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a Unidad de Inmunología, Departamento de Bioquímica y Biología Molecular,
b Departamento de Obstetricia y Ginecología, Facultad de Medicina, Universidad de Granada, E-18012 Granada, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, decidua, immunology, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although pregnancy has been considered as an example of semiallogeneic graft acceptance, this graft is not always successful because between one-third and one-half of all human conceptions fail to progress [5]. There is increasing evidence that the immune system participates in the mechanism of fetal elimination during spontaneous abortion [6–8]. In mice and humans, normal pregnancy is related to the local and peripheral production of Th2 cytokines [9, 10], whereas abortion is associated with Th1 cytokine production [10, 11] or with a decrease in Th2 cytokines [12]. Recent findings in mice have shown that maternal T cells tend to reject the semiallogeneic embryo, although under normal conditions this rejection is inhibited by catabolism of tryptophan by the conceptus [7], by clonal deletion of fetal-reactive maternal T cells [13], and by the production of Th2 cytokines [9]. Moreover, human trophoblast expresses HLA-G, a nonclassical human leukocyte antigen (HLA) class I molecule that binds the inhibitory receptors of cytotoxicity. The high expression of these receptors by normal decidual NK cells [14], and also probably by T cells [15], leads to the protection of the trophoblast against cell cytolysis [14].
These previous findings suggest that decidual T and NK lymphocytes are prone to attack the trophoblast, and probably do so during spontaneous abortion, although their cytocidal activity is down-regulated by different mechanisms during normal pregnancy [7, 9, 13]. To investigate the participation of the immune system in human spontaneous abortion, we compared the lymphocyte populations of decidua and cytotoxic activity of these lymphocytes against the trophoblast in human spontaneous abortion and elective termination of pregnancy (ETP).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fifty-three samples from first-trimester spontaneous abortion (in women aged 21–32 yr; gestational age, 8 wk ± 10 days) were collected at the Departamento de Obstetricia y Ginecología, Hospital Universitario San Cecilio, Granada. Anembryonic pregnancies or fetal death was confirmed by ultrasonography. Fifty-five specimens from ETP (age range, 23–35 yr; gestational age, 8 wk 2 days ± 7 days) were obtained at the Clínica El Sur (Málaga) and Gineclínica (Granada). Samples were collected by vaginal curettage; in spontaneous abortion, curettage was carried out within 24 h after diagnosis. None of the abortions was pharmacologically induced. Both groups received the same exclusionary criteria: women receiving any medication or with infectious, autoimmune, or other systemic or local diseases, were excluded. Informed consent was obtained from each patient. Blood progesterone from patients with spontaneous abortion was quantified with a commercial enzyme immunoassay (Boehringer-Mannheim, Mannheim, Germany). This research was approved by the ethics committee of the Hospital Universitario San Cecilio, Granada.
Extraction of Decidual Lymphocytes
Samples of decidua from different patients were not mixed in order to avoid the induction of allogeneic reaction of leukocytes. The method of extraction has been described elsewhere [16]. Briefly, samples from decidua of spontaneous abortion or ETP were thoroughly washed in PBS. Decidual fragments were finely minced in a small volume of RPMI 1640 (Sigma, St. Louis, MO) and then pushed through a 53-µm sieve (Gallenkamp, Loughborough, U.K.). The resultant cell suspension was washed with RPMI and layered on an equivalent volume of Lymphoprep (Flow Laboratories, Hertsfordshire, U.K.) at room temperature, and centrifuged for 20 min at 600 x g. The cells were collected from the interface, suspended in RPMI, and washed. The cells were then suspended in complete culture medium (RPMI 1640, 10% fetal calf serum [FCS], 100 U/ml penicillin, and 50 g/ml gentamicin), and incubated for 2 h at 37°C in an atmosphere of 5% CO2 to allow adherent cells to attach to the plastic. The supernatant containing decidual lymphocytes was then collected, and the cells were washed and suspended in PBS for flow cytometric analysis. Lymphocyte viability was microscopically determined by trypan blue exclusion. Only samples with more than 90% viable lymphocytes were used.
To obtain peripheral blood lymphocytes (PBLs), blood samples were taken from healthy volunteers. We diluted each sample in the same volume of 0.25% PBS-EDTA, and centrifuged them on Lymphoprep. Thereafter, we followed the same steps that we used for decidual lymphocytes.
Flow Cytometry
One hundred microliters of a suspension of 1 x 106/ml of decidual lymphocytes in PBS was incubated with 10 or 20 µl of the appropriate monoclonal antibody (Table 1) for 30 min at 4°C in the dark. Cells were washed, suspended in 1 ml of PBS, and immediately analyzed in a flow cytometer (Ortho Cytoron Absolute; Ortho Diagnostic Systems). To identify dead cells we incubated lymphocytes with propidium iodide (Sigma). Although the isolated cells were mostly lymphocytes, cells were studied in the electrically gated lymphocyte cluster. The percentage of cells that were antibody-positive was calculated by comparing then with the appropriate isotype control.
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Cell Line
JEG-3, an extravillous trophoblast (EVT) choriocarcinoma cell line, was maintained in complete culture medium RPMI 1640 containing 5% FCS, and cells were used as targets when they were in the log phase of growth.
Culture of Lymphocytes with Interleukin-2
Decidual lymphocytes or PBLs were cultured for 4 days in complete medium containing 100 U/ml of interleukin-2 (IL-2; Sigma). After this period of incubation, cells were washed and suspended in complete culture medium for cytotoxicity studies.
51Cr-Release Assay
JEG-3 cells were removed from the flask with 0.05% trypsin/0.02% EDTA, washed, and suspended in complete culture medium. Cells (2 x 104) were added to each well of a 96-well flat-bottomed plate (Becton Dickinson, San Jose, CA) and left to incubate overnight at 37°C in 5% CO2. The plates were washed twice with medium, and 3 µCi of Na251CrO4 (Amersham, Buckinghamshire, U.K.) in 35 µl of culture medium was added to each well. After overnight labeling in a moist atmosphere of 5% CO2 at 37°C, the cells were washed three times, and then 100 µl of complete culture medium was added. Effector cells from decidua or PBLs were added in a volume of 100 µl of culture medium to obtain different effector:target ratios. After 5 h of incubation at 37°C, 100 µl of supernatant was removed from each well and counted with a gamma counter. Percentage cytotoxicity was calculated according to the formula
This and the following experiments were carried out in triplicate.
DNA Fragment Assay
To investigate the induction of apoptosis in target cells we studied DNA fragmentation as an early sign of this phenomenon. One hundred microliters of complete medium containing 2 x 104 JEG-3 cells was added to each well of a 96-well flat-bottomed plate. Then, 0.5 µCi of [3H]thymidine ([3H]TdR; Amersham) was added per well, and the plates were incubated overnight at 37°C in 5% CO2. The supernantants were discarded and 100 µl of complete culture medium was added. Different amounts of effector cells from decidua or PBLs were added in a volume of 100 µl of culture medium to obtain different effector:target ratios, as shown in the figures. After 5 h of incubation at 37°C in 5% CO2, the plates were centrifuged at 400 x g for 10 min, and supernatants were replaced with 200 µl of hypotonic lysing buffer (10 mM Tris, 1 mM EDTA pH 7.5) containing 0.2% Triton X-100. Twenty-five microliters of supernatant was carefully removed from each well and counted in a ß-scintillation counter. Wells with target cells and no effectors were used to determine spontaneous release (spontan. cpm). A solution of 0.1% SDS was used as a positive control for DNA fragmentation. To determine maximal cpm (max. cpm), wells with target cells and no effectors were harvested onto glass fiber filters using a cell harvester (Skatron Instruments AS, Lier, Norway) and counted in a ß-scintillation counter. The results were expressed according to the formula
Our [3H]TdR-release assay was based on the methods described of Matzinger [17] and Duke et al. [18]. As in the Matzinger test [17], we carried out the assay in a microtiter plate format; however, as in classical DNA fragment assays [18], radioactivity was measured directly in the supernatant after centrifugation. The direct measurement of DNA fragments makes the assay more sensitive.
Light Microscopy
Fifty thousand JEG-3 cells were cultured on a Lab-Tek chamber slide system (Nalge Nunc International, Naperville, IL) and allowed to attach; the slide was then washed with PBS, and 1.5 x 106 decidual lymphocytes in 500 µl of culture medium was added. After 5 h of incubation at 37°C in 5% CO2, the slide was washed with PBS, fixed with methanol, stained with hematoxylin and eosin, and then mounted under coverslips with dibutyl phthalate xylene mountant for microscopy. Each preparation was examined at a magnification of 1000x oil immersion with a 100x objective lens and a 10x eyepiece. JEG-3 cells were much larger than lymphocytes, therefore, the two types of cell were easily distinguished. Apoptotic JEG-3 cells were identified by the condensation of nuclear heterochromatin into a crescent apposed to the nuclear membrane, and later into a single body or multiple-dense bodies. Apoptotic and nonapoptotic JEG-3 cells were counted and the results were expressed as percentages of apoptotic JEG-3 cells.
Transmission Electron Microscopy
For transmission electron microscopy, we cultured decidual lymphocytes with JEG-3 cells on a slide as we did for light microscopy. The preparation was fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate/0.1% sucrose buffer. Cells were washed in the same buffer and postfixed in 2% osmium tetroxide, dehydrated through a graded acetone series, and embedded in Epon 812. Thin sections were cut on a Reichert-Jung Ultracut E ultramicrotome (Vienna, Austria), stained with lead citrate, and visualized and photographed in a transmission electron microscope (10C; Carl Zeiss, Inc., Oberkochen, Germany). Apoptotic cells were identified mainly by the nuclear alterations described in the previous section of light microscopy, and by the formation of apoptotic bodies. We also confirmed the integrity of the plasma membrane.
Statistical Analysis
Proportions of decidual lymphocytes and percentages of apoptotic cells were compared with the Student t-test. A P value of < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 shows the comparison between the lymphocyte populations from spontaneous abortion deciduas and presumed normal deciduas (i.e., ETP). No significant differences were found between the two types of decidua in the population of CD20+ B cells or in the NK populations of CD56+, CD16+, CD56+CD16+, CD56+CD16- and CD56+CD8+ lymphocytes. However, the number of CD3+, TCR
ß+, and CD3+TCRß+ T cells were significantly higher in spontaneous abortion. This increase was determined mainly by T helper cells, as CD4+ lymphocytes were also significantly elevated in spontaneous abortion compared with those in ETP. CD8+ lymphocytes (CD3+CD8+ or CD8+TCRß) also appeared to contribute to the increase in decidual T lymphocytes in spontaneous abortion, although the differences in the proportions of these populations were not statistically significant. We found no differences in the populations of T
lymphocytes. Decidual lymphocytes from spontaneous abortion seemed to be activated because the percentages of lymphocytes expressing the activation markers CD25, CD38, and CD69 were elevated in spontaneous abortion. Populations of activated T cells (CD3+CD25+ and CD4+CD69+) and probably activated NK cells (CD56+CD25+ and CD56+HLA-DR+) were significantly higher in spontaneous abortion. A small population of CD56+TCRß+ cells, which may correspond to natural T (NT) cells [19] was also significantly elevated in spontaneous abortion in comparison with ETP.
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We did not observe significant differences between embryonic and anembryonic pregnancies in the lymphocyte populations of spontaneous abortion deciduas (not shown), suggesting that the presence or absence of embryo is not a determining factor in the variations in decidual lymphocyte proportions. Although serum levels of progesterone were significantly lower in patients that had spontaneous abortion (10.3 ± 10.4 ng/ml) than in ETP (26.7 ± 6.2 ng/ml) (P < 0.01), we did not find any correlation between these levels and the proportions of spontaneous abortion decidual lymphocytes (not shown).
Cytotoxicity of Decidual Lymphocytes from ETPand Spontaneous Abortion Against Extravillous Cytotrophoblast JEG-3 Cells
Neither decidual lymphocytes from ETP, spontaneous abortion, nor PBLs showed spontaneous cytotoxicity against JEG-3 cells in the 51Cr-release assay (which identifies necrosis). These target cells were, however, lysed when the lymphocytes were previously stimulated with IL-2 (Fig. 2). Decidual lymphocytes from spontaneous abortion, but not PBLs or decidual lymphocytes from ETP, spontaneously induced apoptosis in JEG-3 cells, as determined by the DNA fragment assay (Fig. 3).
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Quantification of Apoptotic JEG-3 Cells Treatedwith Decidual Lymphocytes from ETPand Spontaneous Abortion
JEG-3 cells cultured with decidual lymphocytes from either ETP or spontaneous abortion were stained with hematoxylin and eosin, and the proportions of apoptotic JEG-3 cells were determined by light microscopy. The proportions of apoptotic cells were significantly higher in the preparations of JEG-3 cells cultured with decidual lymphocytes from spontaneous abortion than with those from ETP (P = 1.86 x 10-5) (Fig. 4).
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Ultrastructure of JEG-3 Cells Treated with Decidual Lymphocytes from ETP and Spontaneous Abortion
JEG-3 cells cultured with decidual lymphocytes from ETP or spontaneous abortion were observed with electron microscopy. Most JEG-3 cells showed a normal morphology after culture with decidual lymphocytes from ETP (Fig. 5A). However, when JEG-3 cells were cultured with decidual lymphocytes from spontaneous abortion, we detected lymphocytes that sent membrane prolongations to or that interacted with JEG-3 cells (Fig. 5, B–E). Some of the JEG-3 cells exhibited clear signs of apoptosis, although their plasma membrane remained intact (Fig. 5, D–F): condensation and shrinkage of nuclear material, chromatin aggregation, pyknosis of the nuclei (Fig. 5, E and F), compacted cytoplasm containing numerous vacuoles (Fig. 5, D–F), cell blebbing, and cell fragmentation with the formation of apoptotic bodies (Fig. 5E). No signs of necrosis were evidenced.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although decidual lymphocytes constitutively express activation antigens [4, 20], the significant increase in the proportions of decidual lymphocytes expressing these antigens (CD25, CD38, and CD69) in spontaneous abortion (Fig. 1) further supports that a greater degree of immune activation occurs in this situation [21–23]. In mice and humans, normal pregnancy is related to the local and peripheral production of Th2 cytokines [9, 10], whereas abortion is associated with Th1 cytokine production [10, 11] or with a decrease in Th2 cytokines [12]. It is therefore probable that the increase in CD4+ cells detected by us in spontaneous abortion decidua corresponds to Th1 cells. A small population of cells with a peculiar phenotype (CD56+TCRß+) was also significantly higher in spontaneous abortion in comparison with ETP (Fig. 1). This phenotype may correspond to that of CD3+CD56+ natural T (NT) cells, detected in human liver, which produce mainly Th1 cytokines [19]. In this connection, decidual CD56+TCRß+ cells may also be a source of Th1 cytokines in human spontaneous abortion. It is interesting that decidual NTs, which secrete Th1 cytokines, have been shown to be involved in murine abortion [24].
An increase in Th1 cytokines may directly induce cytotoxicity or activate cytotoxic cells, which may lead to destruction of the trophoblast [6]. Although recent results have shown that human decidual lymphocytes may play a role in placental detachment during parturition [25], these lymphocytes from normal pregnancies were unable to spontaneously lyse the trophoblast in vitro [4, 16]. Nevertheless, they became lytic for the trophoblast after stimulation with IL-2 [3, 4] (Fig. 2). This suggested that decidual lymphocytes were potentially cytotoxic, although in normal pregnancies the preponderance of Th2 cytokines probably blocks Th1 cells, and hence inhibits cytotoxicity against the trophoblast [6, 9]. On the contrary, in spontaneous abortion we expected Th1-activated decidual lymphocytes to lyse trophoblast cells. Nevertheless, we found that neither the decidual lymphocytes of spontaneous abortion nor those of ETP or PBLs could lyse the JEG-3 EVT cell line in the 51Cr-release assay. On the other hand, decidual lymphocytes of spontaneous abortion, unlike decidual lymphocytes of ETP or PBLs, spontaneously induced DNA fragment release in JEG-3 (Fig. 3). In this connection, using histological methods, we confirmed the incidence of a significantly higher proportion of JEG-3 cells showing morphological signs of apoptosis, but not of necrosis, when these cells were cultured with decidual lymphocytes from spontaneous abortion (Figs. 4 and 5).
These data show that decidual lymphocytes of spontaneous abortion induce apoptosis, but not necrosis, in the EVT. Our results are consistent with those of Kokawa et al. [26], who also detected (by molecular biochemical techniques) an increase in low molecular weight DNA fragments in the trophoblast of spontaneous abortion in comparison with ETP. Kokawa et al. [26] observed a limited but detectable cleavage of DNA in the trophoblast of normal pregnancies. We also detected a proportion of JEG-3 cells with signs of apoptosis (2.96 ± 1.73) when these cells were cultured with decidual lymphocytes from ETP (Fig. 4). Nevertheless, we observed no activity in the DNA fragment assay when decidual lymphocytes of ETP were used as effectors against JEG-3 cells (Fig. 3). This apparent discrepancy may be attributable to the fact that the incidence of spontaneous [3H]TdR release by JEG-3 cells during this assay might have interfered with the detection of the DNA fragments induced by the decidual lymphocytes from ETP.
Several authors [27, 28] who used hematoxylin and eosin staining have also reported apoptosis in trophoblast from normal pregnancies. Our percentages for ETP were much higher than those reported by Smith et al. [27], and higher than but close to those of Chan et al. [28]. The differences are probably because we used an in vitro system with an EVT cell line, whereas those authors determined apoptosis in sections of placenta. Nevertheless, other authors who used the in situ DNA ligation method reported proportions higher than ours in first-trimester normal placenta [29]. The incidence of apoptosis during normal pregnancy suggests that apoptosis is a physiological mechanism by which excessive trophoblast expansion is controlled by decidual lymphocytes during normal pregnancies [29]. Our results and those of Kokawa et al. [26] suggest that during spontaneous abortion, this physiological mechanism of control is more intensely activated, leading to a more extensive destruction of the trophoblast by apoptosis, which results in the end of pregnancy. The mechanism by which apoptosis is induced in the trophoblast remains to be determined. Th1 cytokines such as tumor necrosis factor are directly cytotoxic to the trophoblast [30]; decidual lymphocytes such as NK and CD8+ cytotoxic T lymphocytes (CTLs) may also be activated by Th1 cytokines. In fact, the small CD56+CD25+ and CD56+HLA-DR+ subpopulations that are significantly higher in spontaneous abortion decidua in comparison with ETP (Fig. 1) may correspond to activated NK cells, which may also contribute to trophoblast elimination. Furthermore, the expression of killer inhibitory receptors (KIRs) by decidual NK cells, which in normal pregnancies is high [14], is reduced in human spontaneous abortion. This probably leads to the disinhibition of cell cytotoxicity against the trophoblast [22].
Most spontaneous abortions occur because of chromosomal abnormalities in the embryo. The mechanism that links this (or any) nonimmunological cause to the immune response of the decidua against the trophoblast remains to be elucidated. We have found that the presence of an embryo does not appear to be an important factor in the immune response because in our study there was no significant difference in the proportions of decidual lymphocytes between embryonic and anembryonic spontaneous abortion (not shown). Progesterone up-regulates the production of Th2 cytokines by lymphocytes [31], inhibits the production of Th1 cytokines [32], and appears to down-regulate CTL activity in the uterus [33]. In our cases of spontaneous abortion, we observed a significant decrease in progesterone concentration in peripheral blood from patients who had undergone spontaneous abortion. The reduced secretion of progesterone may trigger a Th1 response, which in turn, would activate cytotoxicity against the trophoblast. Nevertheless, we found no correlation between the serum concentrations of progesterone and any of the proportions of decidual lymphocytes in spontaneous abortion (not shown). It is probable that local rather than peripheral concentrations of progesterone correlate better with these proportions.
The Th1-Th2 balance appears to be a physiological mechanism that may lead either to Th2 cytokine production and successful pregnancy or Th1 cytokine production and spontaneous abortion [6]. It seems that decidual lymphocytes are always prone to kill the trophoblast, although their activity is blocked under normal conditions [7, 9, 10, 12–15]. Maternal immune activity against the trophoblast may be an efficient biological mechanism to eliminate a fetus when any gestational problem arises; this would select only normal fetuses to develop to term. Conception rates in mammals are high enough to allow for such a mechanism of natural selection.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 34958249015; engarcia{at}ugr.es
Received: 22 June 2001.
First decision: 11 July 2001.
Accepted: 17 May 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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26 mice. J Exp Med 1998 187:217-22314 NKT cells in abortion. Proc Natl Acad Sci U S A 2000 97:740-744This article has been cited by other articles:
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 O. Blanco, I. Tirado, R. Munoz-Fernandez, A. C. Abadia-Molina, J. M. Garcia-Pacheco, J. Pena, and E. G. Olivares Human decidual stromal cells express HLA-G Effects of cytokines and decidualization Hum. Reprod., January 1, 2008; 23(1): 144 - 152. [Abstract] [Full Text] [PDF]
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 H. J. A. Carp and Y. Shoenfeld Recurrent spontaneous abortions in antiphospholipid syndrome: natural killer cells an additional mechanism in a multi factorial process Rheumatology, October 1, 2007; 46(10): 1517 - 1519. [Full Text] [PDF]
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M.-R. Du, W.-H. Zhou, F.-T. Yan, X.-Y. Zhu, Y.-Y. He, J.-Y. Yang, and D.-J. Li Cyclosporine A induces titin expression via MAPK/ERK signalling and improves proliferative and invasive potential of human trophoblast cells Hum. Reprod., September 1, 2007; 22(9): 2528 - 2537. [Abstract] [Full Text] [PDF]
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M. Kimatrai, C. Oliver, A. C. Abadia-Molina, J. M. Garcia-Pacheco, and E. G. Olivares Contractile Activity of Human Decidual Stromal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 844 - 849. [Abstract] [Full Text] [PDF]
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