Effects And Mechanisms Of Mifepristone As Glucocorticoid Antagonist In The Regulation Of Human Uterine Natural Killer Cells

Mifepristone (RU486) is a synthetic19-norsteroid, which is a potent antagonist of progesterone and glucocorticoid. When it was first found, mifepristone has been used as an abortion drug. Accumulating evidences from the basic and clinical researches demonstrated a variety of potential applications for mifepristone in the areas of gynecology, endocrinology, oncology, and immunology. Recently, several studies have shown that low-dose mifepristone retards endometrial development to achieve the purpose of contraception, so-called endometrial contraception. However, the exact mechanisms responsible for contraception by mifepristone are not fully understood.Part I Effects of human uterine stromal cells on uNK-cell survival and function in vitro Objective:To investigate the effects of human uterine stromal cells on uNK-cell survival, proliferation and cytotoxicity in vitro.Methods:Human endometrial stromal cells were separated in vitro. The conditioned medium was derived from the endometrial stromal cells in proliferative phase, secretory phase, and early pregnancy. Uterine natural killer (uNK) cells were separated and purified from human decidual samples of early pregnancy and analysed by flow cytometer. The effects of stromal cell-derived conditioned medium on the morphology of uNK cells were detected by light microscope. uNK-cell proliferation and cytotoxicity to target cells K562were examined by mitochondrial lactate dehydrogenase-based MTS staining and flow cytometry.Results:In current study, the high purity of uNK cells could be purified by using the human NK-cell immune magnetic beads isolation system (94.52±2.44%). Recombination IL-2could maintain uNK-cell survival and promote uNK-cell cytotoxicity. Conditioned medium from stromal cells at different physiological stages could not maintain uNK-cell survival in vitro; conditioned medium from stromal cells in both the secretory phase and early pregnancy could promote uNK-cell proliferation (1.12±0.03and1.22±0.06). In contrast with the control group (63.39±5.18%), conditioned medium in all groups were able to inhibit uNK-cell cytotoxicity (46.98±3.58%,45.28±4.05%,43.72±1.67%). However, there was no significant difference among the proliferation, secretory, and decidua groups.Conclusion:Human endometrial stromal cells could not maintain the uNK-cell survival, but may be involved in the regulation of uNK-cell functions through influencing proliferation and cytotoxicity. Part Ⅱ Effects of mifepristone on the bioactivity of human uNK cellsObjective:To investigate the influences of different doses of mifepristone on the proliferation, apoptosis, IFN-γ secretion, and the expression of perforin and granzyme-B in human uNK cells.Methods:Human uNK cells were purified from decidual tissue and treated with different concentrations of mifepristone in vitro. The cell proliferation was examined by using MTS staining; flow cytometer was used to evaluate the apoptosis, cytotoxicity and expression of perforin and granzyme-B; ELISA assay was used to detect the secretion level of IFN-γ.Results:We found that mifepristone (0,65,200and1000nmol/L) dose not directly affect the proliferation, apoptosis, and IFN-γ secretion of human uNK cells.65nmol/L and200nmol/L mifepristone had no significant influence on uNK cell-mediated cytotoxicity (68.92±5.96%,69.62±4.22%and62.24±4.39%, p>0.05), which could be significantly augmented by1000nmol/L mifepristone (62.24±4.39%and73.16±4.27%, p<0.05). In addition,1000nmol/L mifepristone significantly promoted the expression level of perforin (49.13±0.03%and36.23±0.05%, p<0.05) but not granzyme-B (50.50±0.02%and45.97±0.03%, p>0.05).Conclusion:Our current study indicated that mifepristone dose not directly affect the proliferation, apoptosis, and IFN-y secretion of uNK cells, but augments uNK-cell cytotoxicity and this effect is probably due to the increased expression of perforin by mifepristone. Part Ⅲ The roles and mechanisms of mifepristone in the regulation of human uNK-cell cytotoxicityObjective:To explored whether the increased cytotoxicity and perforin expression in uNK cells by mifepristone is due to either anti-progesterone or anti-glucocorticoid activity, and also investigated relevant changes in the mitogen-activated protein kinase (MAPK) pathway and evaluated the roles of ERKl/2activity in mifepristone-induced changes of human uNK-cell cytotoxicity and perforin expression.Methods:Human uNK cells were purified from decidual tissue and treated with different concentration of progesterone, cortisol and mifepristone in vitro. Flow cytometer was used to examine the cytotoxicity and perforin expression of uNK cells; Western blotting was used to detect the phosphorylation level of ERK1/2, p38and JNK. A specific ERK1/2inhibitor of the MAPK pathway, PD98059, was used to evaluate the roles of ERK1/2activity in the mifepristone-induced changes of human uNK-cell cytotoxicity and perforin expression.Results:Progesterone had no effects on the cytotoxicity of human uNK cells and cortisol (1×10-6M and1×10-5M) significantly decreased in the cytotoxicity of uNK cells (55.07±4.04%,56.20±3.11%and62.30±2.69%, p<0.05). Mifepristone (1×10-6M) increased the uNK cell-mediated cytotoxicity (62.30±2.69%and73.16±4.26%, p<0.05) and perforin expression (36.23±4.84%and49.13±2.92%, p<0.05) and these effects could be reversed by cortisol (1×10-6M)(73.16±4.26%3and66.92±2.87%, p<0.05;49.13±2.92%and33.14±3.45%, p<0.05). Mifepristone increased the phosphorylation levels of ERK1/2but not p38and JNK in uNK cells; cortisol significantly suppressed the mifepristone-induced phosphorylation of ERK (0.87±0.09and0.48±0.07, p<0.05). After pretreatment with PD98059, mifepristone had no effect on uNK cell-mediated cytotoxicity and perforin expression, with or without cortisol.Conclusion:Mifepristone augments the uNK-cell cytotoxicity and increases the perforin expression through the antagonism of glucocorticoid, and induces the phosphorylation of ERK1/2in human uNK cells. ERK1/2pathway may be involved in the immune regulation of human uNK cells by mifepristone.