Mifepristone Induces Maturation Of Human Monocyte-derived Dendritic Cells

The survival of the semi-allogeneic fetus has been an immunological paradox. The mechanism by which the fetus is not rejected by maternal immune system is unclear. It is generally accepted that fetal survival depends on the local immunity at the maternal-fetal interface. Human endometrium is richly populated by maternal leukocytes, including uterine naturally killer (uNK) cell, T cell, macrophage and DCs. Considerable studies have been focused on the role of uNK and T cell in maternal-fetal tolerance. Recent evidence suggests that DCs are critical mediators of immunological interactions at the maternal-fetal interaction.DCs are the most potent antigen-presenting cells with a unique ability to induce primary immune responses. The capacity of DCs to activate T cells is 1,000 times more than macrophages and B cells. Human mucosal surfaces contain specialized DCs capable of sensing these external stimuli and mounting appropriate local responses depending on the nature of the elements they encounter. Emerging evidence indicates that DCs are responsible for the establishment of tolerance as well as immunity. Generally, immature DCs induce tolerance and mature DCs induce immunity. Although small numbers in endometrium and decidua, recent evidence suggests that DCs are involved in this balance between immune defence and maternal-fetal tolerance.Mifepristone (Ru486), a potent progesterone and glucocorticoid receptor antagonist, is widely used as an emergency contraceptive and its dosage is gradually reduced. A single dose of 10 mg mifepristone is one of the most effective hormonal methods for emergency contraception, with little side effects. Recently, mifepristone has shown the potential to be used as a novel estrogen-free contraceptive which disturbs endometrial development enough to prevent implantation without affecting ovulation. Daily dose of 1 mg mifepristone, reaching a steady plasma level of 65 nmol/L with no impairment on ovulation, was effective to prevent pregnancy and is called "endometrium contraception". But the exact mechanism of endometrial contraception, especially immunological mechanism, remains unknown.In current study, we first investigated endometrial dendritic cell populations during the normal menstrual cycle by immunohistochemistry and explored the role of DCs in the establishment of maternal-fetal tolerance. Then we investigated the effect of mifepristone on the phenotypic and functional maturation of human moncyte-derived DCs, and consequently, to elucidate the mechanism of mifepristone as an anti-implantation contraceptive or emergency contraceptive.PartⅠEndometrial dendritic cells populations during the normal menstrual cycleObjective:To investigate the number and distribution of dendritic cells in normal endometrium of reproductive age during the normal menstrual cycle. And to explore the role of DCs in the establishment of maternal-fetal tolerance. Methods:Normal endometrial sample weres collected from forty women of reproductive age,20 in the proliferative phase (d6-10) and 20 in the "window of implantation" (d20-24). Endometrial specimens were stained with hematoxylin and eosin for histological examination. And the expression of CD1a and CD83 was evaluated by immunohistochemistry.Results:1. CD1a positive dendritic cells were found in all cases of the "window of implantation" and most cases of the proliferative phase (18/20,90%).2. The density of CD1a+DCs in the "window of implantation" was 18.2±5.76 cells/mm2, significantly higher than that in the proliferative phase (6.5±4.05 cells/mm2, P＜0.05).3. Serum progesterone level was correlated with the density of CD1a+ DCs.4. No CD83+ DCs were found in human emdometrium.Conclusion:1. Human endometrium contains CD1a+ DCs, but no CD83+ mature DCs.2. The number and maturation of DCs may be regulated by ovarian hormones.3. The highly coordinated cyclical changes in DCs populations may be important for the establishment of maternal-fetal tolerance. PartⅡExpansion of DCs from human peripheral blood in vitroObjective:To generate large numbers of DCs from CD14+ monocytes in vitro.Methods:Peripheral blood mononuclear cells (PBMC) from healthy female donors (buffy coats obtained from the local central blood bank) were isolated by Ficoll-Hypaque density gradient separation. CD14+ monocytes were enriched with bead-labeled anti-CD 14 mAb on a high-gradient magnetic cell sorting system (MACS). Purified CD14+ monocytes were cultured in PRMI1640 medium containing 100 ng/ml rhGM-CSF and 20 ng/ml rhIL-4 for 6days. LPS (100 ng/ml) was added for another 48 hours to induce maturation. The morphology was monitored by light microscopy and scanning electron microscopy, and the immunophenotypes were determined by flow cytometry. Allogeneic mixed leukocyte reactions were used for immune functional assays.Results:1. Light microscopy and scanning electron microscopy showed typical DCs morphology after 6 days.2. Freshly isolated monocytes were CD14+CD1a-.After 6 days, cells showed an immature DCs phenotype (CD14-CD1a+ HLA-DRlowCD83-) and induced little or no allogeneic T cells proliferation in MLR.3. Addition of LPS to immature DCs resulted in a marked increase in the expression of HLA-DR, as well as CD83, indicative of DCs maturation. And mature DCs stimulated allogeneic T cell proliferation markedly in a stimulator/responder ratio-dependent manner. Conclusion: This study developed a simple way to generate large numbers DCs from PBMC with rhGM-CSF and rhIL-4. With analyzing morphology, phenotypes and function, we confirmed these cells were typical DCs.PartⅢMifepristone induces maturation of human monocyte-derived dendritic cellsObjective:To investigate the effect of mifepristone on the phenotypic and functional maturation of human moncyte-derived DCs, and consequently, to elucidate the mechanism of mifepristone as an anti-implantation contraceptive or emergency contraceptive.Methods:DCs were generated from human peripheral blood CD14+ cells cultured with GM-CSF and IL-4. On day 6, immature DCs were left untreated (negative control), stimulated with various concentrations of mifepristone (20 nmol/L,65 nmol/L,200 nmol/L and 1800 nmol/L), DMSO (solvent control), or LPS (posotive control,100 ng/mL). After 48 hours, DCs were harvested and stained with mAbs against CD1a, CD83 and HLA-DR to determine their phenotype by flow cytometric analysis. The cytokine levels secreted by DCs were determined by ELISA and the allostimulatory capacity of DCs was measured by MLR.Results:1. Flow cytometric analysis revealed that mifepristone upregulated the surface expression of MHC classⅡmolecules and specific molecule CD83.2. Compared with negative control, mifepristone enhanced the secretion of IL-12p70 by DCs significantly (P＜0.05). Up to 200 nmol/L, the IL-12p70 production by DCs was upregulated by mifepristone in a dose-dependent manner.3. Compared with negative control, mifepristone-treated DCs strongly stimulates the proliferation of allogeneic T cell in a mixed lymphocyte reaction (P＜0.05). Up to 200 nmol/L, the capacity of stimulating the proliferation of T cell was upregulated by mifepristone in a dose-dependent manner.Conclusion:Mifepristone induced a distinct phenotypic and functional maturation of DCs in vitro. Similar mechanisms may be effective in vivo, providing an explanation for the possible mechanism of mifepristone as a contraceptive agent, especially as an anti-implantation contraceptive.