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Laboratory for Reproductive Immunology,3 Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai 200011, China
Department of Obstetrics & Gynecology,4 The Affiliated Hospital, Hainan Medical College, Haikou 570102, China
ABSTRACT
The embryo expresses paternal antigens foreign to the mother, and therefore has been viewed as a natural allograft. Cyclosporin A (CsA) is an immunosuppressant for preventing allograft rejection. Little is known, however, about the modulating effect of CsA on the materno-fetal relationship. In this study, pregnant CBA/J female mice mated with DBA/2 or BALB/c male mice as abortion-prone and normal pregnancy matings were administered, respectively, with CsA at Day 4 of gestation. We demonstrated that the administration of CsA at the window of implantation resulted in maternal T-cell tolerance to paternal antigen, and it improved pregnancy outcome in the CBA/J
DBA/2 abortion-prone matings. CsA administration enhanced Th2 and reduced Th1 cytokine production at the materno-fetal interface, and it expanded peripheral CD4+CD25+ FOXP3+ regulatory T cells in abortion-prone matings, implying development of Th2 bias and regulatory T cells. On the other hand, we observed that treatment with CsA led to enhanced growth and invasiveness of trophoblasts in the abortion-prone matings. Together, these findings indicate that CsA in lower dosages can induce materno-fetal tolerance and improve the biologic functions of trophoblast cells in the abortion-prone matings, leading to a successful pregnancy, which is useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.
cytokines,, immunology, placenta, pregnancy, trophoblast
An embryo is a mating product of histoincompatible individuals in an outbred population. It can be viewed as a semi-allogeneic graft in a natural pregnancy that has to be tolerated through the entire gestational period [1]. Maternal immune rejection against the fetus is postulated to be an important cause of unexplained pregnancy wastage [2]. Thus, maintaining the maternal immune tolerance toward the fetal semi-allograft during pregnancy, which has been referred to as immune pregnancy pause, is a main concern for treating the unexplained pregnancy loss.
Cyclosporin A (CsA) is a powerful immunosuppressive that is widely used to prevent organ rejection and to treat certain autoimmune diseases [3, 4]. Most effects of CsA are exerted through the formation of a specific complex with cyclophilins in the cytoplasm, and the cyclophilin/CsA complex binds to calcineurin and inhibits its serine-threonine protein phosphatase activity. This inhibition is critical for the antigen receptor signaling in immunocompetent cells, leading to inactivation of lymphokine genes essential for T-cell proliferation and activation, ultimately resulting in immunosuppression [5]. The clinical application of CsA has revolutionized organ transplantation and improved the therapeutic management of some autoimmune diseases.
Paradigms from transplantation immunology provide models for investigating the materno-fetal relationship with respect to the influence of maternal-fetal human leukocyte antigen compatibility on pregnancy outcome, and the development of prophylaxis and therapeutics of the pregnancy loss. So we speculated that CsA was able to suppress maternal rejection to the allogeneic fetus.
There is a unique cytokine network at the materno-fetal interface that plays a key role in the maintenance of pregnancy by modulating and integrating immune and endocrine systems. It has been suggested that in a successful pregnancy, a Th2 (a second type of T helper cell) bias is present at the materno-fetal interface and in the periphery, but in an abortion-prone pregnancy a Th1 bias would occur systemically and at the materno-fetal interface [6, 7]. In organ transplantation it has been shown that CsA significantly prolongs allograft survival through the induction of the shift from Th1 to Th2 bias [8]. So we propose that CsA can promote a Th2/3 (a third type of T helper cell) shift from Th1, providing a good milieu for a successful pregnancy at the materno-fetal interface.
Regulatory T cells (Tregs) are increasingly recognized as playing a major role in maternal tolerance to the fetus [9]. Therefore, it is essential that CsA treatment must first be shown to be unable to block Treg generation before clinical application. Although there is now a greater degree of consensus that CsA potentially has a crucial role in the induction and maintenance of transplantation tolerance, the inhibitory or stimulatory effects of CsA on the development of regulatory T cells in vivo remain controversial.
Successful pregnancy, a complex program, requires not only maternal tolerance to an allogeneic fetus, but also the most fascinating properties of trophoblast cells. Trophoblast cells proliferate, migrate, and invade a mother's uterine wall and decidual vasculature in order to nourish the developing fetus in a way that is imitated by malignant tumors. Either insufficient invasion or failure to adequately proliferate can contribute to spontaneous pregnancy loss, intrauterine growth retardation, pregnancy-induced hypertension, or preeclampsia [10–13]. It has been demonstrated that CsA increased growth and invasion in vitro of adenocarcinoma cell line [14]. The first-trimester human trophoblast cells function akin to tumor cells, and the tumorlike properties of trophoblasts are critical for appropriate materno-fetal interactions. So we speculated that CsA could promote the proliferative and invasive capacities of trophoblast cells, which play a key role in implantation and placentation, and therefore is essential for the subsequent development of the embryo.
In the present study we first observed the changes in the fetal resorption rates in abortion-prone matings after treatment with CsA, and then analyzed the Th1/Th2/Th3 cytokine production at the materno-fetal interface. The proliferation and interleukin 2 (IL2) production of maternal splenocytes in response to paternal stimulator cells, splenic CD4+CD25+FOXP3+ T-cell population expansion, and proliferative and invasive abilities of the first-trimester cytotrophoblast cells were also determined. Our data exposed differential regulatory effects of CsA on the maternal immunocompetent cells and embryonic cytotrophoblasts.
Inbred strains of female 8-wk-old CBA/J (H-2k) and male DBA/2 (H-2d) and BALB/c (H-2d) mice were obtained from the Jackson Laboratories (Bar Habor, ME), and subsequently were maintained in the Laboratory Animal Facility of Chinese Academy of Sciences (Shanghai, China). They were usually maintained for 2 wk in the animal facility before use. The housing and handling of the experimental animals were in accordance with the guidelines of the Chinese Council for Animal Care. The CBA/J x DBA/2 matings with very high fetal resorption rates were used as the abortion-prone model, whereas CBA/J x BALB/c matings were used as a normal pregnancy model with low resorption rates. The day of appearance of a copulatory plug was arbitrarily designated as Day 0 of gestation.
It has been shown that CsA can exert potent effects on a wide range of cell types regulating disparate biologic functions depending on the cell type and the applied dosage of CsA [15, 16]. Our previous study [17] demonstrated that CsA at low doses (0.0001–1.0 µmol/L) stimulates proliferation and invasion while inhibiting serum starved-induced apoptosis of human first-trimester cytotrophoblast cells. However, CsA at a high concentration (10 µmol/L) inhibited proliferation and invasion and induced apoptosis of human cytotrophoblasts [17]. Therefore, we treated the mouse with a wide range of CsA doses and measured the blood concentration of CsA 2 h later. The primary results showed that the blood concentrations of CsA at 5, 10, and 15 mg/kg body weight were just within this range (0.1–1.0 µmol/L). In the present study, the pregnant CBA/J females mated with DBA/2 or BALB/c males were intraperitoneally injected with either CsA (C-3662; Sigma, Taufkirchen, Germany) in dosages of 5, 10, and 15 mg/kg body weight or vehicle only (controls), respectively, on Day 4 of gestation, which was the window of murine implantation. These pregnant CBA/J mice were killed on Day 9 (early-middle pregnancy) or Day 14 (middle-late pregnancy) of gestation for collecting placental or decidual tissue, respectively, and counting the embryo resorption.
ELISA for Soluble Cytokine Determination
The pregnant CBA/J mice were killed on Day 9 or 14 of gestation. The uterine horns were opened longitudinally, and the feto-placental unit was separated from the uterine implantation sites. The whole placental and decidual unit was separated individually from the respective embryo and its implantation site, and then single-cell suspensions, which included both placental and decidual cells, were prepared. The cells were suspended at 5 x 106/ml in complete medium and cultured for 24 h in 24-well flat-bottom plates containing 2 ml complete medium. The supernatants then were harvested, and interferon 
(IFNG), IL12, IL4, IL10, and transforming growth factor β-2 (TGFB2) levels were evaluated by ELISA (R&D Systems, Minneapolis, MN).
The spleen was aseptically removed and mechanically teased out of the stroma in 10 ml PBS. The cell suspensions were filtered through 110-µm pore size nylon mesh and then treated with NH4Cl/Tris buffer to remove red blood cells. Thereafter, the cells were washed three times and prepared for in vitro culture in complete medium of RPMI 1640 containing 10% FCS, 1 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Isolation of Murine Trophoblasts
The uterine horns of pregnant CBA/J mice on Day 9 of pregnancy were opened longitudinally, and the fetoplacental unit was separated from the uterine implantation sites. After being washed in cold Hanks balanced salt solution, the ectoplacental cell cone was further removed from the fetoplacental unit, cut into small pieces, and collected in Hanks balanced salt solution containing no Ca2+ and no Mg2+ (Sigma). Murine trophoblasts were obtained as previously described [18].
In 24 h of culture, trophoblast cells were fixed in 4% polyformaldehyde for 20 min at room temperature, washed in PBS, and permeabilized for 4 min in 0.3% Triton X-100/PBS. The cells were incubated with 20% bovine serum in PBS for 30 min to reduce nonspecific binding. The primary antibodies diluted in PBS containing 1% BSA were added. Anti-mouse cytokeratin-7 (MAB3226, RCK105; Chemicon, Temecula, CA) and anti-mouse vimentin monoclonal antibody (MAB1681, LN-6; Chemicon) were used as markers for identification of trophoblasts and nontrophoblasts, respectively. The concentrations of anti-cytokeratin-7 and anti-vimentin were both 1.0 µg/ml. Isotype-matched irrelevant IgG (Sino-America Co. Ltd., Shanghai, China) was used as a control. After incubation with primary antibody overnight at 4°C, the cells were washed in PBS/0.1% Tween and then incubated with horseradish peroxidase-labeled secondary antibody (Sino-America Co. Ltd.) for 2 h at room temperature. The slides were stained with DAB and counterstained with hematoxylin. The experiments were repeated five times.
Flow Cytometry Analysis for CD4+CD25+FOXP3+ Regulatory T Cells
The splenocytes were obtained from pregnant CBA/J mice on Day 9 of gestation. The splenocytes were stained by a standard immunofluorescence assay with various combinations of fluorescence-conjugated antibodies. In brief, the prepared cells were first stained with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD4 (RM4–5) and allophycocyanin-conjugated rat anti-mouse CD25 (PC61.5). After permeabilization, the cells were stained with phycoerythrin-conjugated rat anti-mouse FOXP3 (FJK-16s). Samples were analyzed on a FACSCaliber flow cytometer using CellQuest software (BD Biosciences, San Jose, CA). Statistical analysis was performed using the isotype-matched controls as a reference.
[3H]Thymidine Incorporation for Splenocyte Proliferation
Splenocytes from pregnant CBA/J mice on Day 9 or 14 of gestation were used as responder cells, and the paternal splenocytes were treated with mitomycin C as stimulator cells. The responder cells (2 x 105/well) and the mitomycin C-treated stimulator cells (2 x 105/well) were aliquoted into 96-well, round-bottom microtiter plates (Nunc, Roskilde, Denmark) in a final volume of 200 µl complete medium. PHA (5 µg/ml) was used as positive control. The responder cells cultured with the complete medium alone in 96-well, round-bottom microtiter plates were used as negative control. In a 4-day incubation at 37°C, [3H]thymidine (0.5 µCi/well) incorporation was measured for an additional 6 h. The cells were harvested onto a glass-fiber paper using a semiautomatic cell harvester, and the thymidine incorporation was measured in a liquid scintillation counter.
For measurement of IL2 production, supernatants of the cultured splenocytes were collected after 48 h of culture and were stored at –20°C. IL2 production was analyzed by ELISA (R&D Systems).
On Day 14 of gestation, female mice were killed, the uteri were removed, and the implantation sites were evaluated. The resorbed embryos were identified by their small size, and necrotic hemorrhagic appearance was compared to that of normal embryos. Percentage of resorption was calculated by the following formula: %R = Re / (Re + F) x 100, where R represents the percentage of resorption relative to the total number of effective implantation sites, Re represents the number of the resorbed embryos, and F represents the number of viable embryos, which was as described previously [19].
MTT Assay for Trophoblast Proliferation
The effect of CsA on proliferation of murine first-trimester trophoblast cells was analyzed by the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; Sigma Chemical, St. Louis, MO) assay. For the MTT assay, the isolated trophoblast cells were resuspended in DMEM with 10% fetal bovine serum (FBS) and were seeded at a density of 1 x 104 cells/well in 96-well, flat-bottom microplates. The cells were cultured in DMEM complete medium for 48 h following 12 h of serum starvation. Then, 20 µl MTT stock solution (5 mg/ml) was added to each well. Experiments were stopped in 4 h by adding DMSO, which dissolved the formazan crystal. Absorbance at a wavelength of 540 nm was measured by a microplate reader. The values of the cells from abortion-prone matings were compared to the values from normal pregnancy matings and reported as the proliferation index.
To evaluate the effect of CsA on invasiveness of murine trophoblast cells, transwell inserts (6.5-mm filters, 8-µm pore size; Corning, Corning, NY) containing polycarbonate filters were used. The upper side was coated with 10 µl of 5 mg/ml Matrigel and allowed to gel. The cells (1 x 105 cells) were plated in 200 µl DMEM with 1% FBS to the transwell. The lower chamber was filled with 600 µl DMEM with 10% FBS. After incubation for 48 h at 37°C, 5% CO2, the cells which did not invade into the lower chamber were completely removed from the upper surface of the filter with gentle swabbing. The migrant cells of the lower surface were then fixed in methanol for 10 min at room temperature and stained with hematoxylin. The invasion was determined by counting the number of stained cells on the membranes in 10 randomly selected, nonoverlapping fields at 200x magnification using a light microscope. Each condition was tested in triplicate wells, and experiments were repeated three times.
All values are shown as mean ± SD. Comparisons were done by one-way ANOVA for cytokine production, proliferation, and IL2 production of the pregnant CBA/J splenocytes, expansion of the regulatory T-cell population, proliferation and invasion of mouse trophoblast cells, and number of implantation sites and litter sizes, or by chi-square test for percentages. Differences were accepted as significant at P < 0.05.
CsA Improved Pregnancy Outcome of Abortion-Prone Matings
It was reported previously that the proportion of implantations that aborted spontaneously in DBA/2-mated CBA/J fetal mice ranged from 20% to 40% [20]. In our colony the spontaneous abortion rate was 20.25%. In order to testify whether the fetal loss was decreased by the administration of CsA in vivo, CsA or vehicle was injected intraperitoneally into the pregnant CBA/J females on Day 4 of gestation, which was the window of murine implantation. The implantation sites were evaluated on Day 14 of gestation. The results, shown in Table 1, clearly demonstrate that treatment of CBA/J females with 5 mg/kg or more CsA significantly reduced the resorption rate and increased the implantation rate and the litter size of CBA/J x DBA/2 matings compared with the vehicle control (P < 0.01). The embryo resorption rate was similar to that of the normal pregnancy model (CBA/J x BALB/c matings). Interestingly, there was no significant further effect of CsA on the fetal resorption rate in the successful pregnant CBA/J x BALB/c matings compared with the vehicle control. In contrast, the implantation rate and litter size of the CBA/J x BALB/c matings were decreased with CsA dosage increase. In addition, we found that administration with CsA only once at the window of implantation could slightly increase the weights of fetus and placenta in CBA/J x DBA/2 matings (P > 0.05; data not shown). These findings indicate that treatment with CsA is efficient in preventing maternal rejection to the allogeneic fetus in the abortion-prone matings. However, an increasing trend in fetal resorption with increasing dosage of CsA appeared, which suggests that the dosage of 5 mg/kg is the lowest efficient dose, and a dosage of CsA higher than that might be detrimental to embryo development.
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Administration with CsA Skewed Toward a Th2 Bias at the Materno-fetal Interface
It is known that a successful pregnancy in mice is a Th2/3 phenomenon; abnormal elevations of Th1 cytokines are associated with spontaneous abortions and impaired fetal development. Therefore, we next determined whether an increase of fetal viability by treatment with CsA was associated with changes in cytokine expression. We analyzed the effects of CsA on the production of Th1/Th2/Th3 cytokines produced by a mixture of placental and decidual cells isolated from the ectoplacental cell cone by ELISA. The data are summarized in Figure 1. Compared with the vehicle control, treatment with CsA resulted in lower production of IL12 and IFNG and higher production of IL4, IL10, and TGFB2 at the materno-fetal interface of abortion-prone matings. However, no change was observed at the materno-fetal interface of the normal pregnancy matings (data not shown).
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Administration with CsA Induced Maternal Hyporesponsiveness to Paternal Antigen
Based on the findings above, to further understand the inhibitory effects in vivo of CsA treatment, we examined maternal immune responses to paternal antigens. The splenocytes from pregnant CBA/J mice on Day 9 or 14 of gestation were stimulated with mitomycin C-treated paternal DBA/2 or BALB/c splenocytes. Mixed lymphocyte reaction (MLR) demonstrated that administration with CsA strongly inhibited proliferation of CBA/J splenocytes in response to DBA/2 stimulator cells at Day 9 as well as Day 14 of gestation. Moreover, seriously impaired IL2 production was observed compared with the vehicle. The decrease in MLR reactivity was similar to that observed for CBA/J splenocytes in response to BALB/c stimulator cells (Fig. 2). These results indicate that administration with CsA only once on Day 4 of gestation, the window of murine implantation, successfully induces maternal tolerance to the paternal antigens, suggesting that CsA inhibits maternal T-cell activation in vivo, which can persist throughout gestation.
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Administration with CsA Expanded the Maternal Peripheral CD4+CD25+FOXP3+ T-Cell Population
Recent evidence has indicated that regulatory T cells are required for the maternal immune tolerance to the fetus [9]. This prompted us to further investigate the possibility of regulatory T cells being present in the tolerant mothers. We analyzed by flow cytometry the splenic CD4+CD25+ T-cell subset of pregnant CBA/J mice on Day 9 of gestation. Since FOXP3 is important for the development and function of human CD25+CD4+ regulatory T cells, and Treg activity correlates with FOXP3 expression [21–23], we further examined the expression of intracellular FOXP3 in the CD4+ T-cell population. Data summarized from five independent experiments are presented in Figure 3. Compared with the normal pregnancy matings, there was a lower frequency of CD4+CD25+ T cells in the splenocytes of the abortion-prone matings. Since the dosage of 5 mg/kg body weight of CsA was sufficient to improve the pregnancy outcomes of abortion-prone mice, we then chose the lowest efficient dose to analyze the effect of CsA on CD4+CD25+ T-cell expansion. We observed that treatment with CsA resulted in a higher frequency of cells being positive for CD4 and CD25 in the splenocytes in the abortion-prone matings (P < 0.01). Also, frequencies of the FOXP3-expressing cells and CD25+FOXP3+ cells in the CD4+ T-cell population were increased following administration of CsA (P < 0.01). The results above indicate that the administration of CsA expands the maternal CD4+CD25+FOXP3+ T-cell population, suggesting that the regulatory T cells were involved in the maternal tolerance driven by CsA.
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Administration with CsA Promoted the Proliferation and Invasiveness of Murine Trophoblast Cells in Abortion-Prone Matings
In 24 h of culture we characterized the expression of cytokeratin-7 and vimentin in these cells. About 95.3% of the isolated cells stained positive for cytokeratin-7, whereas no cells were found to be stained with anti-vimentin antibody. The purity of the isolated trophoblast cells was 95.3% ± 2.4%.
The capacity of CsA to promote proliferation of the primary cultured trophoblast cells was investigated using MTT assay. The primary trophoblast cells were serum starved for 12 h and then cultured in complete medium for 48 h. The administration of CBA/J with CsA at Day 4 of gestation significantly promoted proliferation of the primary cultured trophoblast cells. The proliferation index was similar to that of normal pregnancy matings (Fig. 4). Since the trophoblasts were about 95% pure, the possibility that the proliferating cells were contaminating cells and not trophoblasts must be excluded. By double staining for proliferating cells (PCNA) and cytokeratin-7 and flow cytometry analysis, we found that administration with CsA significantly increased PCNA expression in cytokeratin-7-positive cells, which confirmed that trophoblasts not contaminating cell types were proliferating in this assay (data not shown).
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To test the effects of CsA administration on murine trophoblast cell invasiveness, a Matrigel-based transwell assay was carried out. The cells were added into the upper chamber, and a serum gradient was used between the upper and lower chambers to accelerate cell invasion. As shown in Figure 5, the invasion of trophoblasts from abortion-prone matings was lower than that from normal pregnancy matings. After administration with CsA, the invasion of the trophoblast cells in the early gestation of abortion-prone matings was improved and the invasiveness of these cells was almost equivalent to that of the normal pregnancy matings. However, the CsA administration at the window of implantation did not influence the invasiveness of trophoblast cells on Day 14 of gestation (P > 0.05; data not shown).
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The mammalian fetus develops complex and intimate associations with maternal uterine tissues and blood supply beginning shortly after blastocyst implantation and continuing throughout gestation [24]. From an immunologic perspective, a successful pregnancy is paradoxical, because interactions between maternal T cells and fetal tissues that express the paternally inherited alloantigens should provoke a maternal immune response to fetal antigens. Consequently, pregnancy success depends on the processes that suppress maternal immunity or attack fetal alloantigens during gestation. Nevertheless, fetal loss caused by immune complications may be considerable. It has been estimated that up to 50% of human pregnancies end in failure due to complications that arise after implantation [25]. The triggers and mediators of fetal damage, however, are still largely poorly understood. Although some pregnancy failures may be explained by genetic or developmental abnormalities, immunologic impacts are considered critical to a successful pregnancy. For many years, treatment of pregnancy loss, especially recurrent spontaneous abortion, has become a medical enigma that has stimulated research. A lot of immunotherapy for recurrent pregnancy loss has been proposed [26]. However, the clinical efficiency is ill defined and even controversial. So it apparently is urgent that a more effective and safe therapy to solve this matter is found.
CsA has exerted an immense effect on prolonging the survival of allografts in the host as a potent immunosuppressive agent since its clinical application in the 1970s. CsA restrains the proliferation of T cells by blocking Ca2+-associated signaling events and inhibits IL2 production [5], leading to the inhibition of rejection cascade mechanisms. In addition to suppressing mature T cells in the periphery, CsA has a powerful influence on the thymus [27]. Recently, a series of evidence has suggested that CsA exerts its immune modulations not only on T cells but also on antigen-presenting cells, such as macrophages [28] and dendritic cells (DCs) [29, 30]. CsA inhibited IL12 production as well as the upregulation of co-stimulatory molecules and CD40L in DCs, but it augmented the IL10 production from the LPS-stimulated CD11c+ subset. It has been suggested that the therapeutic effect of CsA is achieved by correcting a Th1/Th2 imbalance, which may be involved in the pathogenesis of allograft rejection [31] and rheumatoid arthritis [32]. In addition, CsA can impair DC migration and inhibit DC maturation [33].
Since the embryo expresses paternal antigens foreign to the mother, it therefore has been viewed as a natural allograft [34]. The success from transplantation immunology has provided models for the development of treatments to prevent from fetal loss. However, although CsA has been implicated as pivotal players in the prolongation of graft survival, the capacity and possible mechanisms of CsA to suppress maternal immune responses on the allogeneic fetus have received little attention. So it is meaningful to explore the immunoregulatory roles of CsA both in the maintenance of materno-fetal tolerance and its application as a new strategy in the treatment of recurrent spontaneous abortion and other pregnancy complications.
Here we observed that the embryo resorption rate in abortion-prone matings was significantly reduced by administration with CsA only once during peri-implantation and that this effect can persist throughout gestation, which was consistent with the increasing evidence that the parameters with characteristics of a maternal immune response during murine pregnancy are established early in gestation. Moreover, the administration with CsA strongly inhibited the proliferation of maternal splenocytes in response to the paternal stimulator cells, and this was accompanied by seriously impaired IL2 production, which substantiated in vivo inhibitory effects of CsA in abortion-prone mice. The present study indicates that CsA is effective in suppressing maternal rejection to the allogeneic fetus in abortion-prone matings. Interestingly, the administration with CsA only once on Day 4 of gestation, the window of murine implantation, had no further improvement on pregnancy outcome in the normal pregnancy model. Therefore, the low resorptions in the CBA/J x BALB/c matings probably are due to chromosomal errors [35], which cannot be corrected by the administration with CsA, so the embryo resorption rate did not alter significantly.
It has been shown that gestation is associated with a transient depression of maternal cell-mediated immunity in order to protect the semi-allogeneic embryo from rejection. The hallmark of this immune tolerance is a profound modulation of T-cell responses, best characterized by a shift from a Th1- to a Th2-type cytokine response. It has been demonstrated that a failure in Th2/3-type cytokine responses is associated with recurrent abortions, complications, and poor outcome of pregnancy [6]. In agreement with previous reports, decreased concentrations of Th2/3-type cytokines and increased concentrations of Th1-type cytokines were found on Days 9 and 14 of gestation in abortion-prone matings compared with successful pregnancy matings. Administration with CsA enhanced Th2/3 and reduced Th1 cytokine production at the materno-fetal interface in abortion-prone matings. Furthermore, there was a similar trend in decreasing resorption rate and increasing Th2/3 production with an increasing dosage of CsA, implying that the development of Th2/3 cells might contribute to the CsA-mediated maternal tolerance to the fetus and abortion prevention. Interestingly, no significant change of cytokine production was found in the successful pregnancy model after treatment with CsA (data not shown). It is suggested that the original physiologic Th2/3 predominance at the materno-fetal interface of the normal pregnancy remains undisturbed by the administration of CsA.
The CD4+CD25+ Tregs have recently been described as a unique subpopulation of T cells. They were confirmed as playing a major role in the prevention of autoimmunity and the tolerance allogeneic organ grafts [36, 37]. Aluvihare et al. reported that the absence of the CD4+CD25+ Treg led to a failure of gestation due to immunologic rejection of the fetus, suggesting that the CD4+CD25+ Tregs mediate maternal tolerance to the fetus [9]. Other authors confirmed the important role of Tregs in human pregnancy [38]. In accordance with the data above, our study showed a decrease in the percentage of splenic CD4+CD25+ cells in abortion-prone matings, and treatment of the abortion-prone matings with CsA significantly increased fetal viability as well as the percentage of splenic CD4+CD25+ cells, suggesting that the regulatory T cells are involved in the maintenance of materno-fetal tolerance driven by CsA. CD25 is not an absolute marker for naturally occurring Tregs, since it also is expressed at high levels on the activated but otherwise conventional nonregulatory T cells. Since FOXP3 can be a crucial regulatory gene for the development and function of CD25+CD4+ Tregs [21–23], we therefore analyzed FOXP3 as a more accurate Treg marker. Our results showed that the majority of the expanded splenic CD4+CD25+ T cells expressed intracellular FOXP3, suggesting that expansion of the CD4+CD25+FOXP3+ regulatory T-cell subset really has occurred.
During implantation and placentation, adequate proliferation and invasion of trophoblast cells are basic for a successful pregnancy. Insufficient proliferation and invasion or increased apoptosis can contribute to spontaneous pregnancy loss, intrauterine growth retardation, pregnancy-induced hypertension, or preeclampsia. Since treatment with different doses of CsA could decrease embryo resorption rates in abortion-prone matings, we further observed whether CsA improved pregnancy outcome by regulating biologic function of murine cytotrophoblast cells. By using the lowest efficient dose of CsA, we found that CsA treatment in vivo was able to promote growth and invasion in vitro of murine cytotrophoblast cells of the early to middle pregnancy stages. The precise mechanisms regulating trophoblast cell proliferation and invasion are not completely understood.
Olivares reported that the decidual local and peripheral lymphocytes were activated in human spontaneous abortion and preeclampsia [13], leading to extensive destruction of the trophoblast by inducing apoptosis, altering differentiation or proliferation, modifying the secretion of hormones, and hampering trophoblast invasion and vascular remodeling through the abolition of the secretion of prourokinase-type plasminogen activator (pro-UPA) and induction of plasminogen activator inhibitor-1 (PAI1) secretion by trophoblast cells [39–42]. Recently, several studies have shown that cytokines, including IL12 and IFNG, modulate trophoblast invasiveness by biasing the ratio (protease:protease inhibitor) in favor of the inhibitors and upregulating E-cadherin, thereby implying that alteration in cell-cell adhesion and regulation of the proteases and the inhibitors possibly contribute to the anti-invasive role of these cytokines [43]. So it is supposed that suppressing lymphocyte activation and production of Th1-type cytokines might be involved in the modulation of the biologic function of trophoblast cells by CsA. In addition, our previous study showed that CsA could promote proliferation and invasiveness and inhibit apoptosis in vitro of human first-trimester trophoblasts at low concentrations while suppressing proliferation and invasion and inducing apoptosis of the trophoblasts at high concentrations [17]. So it is reasonable to hypothesize that CsA may promote trophoblast growth and invasiveness directly as well as via suppression of lymphocyte activation upon the embryo antigen.
Our data support the notion that CsA is beneficial to pregnancy in the abortion-prone CBA/J x DBA/2 matings via regulation of trophoblast function and induction of materno-fetal immunotolerance, and the findings may provide a new, more rational immunotherapy for certain pathologic pregnancies, such as allogeneic pregnancy wastage, preeclampsia, and so on.
However, there are considerable known side effects of long-term CsA administration. Several reports have suggested that the production of oxygen-free radicals (OFRs) is a common mechanism of CsA toxicity [44–46]. Notably, CsA administration over a long duration could cross the placenta to the fetus, and the increased oxidative stress in the placenta is involved in the pathophysiology of preeclampsia. Our results showed that the litter size was reduced in the CBA x BALB/c mating in response to CsA treatment, which may be the result of the potential cytotoxic side effects of CsA. There has been much concern over the potential detrimental effects of the immunosuppressive drug. To our knowledge, no report is available about CsA usage for prevention of pregnancy loss or preeclampsia. There have been a growing number of case reports on transplant patients receiving CsA who delivered a normal child. Indeed, most follow-up studies showed that the offspring appeared to have normal postnatal growth and development [47, 48]. It was reported that no significant passage of CsA into the fetus occurred after a single injection in pregnant mice [49]. This supports the possibility that a single injection of CsA during the peri-implantation period may prevent abortions and have no side effects on embryo development. Furthermore, the dosage of CsA used in our study is far lower than the dose used in transplantation. In other studies, we have indeed used lower concentrations of CsA (0.1 and 1.0 mg/kg) and have observed essentially the same results as higher doses (data not shown), suggesting that an even lower dose may be more effective and be of some benefit in pregnancy complications with fewer side effects. In addition, our study in human trophoblasts has demonstrated that CsA at lower concentrations (0.0001–1.0 µmol/L) promotes proliferation and invasiveness and inhibits serum depletion-induced apoptosis in human trophoblasts, whereas CsA concentrations of 1.0 µmol/l or higher enhanced the proapoptotic effects, which was consistent with recently accumulated data showing that CsA at a low dosage was cytoprotective, but a high dosage of CsA increased OFR production and proapoptosis [15, 16, 50]. The results from the present study show that the doses of CsA used were just within this range (0.1–1.0 µmol/l; data not shown). So despite the existing concern, CsA is worth further research as a potential therapeutic for some pregnancy complications of maternal-fetal regulation disorder.
FOOTNOTES
1Supported by National Key Research Program of China 2006CB0F1009 to D.J.L.; Shanghai Foundations for Basic Research no. 03JC14016 to D.J.L. and no. 06ZR14120 to M.R.D.; Program for Outstanding Medical Academic Leader to D.J.L.; and 985 Foundations of Fudan University no. 985B36 to D.J.L. The authors have no conflict of interest or financial interests. 
Correspondence: 2FAX: 0086 21 6345 0768; e-mail: djli{at}shmu.edu.cn
Received: 22 August 2006.
First decision: 20 September 2006.
Accepted: 12 January 2007.
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