The Effect Of Mifepristone On The Peripheral Blood Natural Killer Cells Activity And Expression Of CD94/NKG2A And NKG2D During The Secretory Phase

Background: A fetus is considered to be a natural semi-allograft, since half of the fetus histocompatibility antigens come from the father, and trophoblasts share common mechanisms and antigens with tumor cells and viruses. Throughout the implantation process, the embryo is not rejected by the maternal immune system for the existence of complicated immunological regulation. There are two interfaces between maternal immune cells and the fetus. The first interface is between maternal immune cells and the fetal trophoblast in the deciduas. The second immune interface of pregnancy involves interactions between circulating maternal immune cells and the syncytiotrophoblast. The immune interactions at the maternal-fetal interface play an important role in implantation biology. Sergeant et al. argue that maternal-fetal systemic immune interactions in humans might be predominantly NK-cell instead of T-cell mediated. NK cells are CD3(-)CD56(+) and/or CD16(+) cytotoxic lymphocytes. NK cells comprise 5-15% of lymphocytes in peripheral blood. They are a key component of innate immunity, particularly crucial during the early phase of immune responses against certain viruses, parasites, and microbial pathogens, and participate partly in the allograft rejection. NK cells can discriminate between normal cells and abnormal cells (such as virus-infected or tumor cells) by using a repertoire of cell surface activating and inhibitory receptors. The receptors on NK cells control their activation, proliferation, and effector functions and in most instances the inhibitory signals over-ride the triggering ones. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and recognizes HLA-E. CD94/NKG2A appears to function as the failsafe inhibitory receptor because it is expressed on the vast majority of human NK cells. Studies found CD94/NKG2A can inhibit NKG2D-mediated Vav1 phosphorylation. The NKG2D receptor is one of the best-characterized activating receptors expressed on NK cells. CD94/NKG2A andNKG2D can play a crucial role in cytotoxicity, cytokine synthesis, and innate and adaptive immune responses. In the first weeks of pregnancy, there is increased expression of inhibitory receptors (various KIRs including CD94/NKG2A) among peripheral NK cells, reaching a maximum within the third month of gestation, with a subsequent decline to basal levels by the end of pregnancy. An imbalance between inhibitory and activating receptor expression was also found in women with implantation failures.Mifepristone (RU486 ) , an 11β-dimethyl-amino-phenyl derivative of norethindrone, is a potent progesterone and glucocorticoid receptor antagonist. When it was first found, mifepristone is used as an abortion drug. Recently, mifepristone is used as an emergency contraceptive, at gradually reduced dosages. For the past few years, investigations showed that daily low dose mifepristone could inhibit the development of the endometrium to achieve the purpose of contraception, so-called "endometrium contraception" . Some studies found that mifepristone has multiple effects on the immune system. It will suppress normal lymphocyte proliferation and downregulation interleukin-2 receptors (IL-2R). Mifepristone exhibits both agonist and antagonist effects on the release of T-lymphocyte-derived lymphokines. Researchers also found that mifepristone may affect some of the markers of endometrial receptivity, disrupt the attachment of the human blastocyst to the in vitro endometrial construct, and reduces pregnancy rates. In our previous study, we found that low-dose mifepristone increases the number of CD56(+)NK cells and the percentage of CD3(-)CD56(+)CD16(-)NK subset in receptive endometrium. This suggested that mifepristone have an effect on the uNK cells. In the previous studies, some researchers found that peripheral blood NK activity had a negativite influence on the success of pregnancy. Mifepristone is an oral contraceptive drug. It may have some effect on the peripheral blood cells. So we wanted to know if mifepristone influences the fetal implantation by effect on the pbNK. The present study aimed to investigate the effect of mifepristone on pbNK cytotoxicity, as well as the expression of activating receptor NKG2D and inhibitory receptor CD94/NKG2A on NK cells.Methods and Materials:Twenty peripheral blood samples were obtained from healthy women donors: ten at the proliferative phase (7th-10th day of the menstrual cycle) and ten at the mid-secretory phase (19th-24th day of the menstrual cycle). The menstrual cycle phases were defined by self-reporting by subjects. The women (25-35years, mean age 27.8±3.764and body mass index between 19 and 25kg/m2) had regular menstrual cycles (25-30days), normal genital system anatomy and endocrine indexes, and had not received hormone or immunosuppressant therapy for the last 3 months. Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation using Ficoll-Paque plus and the PBMC got from the proliferative phase and part of the PBMC from the implantation phase were directly used for MTT and FCM assay, and the remaining cells from the implantation phase were resuspened with PRMI 1640 medium containing 10% fetal bovine serum for culture (control group, 200nmol/L group and 1800nmol/L group). The control group was treated with 1ml 1640 medium containing 10%FBS. The PBMC in other two groups were incubated with the same medium supplemented with 200nmol/L or 1800nmol/L mifepristone respectively. Incubations were performed in a humidified atmosphere at 37℃in 5% CO₂.Results:pbNK Cytotoxicity Assay and proportions of CD94/NKG2A and NKG2DReceptors on pbNK During the Menstrual CycleTo examine the variety of NK cell cytotoxicity and the expression proportions of CD94/NKG2A and NKG2D receptors on pbNK during the menstrual cycle, we measured the NK cell cytotoxicity and the expression proportions of receptors in PBMCs from 20 healthy donors by MTT. We found that PBMCs from the proliferative phase of the menstrual cycle had significantly higher cytotoxicity percentage than from the secretory phase (81.71±11.5 vs. 60.16±19.2; P=.007). And the expression proportion of inhibitory receptor CD94/NKG2A on the pbNK from the secretory phase is lower than that from the proliferative phase (60.15±31.0 vs. 86.6±9.0; P=.018). The expression proportion of NKG2D from the secretory phase and proliferative phase had no significant differences (P=.114).pbNK Cytotoxicity Assay after Treatment with MifepristoneAfter treating the pbNK cells with mifepristone 24h, the mifepristone group had significantly increasing cytotoxicity compared to the control group (P=.003). But there was no significant difference between the different concentrations of mifepristone groups (P= .602).Proportions of CD94/NKG2A and NKG2D Receptors on pbNK after Mifepristone Treatment.The receptor CD94/NKG2A and NKG2D expression on the pbNK from the secretory phase (day 19-24 of menstrual cycle) was analyzed by Flow cytometric assay. The expression proportion of inhibitory receptor CD94/NKG2A on peripheral NK cells in 200nmol/l mifepristone group was higher than the control group (55.2±16.0vs. 50.7±16.4; P=.013), and also higher than 1800nmol/l mifepristone group(55.2±16.0 vs. 49.6±17.7; P=.003). There was no significant difference between the control group and the 1800nmol/l mifepristone group (P=.436). No significance difference was found of the expression proportion of activating receptor NKG2D on pbNK among the three groups.Conclusion:1. In secretory phase down-regulated CD94/NKG2A, NKG2D and NK cytotoxicity may benefit with embryo implantation. 2. Mifepristone maybe exert its anti-implantation function by increasing NK cytotoxicity.3. The increasing NK cytotoxicity of mifepristone is not related to CD94/NKG2A and NKG2D.