Molecular Human Reproduction, Vol. 8, No. 9, 841-848,
September 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Expression of interleukin (IL)-11 receptor by the human endometrium in vivo and effects of IL-11, IL-6 and LIF on the production of MMP and cytokines by human endometrial cells in vitro
1 Division of Biomedical Sciences/BMRC, Sheffield Hallam University, City Campus and 2 Biomedical Research Unit, Jessop Wing, Tree Root Walk, Sheffield, UK
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Abstract |
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Interleukin (IL)-6, leukaemia inhibitory factor (LIF) and IL-11 belong to the same family of cytokines whose receptors utilize gp130 as the signalling molecule. We have investigated the expression of the IL-11 receptor, IL-11R
, protein in the human endometrium in vivo and the effects of IL-6, LIF and IL-11 on the production of metalloproteinases (MMPs) and cytokines by cultured endometrial epithelial and stromal cells. Immunostaining showed that IL-11R was expressed in both epithelial and stromal cells, with epithelial staining being more intense than stromal staining and little variation in staining in either compartment throughout the cycle. Incubation of both stromal and epithelial cells with IL-6, LIF and IL-11 had no effect on MMP-2, -7, -9, transforming growth factor (TGF)ß or IL-1ß production or cell growth. IL-6 and LIF also had no effect on tumour necrosis factor (TNF) production, but IL-11 caused a dose-dependent decrease in TNF production by epithelial cells. IL-6 receptor, LIF receptor and gp130 were all expressed by cultured stromal and epithelial cells, showing that the lack of effect is not due to lack of expression of the receptor components. The results show that although IL-6, LIF and IL-11 signal through the same molecule, they may have different effects in endometrial cells, suggesting the activation of different signalling pathways, which may ultimately be important in the control of endometrial function. gp130/human endometrium/interleukin 11/interleukin 6/leukaemia inhibitory factor
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Introduction |
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The cytokines interleukin (IL)-6, IL-11 and leukaemia inhibitory factor (LIF) belong to the family of cytokines which also includes ciliary neurothrophic factor, oncostatin M and cardiotrophin 1. They are grouped as a family because of a shared helical bundle structure, shared subunits in their receptor complexes and in some cases, overlapping function. IL-6, IL-11 and LIF are all produced by the human endometrium (Tabibzadeh et al., 1995a
; Laird et al., 1997; Dimitriadis et al., 2000; Cork et al., 2001) and are known to be important in endometrial function and embryo implantation (Robb et al., 1998; Smith et al., 1998; Sanchez-Cuenca et al., 1999). Experiments in mice have shown the importance of both LIF and IL-11 in embryo implantation. Implantation does not occur in LIF knockout mice, although transfer of homozygous LIF-negative blastocysts to pseudopregnant, wild-type mice results in normal implantation and pregnancy outcome, showing that the defect is in the endometrium (Stewart et al., 1992). Female mice with either an inactive or null mutation for the IL-11 receptor chain (IL-11R) are fertile and their blastocysts implant and elicit an initial decidual response. However, only small decidua form and then subsequently degrade, resulting in pregnancy loss (Bilinski et al., 1998; Robb et al., 1998). In the human endometrium, LIF and IL-6 are produced mainly by epithelial cells. Endometrial levels of IL-6 and LIF peak at the time of implantation (Charnock-Jones et al., 1994; Tabibzadeh et al., 1995a) and are decreased in women with infertility and recurrent miscarriage (Laird et al., 1997; Lim et al., 2000; von Wolff et al., 2000). In contrast to IL-6 and LIF, IL-11 is produced by both stromal and epithelial cells, and stromal cell production increases during decidualization (Dimitriadis et al., 2000; Cork et al., 2001).
These cytokines bring about their effect on cells by interacting with specific membrane receptors, all of which associate with the signalling protein gp130. Both the IL-6 receptor (IL-6R) and IL-11R are transmembrane proteins with short intracellular domains that do not contribute to the signalling process. Binding of cytokine to these receptors results in gp130 homodimerization and gp130 activation can also be brought about by cytokine in combination with its soluble receptor. In contrast, the LIF receptor (LIFR) has a large cytoplasmic domain and shows considerable homology to gp130. Binding of LIF to its receptor results in heterodimerization between gp130 and LIFR, which together act as the signalling molecule (Taga and Kishimoto, 1997). Expression of LIFR, IL-6R and gp130 by the human endometrium in vivo has been reported (Tabibzadeh and Babaknia, 1995; Cullinan et al., 1996) with increased expression in epithelial cells and little variation in expression throughout the cycle.
Although the production of IL-6, LIF and IL-11 by the endometrium is well documented, less is reported about their effects on endometrial cell function. In this study, we investigate the effects of these cytokines on matrix metalloproteinase (MMP) and cytokine production by cultured endometrial stromal and epithelial cells. In addition, we show expression of IL-6R, IL-11R, LIFR and gp130 by cultured endometrial stromal and epithelial cells and, as it has not been previously reported, investigate the expression of IL-11R protein in human endometrium obtained throughout the menstrual cycle in vivo.
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Materials and methods |
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Human subjects
Endometrial biopsy samples were obtained throughout the menstrual cycle from normal fertile women attending the Jessop Hospital for Women in Sheffield, UK, for sterilization or hysterectomy for non-endometrial pathology. All women were aged 20–40 years and had regular cycles of 25–35 days. None of the women had taken any steroid hormones for 2 months prior to the study. Informed consent was obtained from all women participating in the study and ethical committee approval was obtained. Samples were either snap-frozen in liquid nitrogen for immunocytochemistry (19 biopsies) or taken to the laboratory for cell culture (11 biopsies). The biopsies used for immunocytochemistry were dated by morphological appearance according to the criteria of Noyes et al. (Noyes et al., 1950) as previously described (Li et al., 1991
) into early proliferative (n = 2), late proliferative (n = 6), early secretory (n = 2), mid-secretory (n = 6) and late secretory (n = 3) phases. For samples used for cell culture, menstrual cycle dating of the sample was determined by the date of the last menstrual period.
Cell culture
Human endometrial epithelial and stromal cells were prepared and cultured as previously described (Laird et al., 1993). The endometrial biopsy samples were collected in Hank’s balanced salt solution containing streptomycin and penicillin (100 µg/ml). The tissue was chopped finely with scissors and incubated at 37°C for 45 min in 5 ml Dulbecco’s modified Eagles medium (DMEM) containing 0.2% collagenase (type 1a) (DMEMC). During the incubation and again at the end of the incubation, the tissue was pipetted gently to disperse the cells. The epithelial cells were separated from stromal cells by centrifugation at 100 g for 5 min. The supernatant containing stromal cells was removed to another container and the pellet, which contained mainly epithelial cells present as glands, was incubated at 37°C for a further 45 min in 5 ml DMEMC. The cells were again dispersed by gentle pipetting and the epithelial cells were pelleted by centrifugation at 100 g. The stromal cells in the supernatant were added to those from the previous supernatant and together these were pelleted by centrifugation at 300 g.
The cells were further purified by unit density sedimentation. Epithelial and stromal cells were resuspended in 2 ml DMEM containing fetal calf serum (10%), glutamine (4 mmol/l) and penicillin and streptomycin (100 µg/ml) (CDMEM), and gently pipetted onto 8 ml of CDMEM in separate tubes and left for 30 min at room temperature. For the epithelial cells, the top 8 ml was discarded and the cells in the lower 2 ml were used for cell culture. For the stromal cells, the top 8 ml were taken for culture and the lower 2 ml discarded. Cells were plated into multiwell plates at a density of 3.2x105 cells per ml. The cells were grown in CDMEM to confluency (usually 48–72 h) at 37°C in an atmosphere of 5% CO2 and 95% air. At confluency the media was changed and replaced with CDMEM containing 3H-thymidine (37kBq/well; Amersham Life Sciences, Buckinghamshire, UK) and either no further supplements or IL-11, IL-6 or LIF (0.1–10 ng/ml; R&D Systems Ltd, Abingdon, Oxon, UK). These concentrations of cytokines were chosen because previous studies have shown that endometrial cells cultured in this way are responsive to other cytokines (particularly IL-1 and TNF) at these concentrations (Laird et al., 1996; Cork et al., 2001).
IL-11 was added to cells prepared from five biopsies obtained on days 7, 10, 18, 21 and 32 of the cycle; IL-6 was added to cells prepared from three biopsies obtained on days 9, 14 and 17 of the cycle; and LIF was added to cells prepared from three biopsies obtained on days 7, 17 and 25 of the cycle. The cells were grown for a further 48 h, after which the supernatants were removed and stored at –20°C for analysis of MMPs and cytokines. The cells were washed in phosphate-buffered saline (PBS) and incubated in cell dissociation solution at 37°C for 15 min and harvested onto filter paper. Cells corresponding to each well were placed in a scintillation tube with 2 ml scintillation fluid and the tritium content was counted using a Beta-counter. Previous immunocytochemical analysis of vimentin (a stromal cell marker), cytokeratin (epithelial cell marker) and CD-45 (leukocyte marker) of cells prepared and grown in this way has shown that the epithelial and stromal cells are essentially pure and free of leukocyte contamination after 48 h in culture (Laird et al., 1993; Tuckerman et al., 2000).
The specific effect of IL-11 on TNF production by epithelial cells was verified in two further experiments on cells prepared from biopsies obtained on days 10 and 17 of the cycle. In these experiments, replicate wells of cells were incubated with IL-11 (1 ng/ml) with and without an IL-11 neutralizing antibody (25 µg/ml MAB618; R&D Systems) in CDMEM media. Control wells containing CDMEM alone and antibody (25 µg/ml) alone were also included. An antibody concentration of 25 µg/ml was used as this was the concentration suggested by the manufacturer to neutralize 1 ng/ml IL-11. Cells were incubated for 48 h as before. After incubation the supernatant was removed and stored at –20°C until assayed for TNF.
Preparation of cultured cells and tissue sections for immunocytochemistry
A proportion of the separated epithelial and stromal cells prepared from three biopsies was plated into chamber slides at a density of 1.28x105 cells per well. After 5 days the media was removed and the cells washed in PBS. Sections (5 µm thick) were cut from frozen endometrial biopsy material. Cultured cells and tissue sections were fixed in 3.7% w/v formaldehyde in PBS for 15 min, washed twice for 5 min in PBS and fixed in methanol (–20°C) for 4 min and acetone (–20°C) for 2 min. After two washes in PBS, slides were stored at –20°C in sugar storage solution (50:50 0.5 mol/l sucrose:MgCl2 in PBS/glycerol) until being used for immunocytochemistry.
Immunocytochemistry
All slides were washed in PBS to remove sugar solution, quenched in 3% (v/v) hydrogen peroxide in methanol for 10 min at room temperature and then blocked for 30 min at room temperature in either PBS containing 10% normal rabbit serum (NRS/PBS) for IL-11R, IL-6R and gp130, or PBS and goat blocking serum from a rabbit ABC staining kit (Santa Cruz Biotechnology Inc., Wembley, London, UK) for LIFR. The slides were then incubated overnight at 4°C with primary antibody. For IL-11R, a goat anti-human IL-11R polyclonal antibody (sc1947; Santa Cruz Biotechnology) (10 µg/ml in NRS/PBS) was used. LIFR was detected using a rabbit anti-human LIFR antibody (sc659; Santa Cruz Biotechnology) (1 µg/ml in PBS containing 10% normal goat serum). IL-6R and gp130 were detected using mouse anti-human antibodies (MCA 1136, MCA822; Serotec Ltd, Oxford, UK) (10 µg/ml in NRS/PBS).
Binding of the primary antibody to antigen was visualized using either a standard peroxidase anti-peroxidase (PAP) method or an avidin–biotin peroxidase method. For the goat and mouse primary antibodies, slides were incubated with either rabbit anti-goat or rabbit anti-mouse second antibody (Dako Ltd, Ely, Cambridgeshire, UK) (1:200 dilution in NRS/PBS) for 30 min at room temperature followed by incubation with either goat or mouse PAP (Dako) (1:100 diluted in PBS) for 40 min at room temperature. For the rabbit primary antibodies, staining was visualized using a rabbit ABC staining system (Santa Cruz Biotechnology) according to the manufacturer’s instructions. All incubations were carried out in a humid environment to prevent evaporation. Slides were washed twice in PBS between each incubation. Colour was developed by incubation with peroxidase substrate, DAB (3,3'-diaminobenzidine tetrahydrochloride; Vector Ltd, Peterborough, UK) for 8 min. Slides were then washed in distilled water for 5 min, counterstained for 10 min with haematoxylin (10%; Vector), dehydrated through 50, 70 and 90% alcohol for 5 min each and then 95% and absolute alcohol for 10 min each before being cleared in Xylene overnight and mounted with a coverslip using DPX (BDH).
For all antibodies, negative reagent controls were stained in parallel with the primary antibody. The IL-11R antibody was blocked by incubation overnight (4°C) with a specific blocking peptide (sc1947P; Santa Cruz Biotechnology). The primary antibody was replaced with either mouse IgG (10 µg/ml) for IL-6R and gp130 or rabbit IgG (1 µg/ml) for LIFR.
For the biopsy samples, staining was repeated on at least three different sections from each biopsy. Two investigators, independent of one another, assessed all staining semiquantitatively and reached mutual agreement over any differences. Intensity of staining was graded on a scale of 0 (–) to 4 (++++), where – represented negative staining, + represented weak staining, ++ represented moderate staining, +++ represented strong staining and ++++ represented very strong staining.
MMP assays
The amounts of MMP-2, -9 and -7 produced from cultured endometrial epithelial and stromal cells were measured using commercially available Biotrak ELISA kits (Amersham) according to the manufacturer’s instructions. These ELISA kits detect pro-MMP-7, pro-MMP-9 and both the pro- and active forms of MMP-2. The sensitivities of the assays were 0.37 ng/ml for MMP-2, 0.6 ng/ml for MMP-9 and 0.16 ng/ml for MMP-7. The intra-assay variation was 5.7, 5.2 and 3.5% and the inter-assay variation was 10.0, 8.8 and 6.9% for MMP-2, -9 and -7 respectively. Samples were diluted 1:50 for MMP-2 and 1:20 for MMP-7 to ensure that the MMP concentration was within the range of the standard curve.
Cytokine assays
The concentrations of IL-1, TNF and TGFß in the cell culture supernatants were measured using commercially available paired antibody kits (Duoset; R&D Systems), according to the manufacturer’s instructions. The sensitivities of the assays were 4, 16 and 30 pg/ml for IL-1, TNF and TGFß respectively. The intra- and inter-assay variation were <10% for all assays. Latent TGFß was activated to the immunoreactive form by acid activation and neutralization. Cell culture supernatant (125 µl) was incubated with 1 mol/l HCl (25 µl) for 10 min at room temperature, followed by neutralization with 0.5 mol/l HEPES in 1.2 mol/l NaOH (25 µl). The samples were then diluted 1:2 to ensure that the values were within the standard curve. IL-11 was measured in the supernatants of the control wells from experiments with cultured epithelial cells using a commercially available Quantikine ELISA kit (R&D Systems) according to the manufacturer’s instructions. The sensitivity of the assay was 8 ng/ml with intra- and inter-assay variations of 2.4 and 6.9%. The samples were diluted 1:5 in calibration diluent to ensure that the cytokine concentration was within the range of the standard curve.
Statistical analysis
Student’s t-test or Mann–Whitney U-test were used to assess differences between concentrations of MMPs and cytokines produced by cells incubated with various concentrations of IL-6, LIF or IL-11 as appropriate.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Expression of IL-11R
in human endometrium in vivoImmunocytochemical staining showed that IL-11R
was present in both the epithelial and stromal cells of the human endometrium. Figure 1 shows staining obtained from four biopsies taken during the proliferative, early secretory, mid-secretory and late secretory phases of the cycle. Expression of IL-11R in the glandular epithelium was strong, with little variation between the different phases of the menstrual cycle. However, slightly more intense staining for IL-11R was seen during the secretory phases compared with the early to mid-proliferative phases of the menstrual cycle. Although luminal epithelium was not observed in sections from all biopsies, when it was observed it stained intensely and showed similar staining to that seen in the glandular epithelium of the same section (Figure 1d). Staining of the stromal compartment also remained relatively constant throughout the cycle, with only a small increase during the late proliferative and early secretory phases. However, stromal cell staining was less intense than that seen in the epithelium at all times.
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Expression of IL-11R, IL-6R, LIFR and gp130 by human endometrial cells in vitro
Figure 1
shows the expression of the subunits of IL-11R, IL-6R, LIFR and the signalling molecule gp130 in endometrial epithelial and stromal cells after 5 days in culture. Similar staining was seen in cells prepared from two biopsies taken at different times in the cycle. Positive staining for all four proteins was seen in both stromal and epithelial cells, which should therefore be capable of responding to IL-11, LIF and IL-6. Although it is not possible to determine the exact position of staining within the cell, it appeared to be cytoplasmic rather than nuclear, as would be expected for membrane proteins.
Basal production of IL-11 by cultured epithelial cells
Basal concentrations of IL-11 in the supernatants of epithelial cells prepared from the 13 biopsies ranged from 37–4434 pg/ml. In cells prepared from all but one biopsy the values were <750 pg/ml. There was no difference in the concentrations of IL-11 in supernatants of cells prepared from proliferative [median (range) 436 pg/ml (60–731)] and secretory [407 pg/ml (37–4423)] endometrium. In the five experiments where IL-11 was added to the cells, basal levels were 37, 668, 243, 60 and 544 pg/ml.
Effects of IL-11, IL-6 and LIF on TNF, IL-1ß and TGFß production by endometrial cells in vitro
Table I shows the range in basal levels of TNF, IL-1ß and TGFß produced by cultured stromal and epithelial cells prepared from all 11 biopsies divided into proliferative and secretory according to the time in the cycle when the biopsy was taken. TNF was not detected in the supernatants from cultured stromal cells but was produced by epithelial cells. The median amount of TNF produced by cells from the proliferative phase of the cycle was higher than that produced by cells prepared from secretory phase endometrium, but because of the large overlap in the ranges, this was not significant. Levels of IL-1ß in stromal cell supernatants were below the detection limit of the assay in cells from all biopsies. Levels of IL-1ß in epithelial cell supernatants were also below the detection limit in cells from five out of 11 biopsies (one out of five biopsies from the proliferative phase and four out of six biopsies from the secretory phase), and in the others, levels were very low (4–21 pg/ml). In contrast, TGFß was produced by both stromal and epithelial cells and more TGFß was present in supernatants from stromal than epithelial cells. However, there were no differences in the amounts of TGFß produced by both epithelial and stromal cells prepared from biopsies obtained during the proliferative and secretory phases of the cycle.
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Incubation of epithelial cells with IL-11 caused a concentration-dependent decrease in TNF
production (Figure 2). When the data from all five experiments were considered together and concentration of TNF considered as a percentage of control values, a significant decrease in TNF was seen at all concentrations of IL-11 used. These experiments were performed on cells prepared from three biopsies obtained in the secretory phase and two obtained in the proliferative phase of the cycle. The percentage inhibition caused by 10 ng/ml IL-11 was no different in cells prepared from biopsies obtained at different times of the cycle (65, 64 and 18% secretory, and 82 and 34% proliferative). The effects of IL-11 on TNF secretion by epithelial cells were verified by incubation of the cells with IL-11 together with an IL-11 neutralizing antibody. Addition of the antibody together with IL-11 prevented the decrease in TNF production seen when cells were incubated with IL-11 alone (Figure 3). A non-significant increase in TNF production was also seen in cells incubated with antibody alone. In contrast to the effect on TNF, incubation of both stromal and epithelial cells with IL-11 had no effect on IL-1ß (data not shown) and TGFß (Figure 2). Incubation of both stromal and epithelial cells prepared from both proliferative and secretory endometrium with IL-6 or LIF also had no effect on the secretion of TNF, IL-1ß or TGFß (data not shown).
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Effects of IL-11, IL-6 and LIF on MMP production by endometrial cells in vitro
Table I
shows the basal production of MMP-2, -9 and -7 by cultured stromal and epithelial cells divided into proliferative and secretory according to the time in the cycle that the biopsy was obtained. MMP-2 was produced by both epithelial and stromal cells, but stromal cells produced more MMP-2 than epithelial cells. Basal MMP-9 production by stromal cells was below the level of detection, while the amounts produced by epithelial cells were higher than those produced by stromal cells, but were less than the amounts of MMP-2 produced by epithelial cells. MMP-2 production by epithelial cells, but not by stromal cells, was significantly greater (P < 0.01) in cells prepared from secretory compared with proliferative endometrium. Levels of epithelial MMP-9 production were the same in cells prepared from secretory and proliferative endometrium. Stromal cells produced no MMP-7 and the amounts produced by epithelial cells were similar to amounts of MMP-2 produced by these cells. MMP-7 production by epithelial cells prepared from secretory endometrium was significantly lower (P < 0.01) than that from proliferative endometrium. Incubation of both stromal and epithelial cells with IL-6, LIF or IL-11 had no effect on the production of MMP-2, -9 or -7 (data not shown). In addition, incubation of stromal and epithelial cells with IL-11, IL-6 or LIF had no effect on 3H-thymidine uptake by these cells.
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Discussion |
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This study has, for the first time, shown the presence of IL-11R
protein in the human endometrium, with greater immunoreactivity in epithelial cells compared with stromal cells and little variation in receptor expression in either compartment through the menstrual cycle. A previous study has suggested that endometrial IL-11R mRNA expression also shows little menstrual cycle variation (Dimitriadis et al., 2000). Our previous work and that of others (Dimitriadis et al., 2000; Cork et al., 2001) has shown the expression and production of IL-11 by the endometrium, and has suggested increased production by stromal cells during decidualization. This, together with the fact that IL-11R knockout mice undergo an impaired decidual reaction which results in pregnancy loss (Bilinski et al., 1998; Robb et al., 1998), suggests that IL-11 may play an important role in the decidualization process in humans. However, the fact that IL-11R is expressed in the endometrium throughout the cycle suggests that IL-11 may also contribute to other aspects of endometrial function.
In this study, we therefore investigated the effects of IL-11 on several aspects of endometrial function including cell growth, MMP production and cytokine production. Endometrial LIF and IL-6 production are also thought to be important in endometrial function (Smith et al., 1998), and as they act via the same signalling pathways (Taga and Kishimoto, 1997) they might be expected to have similar biological functions to IL-11. We have therefore also investigated the effects of these cytokines on endometrial function. Although previous studies have shown the expression of IL-6R, LIFR and gp130 (Tabibzadeh and Babaknia, 1995; Cullinan et al., 1996; Dimitriadis et al., 2000) and this study has shown the expression of IL-11R in human endometrium in vivo, cells in culture are known to change expression of their protein components. To ensure that the cultured stromal and epithelial cells were capable of responding to IL-11, LIF and IL-6, we firstly showed that they expressed all the receptor components required. The expression of LIFR and gp130 protein by cultured stromal cells is in contrast to the reported lack of expression of their mRNA in vivo shown by in-situ hybridization (Cullinan et al., 1996). This may be due to differences in expression of LIFR and gp130 by stromal cells in vivo and in vitro, or to differences in sensitivities of the in-situ hybridization and immunocytochemical techniques.
In this study, incubation of both stromal and epithelial cells with IL-6, LIF and IL-11 had no effect on cell growth as assessed by 3H-thymidine uptake. Previous studies have suggested that IL-6 inhibits stromal cell proliferation (Zarmakoupis et al., 1995), but is only effective in cells prepared from secretory phase endometrium (Yoshioka et al., 1999). In our study, IL-6 was added to cells prepared from three biopsies, only one of which was taken during the early secretory phase of the cycle and therefore cycle-specific effects were impossible to determine. Effects of IL-6, LIF and IL-11 on endometrial epithelial cell growth have not been reported, but a previous study suggested that LIF has no effect on stromal cell growth (Ohata et al., 2001).
IL-11 caused a dose-dependent decrease in TNF production by epithelial cells. This effect was seen in cells prepared from all five biopsies and no differences were seen in cells prepared from proliferative and secretory endometrium. In addition, the inhibitory effect of IL-11 on TNF was abolished by addition of a neutralizing antibody in two further experiments, verifying that the inhibitory effect was due to IL-11. In a previous series of experiments we showed basal levels of IL-11 in epithelial cell supernatants of between 200 and 3000 pg/ml (Cork et al., 2001). Addition of IL-11 at 0.1 and 1 ng/ml may therefore not be expected to have any effect on these cells. However, basal IL-11 levels in the epithelial cell supernatants in these experiments where IL-11 was added were lower (37–750 pg/ml) than previously reported and therefore the cells should be capable of responding to added IL-11. The non-significant increase in TNF production by epithelial cells in the presence of antibody alone may be due to neutralization of the IL-11 produced endogenously by the cells. IL-11 has been shown to decrease pro-inflammatory cytokine levels, including those of TNF, in inflammatory diseases such as psoriasis (Trepicchio et al., 1999). TNF production by the endometrium is greatest in the secretory phase of the cycle (Tabibzadeh et al., 1995b) and is reduced in early pregnancy (Hunt et al., 1996). It is therefore possible that the increased production of IL-11 by decidualized stromal cells (Dimitriadis et al., 2000; Cork et al., 2001) could result in decreased endometrial TNF production during early pregnancy. IL-11 had no effect on IL-1ß or TGFß production by either epithelial or stromal cells, suggesting a differential effect of IL-11 on endometrial epithelial TNF and IL-1 and TGFß production. However, care should be taken in extrapolating from the in-vitro to the in-vivo situation and this differential effect of IL-11 on cytokine production may not reflect the physiological situation.
In agreement with our previous work and with that of others, TNF was only produced by epithelial cells (Hunt et al., 1992; Laird et al., 1996). In contrast, approximately equal amounts of TGFß were produced by stromal and epithelial cells. Only very low levels of IL-1ß were secreted by either stromal or epithelial cells. It is possible that differences in the production of these cytokines by stromal and epithelial cells may be due to differences in cell number. However, the stromal and epithelial cells were plated out at similar densities and used after 48 h when the cultures were similarly confluent. The amount of TNF and IL-1ß in supernatants from epithelial cells was considerably higher than that in the supernatant from stromal cells, and such a large difference is unlikely to be due only to differences in cell number. The presence of IL-1 within endometrial cells in vivo by immunocytochemistry has been shown in numerous studies (Tabibzadeh and Sun, 1992; Simon et al., 1993) and production of IL-1ß mRNA, but not secreted protein, in response to IL-1ß in cultured stromal cells has also been reported (Semer et al., 1991). The lack of secretion of IL-1ß by both endometrial and stromal cells is characteristic of non-monocyte/macrophage cell types and is thought to be due to lack of the enzyme which processes the pro-IL-1ß protein (31 kDa) to the mature, secreted form of IL-1ß (17 kDa) in other cell types (Semer et al., 1991). In contrast to IL-11, IL-6 and LIF had no effect on the production of TNF by epithelial cells, and also had no effect on TGFß production by both stromal and epithelial cells.
Addition of IL-6, LIF and IL-11 to both stromal and epithelial cells had no effect on MMP-2, -9 or -7 production. Measurements of basal MMP production suggest that both epithelial and stromal cells produce MMP-2, as has been suggested by other in-vivo and in-vitro studies (Martelli et al., 1993; Zhang et al., 2000). Our findings that epithelial cells are the major source of MMP-9 also agrees with those reported by others (Jeziorska et al., 1996; Skinner et al., 1999) and in particular it has been shown that basal production of MMP-9 by cultured stromal cells is very low (Huang et al., 1998). Significantly higher amounts of MMP-2 were produced by epithelial cells prepared from secretory endometrium compared with cells from proliferative endometrium. These in-vitro findings agree with the reported increase in MMP-2 mRNA in secretory endometrium in vivo (Irwin et al., 1996). The presence of MMP-7 in supernatants of epithelial, but not stromal, cells agrees with other in-vivo and in-vitro studies (Rodgers et al., 1993; Osteen et al., 1994). The decreased MMP-7 levels in supernatants from cells prepared from secretory endometrium compared with cells from proliferative endometrium suggests that, as for other MMPs, MMP-7 is suppressed during the secretory phase of the cycle (Marbaix et al., 1992). Although there are no reports on the effects of IL-11 on MMP production by other cell types, both IL-6 and LIF have been shown to affect gelatinolytic production by trophoblast cells (Bischof et al., 1995; Meisser et al., 1999) and increased MMP-11 expression has been seen in response to IL-6 in endometrial stromal cells (Singer et al., 1999).
The results of this study together with the work of others have suggested that although IL-6, LIF and IL-11 all act via gp130, they can have different effects within different cells. For example, in this study IL-6 and LIF had little effect on MMP and cytokine production by endometrial stromal and epithelial cells, but effects of IL-6 and LIF on MMP and cytokine production by human cytotrophoblast and articular chondrocytes have been reported (Villiger et al., 1993; Bischof et al., 1995; Nachtigall et al., 1996; Meisser et al., 1999). In addition, in this study IL-11 inhibited TNF production by epithelial cells, while IL-6 and LIF had no effect. We have shown that this lack of effect is not due to the inability of the cells to express the receptor components and therefore may be due to differences in mechanisms of signal activation or expression of other signalling components in the cells. Experiments in mice have shown that, although the LIF receptor is expressed in the uterus on days 3, 4 and 5 of pregnancy, a response is only seen on day 4, further suggesting the need for additional signals and/or feedback mechanisms other than LIF receptor binding for signalling in endometrial cells (Chen et al., 2000). The stoichiometry of the receptor complexes is known to be different for each cytokine. The IL-6 signal transducing receptor is a hexamer consisting of two IL-6 molecules, two IL-6 receptor chains and a homodimer of gp130 and the hexameric LIF signal transducing receptor consists of two repeats of LIF bound to the LIFR/gp130 heterodimer. In contrast, the IL-11/IL-11R complex undergoes dimerization in the presence of a single gp130 molecule and there is no evidence for gp130 dimerization (Neddermann et al., 1996). Gp130 has been shown to activate a number of different signalling pathways. The most studied is the Jak/STAT pathway, whereby activation of gp130 recruits Jaks to its cytoplasmic region leading to recruitment and specific phosphorylation of STAT molecules. Phosphorylation of STATs results in hetero- or homodimerization and translocation to the nucleus where they act as transcription factors and switch on gene expression. STAT3 has been shown to be the major STAT molecule to be activated by gp130 (Taga and Kishimoto, 1997; Funamoto et al., 2000). In addition, gp130 can also activate the MAP kinase pathway (Taga and Kishimoto, 1997; Lai et al., 1999). This pathway involves the GTP-binding protein Ras, followed by activation of a series of protein kinases (Raf, MEK and MAPkinase). Activation of MAP kinase by phosphorylation results in its transfer to the nucleus of the cell and activation of gene transcription.
In summary, this work has shown the expression of IL-11R protein in the human endometrium for the first time. It has also shown differences between the effects of IL-6, LIF and IL-11 on endometrial cell TNF production. Thus, although all these cytokines signal through the gp130 protein, the activation process may be different, resulting in activation of different signalling pathways which may ultimately be important in the control of endometrial function.
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The authors wish to thank research nurse Barbara Anstie for help in collection of the biopsy samples. These studies were supported financially by a grant from the Special Trustees of the United Sheffield Hospitals and a Sheffield Hallam University/University of Sheffield-funded PhD studentship.
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3 To whom the correspondence should be addressed at: Division of Biomedical Sciences, Sheffield Hallam University, City Campus, Howard St, Sheffield S1 1WB, UK. E-mail: s.m.laird{at}shu.ac.uk
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Submitted on September 10, 2001; resubmitted on February 4, 2002; accepted on May 17, 2002.
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