EP4 on DCs.PGE2 (2.5,5,10 nmol/L) enhanced significantly the expression of CD40, CD83, MHC class Ⅱ molecules on DCs. However, PGE2 had no effect on CD80 expression in all of concentrations. PGE2 (1.25,2.5,5,10,20 nmol/L) inhibit significantly the ability of antigen uptake of DCs. DCs stimulated by PGE2 (2.5,5,10 nmol/L) could promote T cell proliferation. PGE2 (2.5,5,10 nmol/L) increased EP4 expression. 2. CP-25 recovered the abnormal function of DCs by regulating the EP4-cAMP signaling pathwayTreatment of CP-25 (10-5,10-6,10-7mol/L) significantly decreased the CD40, CD83, MHC-Ⅱ expression on DCs, suppressed the proliferation of T cell, increased the ability of antigen uptake. CP-25 (10-5,10-6,10-7mol/L) significantly decreased the expression of EP4 receptor on DCs. CP-25 (10-5,10-6,10-7mol/L) increased the production of cAMP. CP-25 (10-6mol/L) significantly decreased the expressions of GRK2 and P-arrestin2 on DCs.Conclusion:1. PGE2 can promote the maturation of DCs through increasing high expression of costimulatory molecules, inhibiting antigen uptake, promoting T cell proliferation.2. The effects of PGE2 on DCs maturation are related to the regulation of EP4-cAMP signaling pathway.3. CP-25 significantly inhibited the maturation of DCs induced by PGE2 through regulating EP4-cAMP sigaling pathway.