1 Center of Immunology and 3 Laboratory of Tumour and Developmental Biology, Institute of Pathology CHU-B23 and 2 Department of Gynaecology and Obstetrics, University of Liege, B-4000 Liege-Sart Tilman, Belgium and 4 Bichat Hospital, Center of Reproductive Medicine, F-75006 Paris, France
5 To whom correspondence should be addressed at: Center of Immunology, Institute of Pathology CHU-B23, University of Liege, B-4000 Liege-Sart Tilman, Belgium. Email: sperrier{at}ulg.ac.be
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Abstract |
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Key words: endometrium/human chorionic gonadotropin/insulin-like growth factors/interleukin 6/leukemia inhibitory factor/transforming growth factor beta
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Introduction |
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Different cytokines participate in the success of implantation and contribute to endometrial receptivity. Targeted inactivation of Lif in mice leads to a complete failure of blastocyst implantation that is rescued after intraperitoneal LIF administration (Stewart et al., 1992). The role of LIF in human implantation is much less well established. The LIF protein and LIF messenger RNA are present in the human endometrium with a maximum level during the implantation window in EEC. At the same time, LIF receptor is expressed by the blastocyst and human endometrium (Charnock-Jones et al., 1994
; Arici et al., 1995
; Cullinan et al., 1996
; Laird et al., 1997
). Endometrial cells produce LIF in vitro but also in vivo. Delage et al. (1995a) demonstrated that the in vitro secretion by cultured endometrial explants obtained from infertile women were significantly lower when compared to fertile women. Laird et al. (1997)
further confirmed that the amount of LIF in flushings obtained from women with unexplained infertility was significantly lower than those from normal fertile women on day LH+10. On the contrary, when measured on day 26 of the cycle, a low concentration of LIF in uterine flushing is predictive of subsequent implantation. An excess of LIF in the luminal fluid at this time could reflect local inflammation which is detrimental for implantation (Ledee-Bataille et al., 2002
). Interleukin 6 (IL-6) is also secreted in the human endometrium following a temporal profile during the menstrual cycle, with levels rising during the luteal phase and menstruation (Tabibzadeh et al., 1995
; Vandermolen and Gu, 1996
). The IL-6 receptor is expressed at the surface of the embryo, the trophoblast and the endometrium (Sharkey et al., 1995
). Blastocyst implantation is not impaired in Il6/ mice, although the development of the conceptus is compromised (Robertson et al., 1992
; Kopf et al., 1994
; Salamonsen et al., 2000
).
The main objective of this study was to further explore the hypothesis that hCG, and growth factors present at the embryonicendometrial interface and known to intervene in embryo development (IGFs and TGF-1), are able to control LIF and IL-6 secretion by human EEC.
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Materials and methods |
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Separation of endometrial epithelial cells from stromal cells
Endometrial biopsy samples were collected in Hanks' balanced salt solution (HBBS) containing penicillin (100 IU/ml) and streptomycin (100 µg/ml) and endometrial epithelial cells (EEC) were isolated following the method described by Laird et al. (1993). The tissues were finely chopped with scissors and digested for 45 min at 37°C in 5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 0.2% collagenase type 1a (Sigma Aldrich, St Louis, MO). During incubation and at the end of incubation, the tissue was gently pipetted every 15 min to disperse the cells. The EEC were separated from stromal cells by centrifugation for 20 min at 100 g. Pellets contained mainly epithelial cells present both as small clumps and as separated cells. The two withdrawn supernatants, containing mainly stromal cells, were pooled and centrifuged at 300 g to pellet the cells. Epithelial and stromal cells were purified by unit density sedimentation. Each fraction of cells was resuspended in 2 ml DMEM containing 10% fetal calf serum (FCS), 1% glutamine and antibiotics, and gently pipetted onto 8 ml of DMEM + 10% FCS in a test tube and left for 30 min at room temperature. Afterwards, epithelial cells were in the lower 2 ml, while stromal cells were in the upper 8 ml of supernatants. Epithelial cells were used for immunocytochemistry, cell culture/cytokine production and for molecular biology. Before culturing in serum-free medium, epithelial cells were centrifuged at 100 g, and the pellets were washed three times with DMEM containing 1% glutamine, antibiotics, 0.3% bovine serum albumin and 25 mIU/ml human insulin. Stromal cells were used only for immunocytochemistry.
Cell cultures
EEC were plated into 24-well plates at 2 x 105 cells per ml and per well and cultured for 72 h in DMEM with 1% glutamine, 0.3% bovine serum albumin and 25 mIU/ml human insulin. Each treatment was applied in a micro volume (50 µl) at the beginning of incubation. Each condition was tested in triplicate. Too few EEC after separation resulted in the exclusion of the explant from some treatments. The cells used for immunocytochemistry were cultured for 24 h on multi-test slides in media containing DMEM with 1% glutamine, 25 mIU/ml human insulin and 10% FCS.
Cytokines, growth factors and antibodies
Recombinant (rec) IL-1 was provided by Preprotech (London, UK) and used at a final concentration range between 109 and 1012 M. Rec IL-1
was obtained from Genzyme (Cambridge, UK) and was used at 109 M. Anti-human IL-1
antibody was provided by R&D Systems (Abingdon, UK) and used at a concentration of 50 µg/ml. Phytohemagglutinin (PHA) was provided by Glaxo-Wellcome (Zebulon, USA) and used at a final concentration of 5 µg/ml. Lipopolysaccharide (LPS) was purchased from Sigma and used at a final concentration of 20 µg/ml. Human chorionic gonadotropin (hCG) (Pregnyl) was provided by Organon (Brussels, Belgium) and was added at a final concentration of 1, 5, 10 and 50 IU/ml. Anti-hCG antibody from Santa Cruz Biotechnology (Santa Cruz, USA) was added at a final concentration of 2 µg/ml. Transforming growth factor-
1 (TGF-
1) was prepared according to the methodology of Cone et al. (1988)
and used at 108, 1010 and 1012 M. Human insulin-like growth factor (IGF)-1 and IGF-2 (R&D Systems) were used at three final concentrations: 108, 1010 and 1012 M. Anti-human cytokeratin (5, 6, 8, 17 and 19) mAb (clone MNF 116, code no. M0821) and anti-vimentin V9 were from Dako (Copenhagen, Denmark). mAb anti-CD45RB was obtained from Dako. Second antibodies (Jackson, Pennsylvania, USA) were fluorescein (FITC)-conjugated goat anti-mouse IgG (second antibody A) and rhodamine (TRITC)-conjugated goat anti-rabbit IgG (second antibody B).
Cytokine immunoassays
Supernatants of cultured EEC were collected for assay after 72 h. Collected media were stored at 20°C until cytokine measurements. All cytokine (IL-6 and LIF) concentrations were measured with specific ELISAs (Biosource-Europe, Nivelles, Belgium) according to appropriate instructions (De Groote et al., 1992). Sensitivity was 20 pg/ml for LIF and 3 pg/ml for IL-6. Range was 1508200 pg/ml for LIF and 8.5540 pg/ml for IL-6. Inter and intra-assay coefficients of variation (CV) for LIF were <8.4% and <5%, respectively. Inter and intra-assay CV for IL-6 were <9.3% and <7.7%. For LIF assays, results obtained were usually in the standard curve and a preliminary dilution was not necessary. When some rare results were not in the standard curve, a new assay was performed with diluted supernatants. For IL-6 assay, the supernatants were usually diluted (systematically for the IL-1
condition) to ensure that they were in the limits of the standard curve.
Proliferation test
Proliferation tests were performed on EEC cultured for 72 h from three different endometria, using a 5-bromo-2'deoxyuridine (BrdU) cell proliferation colorimetric immunoassay provided by Roche, according to the manufacturer's instructions.
Immunocytochemistry
Multitest slides were kept for 24 h in FCSepithelial or stromal cell medium to allow cell plating (5000 cells/well) and then stained after 20°C methanol fixation (Robert et al., 1991; Martens et al., 1996
). Slides were first incubated with 10% normal goat serum to prevent non-specific binding of goat second antibody to mouse or rabbit Ig. Immunostaining was performed with the following reagents: anti-human cytokeratin mAb MNF 116 diluted at 1:50, anti-vimentin V9 mAb diluted at 1:500, or anti-CD45RB mAb diluted at 1:100, followed by second antibody A or B. The slides were mounted with glycerol/gelatin medium and observed using an Olympus fluorescence microscope. Images were collected with 20x and 40x objectives. A mercury source was used for fluorescein (488 nm) and rhodamine (568 nm).
Molecular biology
RNA extraction
EEC samples isolated from seven different endometrial biospies were frozen in liquid nitrogen (or at 70°C). Total RNA was extracted using an RNeasy Mini Kit® from Qiagen (Valencia, USA) according to the manufacturer's instructions. Samples were treated by RNase free DNase (Roche, Indianapolis, USA) to remove potentially contaminating DNA. The integrity of extracted RNA was assessed by electrophoresis in ethidium bromide-stained 1% agarose MOPS/EDTA gel.
RTPCR
Two hundred and fifty nanograms of total RNA were subjected to reverse-transcriptase using First Strand cDNA Synthesis Kit (Roche). In this method, RNA is reverse transcribed by AMV into single-stranded cDNA. The cDNA was secondarily amplified by FastStart Taq DNA Polymerase (Roche) using the primers corresponding to human hCG/LHR cDNA sequence (upper primer 5'-GGCCGAAAACCTTGGATATTT-3', position 676696; and lower primer 5'-GTAAGTCAACGTGGCCTCCAG-3', position 813833). The expected PCR product was 158 bp in length. Human -actin PCR primer pair (R&D Systems, Abingdon, UK) was used, resulting in a PCR product of 528 bp. Fifty microlitres of PCR reaction mixture contained 20 pmol of each oligonucleotide primer and 2 IU of Taq DNA polymerase. The reaction was started at 94°C for 5 min and run for 40 cycles (45 sec at 94°C, 45 sec at 61°C and 1 min at 72°C). Aliquots of 15 µl of amplified fragments were visualized in ethidium bromide-stained 2% agarose gel. The size of DNA species generated was determined using EZ load precision Molecular Mass Standard (Bio-Rad, Hercules, USA) and a 100 bp ladder (Pharmacia Biotech, Uppsala, Sweden). RTPCR products were sequenced using ABI prism BigDye Terminator Cycle Sequencing Ready Reaction Kit protocol (Genomexpress, Meylan, France) to ensure hCG/LHR identity.
Mathematical and statistical analyses
Because of individual variability, all data have been normalized and expressed as the percentage of the mean of basal values (mean ±SEM). For each experiment (except when specified), statistical analysis of the response to any kind of treatment was performed for the difference with basal value. The significance of differences was evaluated using unpaired nonparametric MannWhitney test with confidence intervals of 95%. Statistical analyses were performed using GraphPad Prism software.
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Results |
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Regulation of endometrial cytokine production by hCG, IGFs and TGF-1
Results were analysed according to the phase of the menstrual cycle: group A for proliferative phase and group B for secretory phase. The proliferative effect of the different reagents tested on EEC cultured for 72 h was assessed in triplicate by a BrdU cell proliferation test provided by Roche (Table II). No significant proliferative effect was found for hCG (50 IU/ml), IGF-1 (108 M), IGF-2 (108 M), TGF-1 (108 M) or IL-1
(109 M).
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Discussion |
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LIF is known to stimulate hCG production by trophoblast (Nishino et al., 1990; Sawai et al., 1995
). In our cultures of human EEC, hCG stimulated LIF production in a concentration-dependent way, suggesting that the blastocyst may influence its own implantation through hCG. This positive effect of hCG on LIF production was specific since it was blocked by a neutralizing anti-hCG antibody and not mediated by a local action of IL-1
since it was not blocked by an anti-IL-1
antibody. Our results also confirm and add to the in vivo results of Licht et al. (2001)
who observed an increase of LIF levels in uterine fluids after microdialysis of the uterine cavity with hCG. The expression of hCG/LHR by human endometrial epithelium in proliferative and secretory phase was evidenced by RTPCR. Some controversy about the classical LH/hCGR expression in the human endometrium was raised by Stewart et al. (1999)
and Stewart (2001)
. Several groups have however shown the presence of hCG/LHR in extragonadal human, rabbit or mouse female reproductive organs (Ziecik et al., 1986
; Jensen and Odell, 1988
; Bonnamy et al., 1990
; Reshef et al., 1990
; Bhattacharya et al., 1993
; Bonnamy et al., 1993
; Derecka et al., 1995
; Zhang et al., 2001
). Moreover, using nested RTPCR, Licht et al. (2003)
recently demonstrated the expression of full-length hCG/LHR mRNA in human endometrium regulated by alternative splicing. In our study, we detected hCG/LHR transcripts on EEC by non-nested RTPCR assays. Despite not observing an effect of cycle stage on the ability of hCG to regulate LIF and IL-6 in EEC cultures, the hCG/LHR expression seems to be lower in the proliferative phase compared to the secretory phase. Further Real Time RTPCR experiments are necessary to exactly quantify the amount of hCG/LHR expression throughout the cycle. As suggested by Fazleabas et al. (1999)
in the Papio anubis endometrium, our results confirm that hCG, besides its endocrine role in maintaining progesterone production by the corpus luteum, also exerts a paracrine action on the human endometrial epithelium.
Previous studies have shown that IL-6 interacts with IL-6 receptor on the trophoblast, resulting in hCG release (Nishino et al., 1990; Masuhiro et al., 1991
; Li et al., 1992
). Our results show that hCG interacts with hCG/LH-R on EEC, resulting in IL-6 inhibition. Further experiments are necessary to elucidate whether hCG could also have such a negative feed-back on IL-6 secretion by trophoblast. Through the inhibition of the pro-inflammatory endometrial IL-6, hCG may play a new role as an immune regulator at the embryonicendometrial interface during implantation. In our cultures of purified EEC from 28 fertile women, we did not obtain the same results as Uzumcu et al. (1998)
who used a mixed population of endometrial cells from six IVF patients, and who observed that hCG (100 and 500 IU/ml) stimulated IL-6 production. The role of hCG as immunosuppressive factor was already suggested by its inhibition of PHA- or concanavalin A-mediated proliferation of human lymphocytes in vitro. Moreover, hCG inhibits IL-2 and simultaneously stimulates sIL-2R (a soluble form of the IL-2R which modulates IL-2 bioavailability) secretion by cultures of human peripheral blood mononuclear cells, suggesting a direct role for hCG in regulation of lymphocyte biological activity (Nakayama et al., 2002
). Khan et al. (2001)
also confirmed the regulatory role of hCG on Th1 cell activity since the treatment of non-obese diabetic (NOD) mice with hCG before the onset of clinical symptoms dramatically inhibited the development of diabetes for a prolonged period of time. Transfer of spleen cells from the hCG-treated NOD mice into immunodeficient NOD/SCID mice blocked the development of diabetes in these mice (Khan et al., 2001
). Finally, recent results of Kayisli et al.
suggested that hCG could be a link in the development of maternal immunotolerance and may facilitate trophoblast invasion by regulating proapoptotic molecules such as FasFas ligand system in endometrial cells (Kayisli et al., 2003
).
IGFs induced a marked increase of LIF secretion without any significant effect on IL-6. In the endometrium, IGFs and their binding proteins (IGFBPs 16) are derived from stromal cells, whereas the epithelium predominantly expresses IGF receptors (IGF type 1 receptor or IGF-1R, IGF type 2 receptor or M6P/IGF-2R) (Rutanen, 1998). IGFs and predominantly IGF-2 are also detected in many fetal tissues from the first trimester (Han and Carter, 2000
). In mouse embryos, increasing Igf1 mRNA levels are observed between the 8-cell and blastocyst stages (Doherty et al., 1994
), while Igf2 expression has been detected from the 2-cell stage. In addition to a paracrine/autocrine control of the cycling endometrial development, the IGFs and their binding proteins appear to play a crucial role during implantation and establishment of pregnancy (Nayak and Giudice, 2003
). It is increasingly evident that disturbances in the IGF system occur in complications of human pregnancy, such as intrauterine growth restriction, as well as pre-eclampsia (Irwin et al., 1999
). In our experimental model, we show that, in the presence of IGF-1 and IGF-2, pro-implantatory LIF production by EEC is increased in vitro. The in vivo source of IGF-1 and -2 might be endometrial stromal cells, but also the pre-implantatory embryo. Indeed, IGF1 and IGF2 mRNA are expressed by human trophoblast, IGF-2 being the most abundantly expressed.
The TGF-1 isoform markedly enhanced LIF secretion while, like hCG, it reduced IL-6 release by cultured epithelial cells from the human endometrium. The TGF-
superfamily related glycoproteins are well known for their role in the induction of embryonic mesoderm and also for their immunosuppressive properties. In mammals, there are four isoforms of TGF-
: TGF-
1, -
2, -
3 and -
4 (Tabibzadeh et al., 1998
). The TGF-
superfamily involves many members, such as bone morphogenic proteins, inhibins, activins and Müllerian inhibitory factor. TGF-
is known to induce in vitro cytotrophoblast differentiation into syncytiotrophoblast, controlling then the process of placentation. The four isoforms of TGF-
are present in the endometrial epithelium and stroma throughout the menstrual cycle in a time-dependent manner, their expression being the highest in the secretory phase (Kauma et al., 1990
; Casslén et al., 1998
; Godkin and Doré, 1998
; Tabibzadeh et al., 1998
). Our data show that, besides its mesoderm-inducing properties within the embryo, TGF-
1 may also contribute both to implantation by enhancing endometrial LIF production, and to maternal tolerance of the fetal graft by inhibiting IL-6 release. This tolerogenic action of the TGF-
superfamily had already been suggested by expression of a TGF-
2-related molecule in the murine decidua (Clark et al., 1995
; Merali et al., 1996
). Our results are in line with Arici et al. (1995)
who demonstrated that TGF-
and IL-1
are potent inducers of LIF expression in endometrial stromal cell culture.
Although IL-6 and LIF signal through the same molecule gp 130, they may exert different effects in endometrial cells, suggesting the activation of different intracellular signalling pathways, which may ultimately be important in the control of endometrial function (Ernst and Jenkins, 2004). This was also suggested by Cork et al. (2001)
on epithelial and stromal cells since LIF and IL-6 had no effect on tumour necrosis factor (TNF-
) production, whereas IL-11, another member of the family, caused a decrease in TNF-
production (Cork et al., 2001
).
In conclusion, this study shows that hCG treatment of human EEC in culture increases LIF secretion and inhibits IL-6 secretion in a concentration-dependent manner. Other growth factors present at the embryonicendometrial interface (IGFs and TGF-1) also promote endometrial secretion of the pro-implantatory LIF, while TGF-
1 strongly inhibits endometrial production of the pro-inflammatory IL-6. These observations suggest that the human blastocyst may contribute both to its implantation through an increase of endometrial LIF secretion and to its tolerance through an inhibition of endometrial IL-6 production.
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Acknowledgements |
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Submitted on March 22, 2004; resubmitted on June 4, 2004; accepted on July 13, 2004.