Molecular Human Reproduction, Vol. 8, No. 7, 636-643,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells
1 Prince Henry's Institute of Medical Research, P.O.Box 3168, Clayton, Victoria 3168 and 2 The Walter and Eliza Hall Institute of Medical Research and Cooperative Research Centre for Cellular Growth Factors, PO Royal Melbourne Hospital, Victoria, Australia
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Differentiation of endometrial stromal cells into decidual cells is crucial for embryo implantation and placentation. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus. We examined the role of IL-11 during progesterone-induced decidualization of human endometrial stromal cells over a 10–12 day period, using prolactin (PRL) production as a decidual marker. These cells produced biologically active IL-11 and expressed IL-11, IL-11R
and PRL mRNA during decidualization. Neutralization of endogenous IL-11 with an anti-human (hu)IL-11 antibody (AB) reduced production of PRL from day 8 and insulin-like growth factor binding protein (IGFBP)-1, another marker of decidualization, from day 10 of culture. Following AB washout, PRL and IGFBP-1 secretion increased. Addition of recombinant (r)huIL-11 (10 or 100 ng/ml) to endometrial stromal cells increased secretion of PRL from day 4 and IGFBP-1 from day 6 compared with progesterone alone. Morphological signs of differentiation accompanied biochemical differentiation in the progesterone-treated cells and were further induced by exogenous rhuIL-11. Our observations demonstrate that human endometrial stromal cells produce biologically active IL-11, which promotes progesterone-induced decidualization. These results suggest that IL-11 has both paracrine and autocrine actions on human endometrial stromal cells and plays an important role in preparing the human endometrium for implantation.
decidualization/endometrial stromal cells/IL-11/IL-11R/implantation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Embryo implantation and development of a functional placenta are critical steps in the establishment of pregnancy. During the secretory phase of the menstrual cycle, human endometrial stromal cells spontaneously differentiate into decidualized stromal cells which are morphologically and biochemically distinct. If pregnancy ensues, decidualization proceeds further and provides the maternally derived component of the placenta. The molecular interactions that regulate the formation, maintenance and remodelling of decidua are poorly understood although many factors such as progesterone (Huang et al., 1987
), corticotrophin-releasing factor (Ferrari et al., 1995; Zoumakis et al., 2000), prostaglandin (PG), estradiol (E2) and other pathways linked to the cAMP pathway are known to be involved (Tang and Gurpide, 1993).
Cytokines play important roles in the events involved in implantation. The analysis of mice with alterations in genes encoding cytokines and their receptors has shown that some cytokines are absolutely required for female fertility (Salamonsen et al., 2000). Interleukin (IL)-11 is a cytokine with pleiotropic actions on a range of different tissues (Du and Williams, 1997). It signals via a heterodimeric receptor complex composed of an IL-11 receptor chain (IL-11R) and the signalling component gp130. IL-11 is one of the few molecules which has been shown to be obligatory for implantation. Female mice with either a null mutation of the gene encoding IL-11R (Robb et al., 1998) or a hypomorphic IL-11R allele were found to be infertile due to defective decidualization during the endometrial response to the implanting blastocyst (Billinski et al., 1998).
Recent evidence has indicated that IL-11 may be involved in human female fertility and more specifically in the decidualization of human endometrial stromal cells. Two studies have demonstrated intense IL-11 immunostaining in the human endometrium during the secretory phase, which includes the `implantation window' (Dimitriadis et al., 2000; Cork et al., 2001). Immunoreactive IL-11 is found in all the major cell types in secretory phase endometrium with greatest intensity in the decidual cells. IL-11 mRNA is likewise expressed by all the major cell types, and both components of the IL-11 receptor (IL-11R and gp130) are expressed in endometrial tissue throughout the menstrual cycle (Dimitriadis et al., 2000). Furthermore, IL-11 mRNA has been shown by gene microarray analysis to be up-regulated during in-vitro decidualization of endometrial stromal cells (Popovici et al., 2000).
Numerous studies have demonstrated that human endometrial stromal cells decidualize in vitro in the presence of certain hormones and growth factors (Tseng et al., 1992; Frank et al., 1994). The decidual cells are typically characterized by their secretory products, particularly prolactin (PRL) and insulin-like growth factor binding protein (IGFBP)-1 (Tabanelli et al., 1992) and by their pavement-like morphology (Tabanelli et al., 1992). Decidualization of endometrial stromal cells can be induced in vitro by the addition of progesterone to cultures treated with E2 (Huang et al., 1987). We postulated that IL-11 produced by endometrial cells during the secretory phase of the human menstrual cycle may modulate the decidualization process in an autocrine or paracrine manner.
The present study examined the role of IL-11 during progesterone-induced decidualization in vitro. Cultured endometrial stromal cells were shown to secrete biologically active IL-11. A role for both exogenous and endogenous IL-11 in the decidualization of endometrial stromal cells was established by measuring the secretion of PRL and IGFBP-1 in cultures supplemented with IL-11 or in which IL-11 action was inhibited by the addition of a neutralizing antibody. We also demonstrated the expression of IL-11R mRNA transcripts and the presence of IL-11R protein in cultured endometrial stromal cells during decidualization.
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Materials and methods |
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Tissue collection
Endometrial tissue was obtained by curettage between days 8–24 of the menstrual cycle from women with regular menstrual cycles and no apparent endometrial dysfunction. Approval was obtained from the Human Ethics Committee at Monash Medical Centre, Melbourne, Australia. Histological dating of the menstrual cycle was performed by an experienced gynaecological pathologist according to the method of Noyes et al. (Noyes et al., 1950
). Samples for cell culture were collected into Dulbecco's modified Eagle's medium (DMEM; Trace Biosciences, Sydney, Australia).
Culture of endometrial explants
Endometrial tissue was divided into two equal wet weight portions (each 
23 mg), placed in a 1:1 mixture of DMEM and Ham's F12 medium (Trace Biosciences) and 1% antibiotics (penicillin, streptomycin and fungizone; Commonwealth Serum Laboratories, Melbourne, Australia) and finely chopped. The medium was then replaced with 1 ml of either DMEM/F12 (1:1) or DMEM/F12 (1:1)/10% charcoal-treated (CT) fetal calf serum (FCS; Trace Biosciences) and 1% antibiotics. After 24 h at 37°C in a humidified tissue culture incubator containing 5% CO2 in air, tissue explants and medium were centrifuged at 160 g and the supernatant was collected and stored at –20°C.
Isolation and culture of human endometrial stromal cells
Cells were prepared from endometrial tissue as described previously (Zhang and Salamonsen, 1997). Briefly, tissue was finely minced with scissors and digested by bacterial collagenase type III (Worthington Biochemical Corporation, Freehold, NJ, USA) at a concentration of 45 IU/ml, in the presence of 3.5 µg/ml deoxyribonuclease (Boehringer Mannheim Biochemica, Mannheim, Germany). After 30–45 min of agitation at 37°C, the digested tissue was filtered sequentially through 45 and 10 µm nylon filters to remove glands, layered onto Ficoll-Paque (Pharmacia, Uppsala, Sweden) and centrifuged at 160 g to pellet erythrocytes. Stromal cells were collected from the Ficoll interface and resuspended in a 1:1 mixture of DMEM/F12 with 10% CT–FCS and 1% antibiotics. The cells were transferred to 24-well dishes (2.5x105 cells/well) and grown for 2–4 days (until confluent) with medium changes every 48 h.
Once confluent, the cells were washed with DMEM/F12 (1:1) and the medium replaced with serum-free medium containing either transferrin, sodium selenite and linoleic acid (TSL) or serum free mix (SFM); SFM (final concentration) consisted of insulin (5 µg/ml; human actrapid; Novo-Nordisk Pharmaceuticals Pty Ltd, Sydney, Australia), transferrin (10 µg/ml; Sigma Chemical Company, St Louis, MO, USA), sodium selenite (25 ng/ml; Sigma), linoleic acid (10 nmol/l; Sigma) and bovine serum albumin (BSA 0.1%; Sigma). TSL (final concentration) consisted of transferrin (10 µg/ml), sodium selenite (25 ng/ml) and linoleic acid (10 nmol/l).
Cells were maintained in medium containing either TSL or SFM for 4 days with a medium change at 48 h. After 4 days, fresh medium was added (experimental day 0) with the additions of either estradiol 17ß 10-8 mol/l (Sigma) with or without progesterone 10-7 mol/l (P-0130; Sigma), or E2 + progesterone with either anti-human (hu)IL-11 (10 µg/ml; AF-218-NA, R&D Systems Inc., Minneapolis, MN, USA) or recombinant (r) huIL-11 (1, 10 or 100 ng/ml; Genetics Institute, Cambridge, MA, USA). Supernatant was harvested every 48 h, centrifuged at 160 g to remove any non-adherent cells and stored at –20°C. On day 12, cell viability was assayed by Trypan Blue (Trace Biosciences) exclusion assay. Each experiment was repeated at least three times with different cell preparations. For immunohistochemistry, cells were grown on poly-L-lysine-treated glass slides.
Endometrial stromal cell purity
Stromal cell preparations used in all experiments were >97% pure as assessed by immunostaining for cytokeratin (using antibody SC-8018; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and vimentin (using antibody SC-6260; Santa Cruz Biotechnology) using standard techniques.
PRL, IGFBP-1 and IL-11 ELISA assays
The production of immunoreactive (IR) PRL, IGFBP-1 and IL-11 by cultured endometrial stromal cells was measured quantitatively using ELISA kits (respectively Bioclone Aust. Pty Ltd, Marrickville, NSW, Australia; Diagnostics Systems Laboratories, Inc., Webster, TX, USA; R&D Systems Inc.) according to the manufacturer's instructions. The conditioned media collected from cell cultures were thawed and concentrated 9-fold using a vacuum dryer prior to assay in duplicate. The lower detection limits of the assays are 50 mIU/l, 0.8 ng/ml and 15 pg/ml for PRL, IGFBP-1 and IL-11 respectively. The coefficient of variation within assays was 3.1, 2.2 and 1.8% and the coefficient of variation between assays was 6.7, 7.1 and 5.5% for PRL, IGFBP-1 and IL-11 respectively. Medium containing CT–FCS, SFM or TSL kept under the same culture conditions in the absence of cells and concentrated appropriately did not show measurable levels of IR-PRL, IR-IGFBP-1 or IR-IL-11 (data not shown).
Bioassay for IL-11
The bioactivity of IL-11 was measured in a human proliferation assay using Ba/F3 cells transfected with human IL-11R and human gp130 cDNAs (Ba/F3 huIL-11R/gp130) expressed in the expression vector-pEFBOS (Mizushima and Nagata, 1990) as previously described (Metcalf et al., 1994, 1995). Ba/F3 huIL-11R/gp130 cells are dependent on IL-11 for survival. Ba/F3 huIL-11R/gp130 cells were washed three times in DMEM containing 10% FCS and resuspended at a concentration of 20 000 cells/ml in the same medium. Aliquots (10 µl) of cell suspension containing 200 cells were then placed in culture wells with 2-fold serial dilutions of IL-11 (starting concentration: 100 ng/ml) or test culture supernatants in a total volume of 20 µl. Normal saline was included as a negative control. After 48 h incubation at 37°C in a humidified atmosphere containing 10% CO2 in air, viable cells were counted using an inverted phase contrast microscope. IL-11 concentrations in the supernatants were determined from the standard curve. The lower detection limit of the assay was typically 50–100 pg/ml. The bioassay specificity for IL-11 was confirmed by the addition of anti-IL-11 neutralizing antibody (15 µg/ml; AF-218-NA; R&D Systems) to wells containing Ba/F3 huIL-11R/gp130 cells and either conditioned media or rhuIL-11. As a control, goat IgG (R&D Systems) was added to the cells at an equivalent concentration.
RT–PCR for IL-11, IL-11R and PRL
Total RNA was isolated from cultured stromal cells using an RNeasyTM Minikit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Semi-quantitative RT–PCR was performed as described previously (Dimitriadis et al., 2000). Samples were treated with DNase I (Boehringer Mannheim) and oligo(dT)-primed first strand cDNA synthesis from 1 µg RNA was performed using avian myeloblastosis reverse transcriptase (Boehringer Mannheim). Aliquots were amplified by PCR with primers for glyceraldehyde phosphate dehydrogenase (GAPDH) using 20 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min. The reaction mixture contained 1xPCR buffer (Boehringer Mannheim), 60 µmol/l of each dNTP, 1.25 IU Taq polymerase (Boehringer Mannheim) preincubated with TaqStart antibody (Clontech, San Diego, CA, USA) at a ratio of 1:1 and 0.1 µmol/l of each specific primer. The product was electrophoresed on a 2% agarose gel, transferred to GeneScreen Plus (Dupont, Wilminton, DE, USA) and the filter hybridized with a 32P end-labelled internal oligonucleotide. The specific primer pairs were as follows: IL-11: 5'-GTGGCCAGATACAGCTGTCGC-3', 5'-GGTAGGACAGTAGGTCCGCTC- 3'; IL-11R: 5'-CAGGGCCTGCGGGTAGAGTCAGTA-3', 5'-CTCCTTTGGTATGGTCCCAGTGCT-3'; PRL: 5'-GAGCCTGATAGTCAGCATATTG-3', 5'-TGGATGTGGGCTTAGCAGTTGT-3'; GAPDH: 5'-ACCACAGTCCATGCCATCAC-3', 5'-TCCACCACCCTGTTGCTGTA-3'. Reaction conditions for IL-11, IL-11R and PRL were as follows. IL-11: 32 cycles at 94°C for 30 s, 62°C for 40 s and 72°C for 1 min; IL-11R: 32 cycles at 94°C for 30 s, 65°C for 40 s and 72°C for 1 min; and PRL: 32 cycles at 94°C for 30 s, 58°C for 40 s and 72°C for 1 min.
Immunohistochemistry for IL-11R and IL-11
Immunohistochemistry of IL-11R was performed on cultured endometrial stromal cells using a mouse anti-huIL-11R antibody (number 4D12) raised against recombinant soluble huIL-11R, produced in baculovirus (unpublished). This antibody detects huIL-11R by Western blot, immunoprecipitation and flow cytometry. A second section on each slide acted as a negative control where normal mouse IgG at the same protein concentration replaced the primary antibody.
Cell monolayers were disrupted by a brief incubation in trypsin–versene solution and cells were then cultured on poly-L-lysine-treated glass slides with their appropriate treatments for 24 h. Cells were fixed with 70% ethanol and allowed to air dry. The cells were rehydrated in distilled water and treated with hydrogen peroxide (3%) to block endogenous peroxide activity. The cells were then washed with Tris-buffered saline (TBS, pH 7.6) for 10 min prior to incubation with 10% normal horse serum/10% FCS in TBS for 30 min followed by incubation with anti-huIL-11R (20 µg/ml) diluted in 10% normal horse serum/10% FCS/TBS at 4°C for 16 h. The tissues were washed in TBS for 5 min then TBS-Tween 20 (0.1%) for 10 min followed by TBS for 5 min prior to incubation with biotinylated horse anti-mouse IgG (1:200; Vector Laboratories Inc., Burlingame, CA, USA) for 1 h at room temperature, followed by streptavidin–biotin–peroxidase complex ABC Elite (Vector) according to the manufacturer's instructions. Peroxidase activity was visualized following the application of diaminobenzidine tetrahydrochloride (DAB) substrate (Zymed Laboratories Inc, San Francisco, USA) for 4 min. Sections were counterstained with Harris haematoxylin, dehydrated and mounted. Microscopy was performed using an Olympus BH2 microscope and cells were photographed using an Olympus camera with ND and LBD2N filters.
IL-11 in endometrial tissue was immunolocalized using anti-huIL-11 (MAB618; R&D Systems Inc.) as previously described (Dimitriadis et al., 2000).
Statistical analysis
Data were expressed as mean ± SEM and analysed by ANOVA followed by Tukey's Post Hoc test. P < 0.05 was considered to be statistically significant.
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Results |
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IL-11 production by human endometrial stromal cells during decidualization
Endometrial stromal cells decidualize in response to progesterone when maintained in serum-free culture conditions with added SFM (Irwin et al., 1989
). By contrast, E2 does not induce decidualization in vitro although it primes endometrial stromal cells to the action of progesterone. The decidualization time-course was conducted in serum-free medium supplemented with SFM and E2 with and without the addition of progesterone. PRL production was used as a marker of decidualization and was significantly increased (P < 0.001) from day 6 of culture in the E2 + progesterone-treated cultures compared with the E2-treated controls (Figure 1), confirming published data. The number of viable cells (mean ± SEM) was not different between the groups at day 10 of culture (E2, 4.3 ± 0.7x105; E2 + progesterone, 4.1 ± 1x105).
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) or E2 + progesterone (
) and medium was changed every 48 h. Supernatant was collected and assayed by ELISA. Data are expressed as mean ± SEM of duplicate wells from three separate experiments. *P < 0.001 versus E2-treated cells. Note that in the case of the E2 treatment, SEM are too low to be visualized.The expression of IL-11, IL-11R
and PRL mRNA transcripts by endometrial stromal cells during decidualization was examined by RT–PCR (Figure 2). IL-11, IL-11R and PRL transcripts were all expressed by cultured endometrial stromal cells. The expression of GAPDH, used as a loading control, was likewise present in all the samples, albeit at variable levels.
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An ELISA assay was used to measure the secretion of IL-11 by the cells. Throughout the 10 days of culture, the levels of IR-IL-11 secreted were not significantly different between non-decidualized and decidualized cells (day 10 of culture: E2, 505 ± 93 pg/ml versus E2 + progesterone, 398 ± 110 pg/ml, n = 3, experiments in duplicate wells). A newly established bioassay was used to demonstrate that the IR-IL-11 produced by both the decidualized and non-decidualized endometrial stromal cells was biologically active in conditioned media from cells treated with E2 and E2 + progesterone (Figure 3
). The addition of anti-IL-11 neutralizing antibody to wells containing Ba/F3 huIL-11R/gp130 cells and either conditioned media or rhuIL-11 fully abolished cell survival (data not shown). Control goat IgG added to the cells had no effect.
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The similar level of secretion of IL-11 from non-decidualized and decidualized stromal cells was a surprising finding, since in intact, fixed endometrial tissue, we have previously shown intense IL-11 immunostaining in decidualized stromal cells and very minimal staining in non-decidualized stromal cells (Dimitriadis et al., 2000
). We therefore tested IL-11 secretion both by endometrial explants and isolated stromal cells with the aim of optimizing culture conditions to examine the function of IL-11 during in-vitro decidualization.
Tissue explants from day 14 endometrial tissue cultured in medium with or without 10% CT–FCS secreted substantial amounts of immunoreactive IR-IL-11 (1.52 ± 0.5 versus 1.55 ± 0.6 ng/ml respectively), whereas immunohistochemical analysis of the same tissue, fixed without culture, showed barely detectable IL-11 and only in the glandular epithelium (data not shown). In this case, the glandular epithelial cells are the more likely source of IR-IL-11; however, the high levels of IR-IL-11 in the culture medium suggest up-regulation of its production in culture. Likewise, freshly isolated endometrial stromal cells secreted IL-11 in culture. However, the levels of IL-11 secreted by the cells differed according to the culture conditions (Figure 4). In the presence of 10% CT–FCS, more IL-11 (P < 0.01) was secreted compared with cells grown in serum-free conditions or serum-free conditions with the addition of either TSL or SFM (Figure 4). The secretion of IL-11 by the cells grown in serum-free conditions with SFM was significantly higher (P < 0.01) than in medium with or without TSL (Figure 4). Thus, IL-11 secretion is likely to be regulated both by trauma (chopping and/or culture) and by culture conditions, particularly the presence of CT–FCS. Viable cell numbers between different treatment groups were not significantly different (data not shown).
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Effect of neutralizing endogenous IL-11 on the decidualization of endometrial stromal cells
To establish whether IL-11 is required for the decidualization of endometrial stromal cells, cells were cultured under conditions that minimized endogenous IL-11 production: TSL replaced SFM in the culture medium and cells were cultured for 4 days prior to the addition of the decidualizing stimulus (E2 + progesterone). A goat IgG affinity-purified neutralizing polyclonal anti-huIL-11 antibody (AB) or equivalent amount of non-immune goat IgG was added to the cells during culture.
As shown previously, endometrial stromal cells treated with E2 secreted very low or undetectable levels of PRL throughout the incubation period (Figure 5A). Cells treated with E2 + progesterone or E2 + progesterone + AB (10 µg/ml, final concentration) secreted significantly more PRL compared with the E2-treated controls from day 6 of the time-course (Figure 5A). Importantly, the addition of AB to the E2 + progesterone-treated cells resulted in significantly lower PRL secretion at days 8 and 10 of culture, although PRL levels remained above the very low secretion by the non-decidualized E2-treated controls. When the AB was washed out at day 10 of culture and replaced with IL-11 (10 ng/ml, final concentration), the secretion of PRL increased and was significantly higher at day 12 of culture compared with the E2 + progesterone-treated cells (Figure 5A).
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) or E2 + progesterone (
) with medium changes every 2 days. A neutralizing polyclonal anti-huIL-11 antibody (AB) was added at a final concentration of 10 µg/ml to an additional set of E2 + progesterone-treated cultures for 10 days (
). Antibody was washed out at day 10 of culture (AB washout) and IL-11 was added at a final concentration of 10 ng/ml. All other treatments were continued throughout the 12 day time course. (A) PRL secretion. Each value represents mean ± SEM of duplicate cultures from four separate experiments. *P < 0.01, E2 versus E2 + progesterone and E2 + progesterone + AB; **P < 0.01, E2 + progesterone versus E2 + progesterone + AB. (B) IGFBP-1 secretion. Each value represents mean ± SEM of duplicate cultures from three separate experiments. *P < 0.01, E2 versus E2 + progesterone and E2 + progesterone + AB. **P < 0.01, E2 + progesterone versus E2 + progesterone + AB.The secretion of IGFBP-1 (another marker of decidualization) by stromal cells was also measured throughout the culture period (Figure 5B
). IGFBP-1 secretion increased in the E2 + progesterone-treated cells from day 8 of culture compared with cells treated with E2 alone. The addition of AB in conjunction with E2 + progesterone resulted in significantly lower IGFBP-1 secretion at day 10 of culture compared with the E2 + progesterone-treated cells. Following the removal of AB at day 10 of culture and the addition of IL-11 to the E2 + progesterone + AB-treated cells, IGFBP-1 secretion increased to the levels of the cells treated with E2 + progesterone only. In those cell cultures in which the AB was not removed at day 10, the production of both PRL and IGFBP-1 remained low at day 12 (data not shown). A concentration of AB <5 µg/ml did not alter the secretion of PRL and IGFBP-1 throughout the decidualization culture period (data not shown). Goat IgG of irrelevant specificity (10 µg/ml) did not inhibit PRL or IGFBP-1 production at any time (data not shown). AB added to the cells alone or to E2-treated cells did not produce changes in PRL and IGFBP-1 secretion compared with E2-treated controls (data not shown). Cells treated with E2 + IL-11 or IL-11 alone secreted PRL at levels comparable with those of cells treated with E2 (data not shown).
The viable cell numbers (mean ± SEM) for the treatment groups E2, E2 + progesterone, E2 + progesterone + AB and E2 + progesterone + AB washout (WO) were not significantly different at day 12 of culture (E2, 5.4 ± 1.5x105; E2 + progesterone, 4.3 ± 0.8x105; E2 + progesterone + AB, 5.1 ± 0.4x105; E2 + progesterone + ABWO, 4.7 ± 0.3x105). Thus, changes in secretion levels of PRL or IGFBP-1 were not due to changes in viable cell number.
Effect of exogenous IL-11 on the decidualization of human endometrial stromal cells
To determine whether exogenous IL-11 could enhance the progesterone-induced decidualization of endometrial stromal cells, increasing concentrations of rhuIL-11 were added to the cells and effects on PRL and IGFBP-1 secretion and on cell morphology were determined. Figure 6A shows PRL secretion by cells treated with E2, E2 + progesterone or E2 + progesterone + IL-11 at final concentrations of 1, 10 and 100 ng/ml throughout the 12 days in culture. PRL secretion by the E2-treated cells remained low, but was significantly higher in the E2 + progesterone-treated cultures compared with E2 alone from day 8 of culture. PRL secretion following the addition of 1 ng/ml IL-11 to the E2 + progesterone-treated cells did not differ from that of E2 + progesterone alone at any corresponding time. However, the addition of 10 ng/ml of IL-11 to E2 + progesterone-treated cells increased the secretion of PRL from day 10 of culture, while IL-11 at 100 ng/ml significantly increased the secretion of PRL from day 4. Therefore, IL-11 treatment of the E2 + progesterone-treated cells resulted in higher PRL secretion at an earlier time in culture compared with cells treated with E2 + progesterone alone.
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The effect on IGFBP-1 secretion by the endometrial stromal cells following the addition of IL-11 to the cells followed a similar pattern to PRL secretion (Figure 6B
). IGFBP-1 secretion by the E2-treated cells remained low and did not change throughout the culture period. IGFBP-1 secretion was significantly higher in the E2 + progesterone-treated cultures compared with E2 alone from day 8 onwards, and this was not altered by the addition of 1 ng/ml IL-11. However, IL-11 (10 ng/ml) increased IGFBP-1 secretion at day 12 of culture, while IL-11 (100 ng/ml) significantly increased IGFBP-1 production from day 6 of culture compared with E2 + progesterone alone. Therefore, similar to its effect on PRL secretion, IL-11 both accelerates and increases the amount of IGFBP-1 secreted by stromal cells during progesterone-induced decidualization, suggesting an increase in the rate of decidualization. The effect of IL-11 on the viability of stromal cells during decidualization was examined. No significant differences in the number of viable cells (mean ± SEM) were found at the end of the 12 day culture between the cultures of cells treated with either E2, E2 + progesterone or E2 + progesterone + IL-11 (1, 10 and 100 ng/ml) [E2, 5.4 ± 1.5x105; E2 + progesterone, 4.3 ± 0.5x105; E2 + progesterone + IL-11 (1 ng/ml) 4.4 ± 1.7x105; E2 + progesterone + IL-11 (10 ng/ml), 4.3 ± 0.8x105; E2 + progesterone + IL-11 (100 ng/ml), 4.9 ± 0.4x105], suggesting that differences in the levels of PRL and IGFBP-1 secretion were not due to changes in cell number.
We immunolocalized IL-11R in the cultured endometrial stromal cells on day 12 of the culture period following different treatments (Figure 7). All cultures treated with E2 alone (Figure 7A), E2 + progesterone (Figure 7B) or E2 + progesterone + IL-11 (10 ng/ml; Figure 7C), showed IL-11R immunoreactivity in only a subset of cells. The morphological characteristics of the cells after different treatments were also assessed. Non-decidualized, E2-treated and confluent endometrial stromal cells remained as spindle-shaped fibroblast-like cells throughout the culture period (Figure 8A). However, cells treated with E2 + progesterone acquired a pavement-like morphology indicative of decidualization by day 10 of culture (Figure 8B), while cells treated with E2 + progesterone + IL-11 (10 ng/ml) also exhibited pavement-like morphology, but there was consistently some loss of distinct cell borders at day 10 of culture (Figure 8C).
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This study demonstrates that IL-11 mRNA and protein are produced by endometrial stromal cells in culture and that the protein is biologically active. Although artefactual secretion of IL-11 by cultured explants and cells is readily induced, the conditions under which the endometrial stromal cells are cultured can be manipulated so that only very low levels of IL-11 are produced, such as seen in vivo. By optimizing cell cultures for low IL-11 production and then exposing them to progesterone as a decidualizing stimulus, a role for IL-11 in advancing decidualization has been demonstrated. Specifically, addition of exogenous IL-11 enhanced the secretion of PRL and IGFBP-1: the amount of each hormone secreted increased and secretion was detectable earlier during the decidualization process than in the absence of exogenous IL-11. Furthermore, addition of anti-IL-11 to the cells reduced progesterone-mediated decidualization. PRL, IL-11 and IL-11R
mRNA transcripts were expressed during the decidualization process. Interestingly, IL-11R immunoreactivity was detected only in a subset of stromal cells and was independent of decidualization.
While immunoreactive IL-11 is detectable in a number of cell types, including decidualized stromal cells in late secretory endometrium, it is low or absent from the non-decidualized stromal cells during the proliferative and early-mid secretory phases (Dimitriadis et al., 2000; Cork et al., 2001). Therefore it was initially perplexing that biologically active IL-11 was secreted in similar quantities by both non-decidualized and decidualized stromal cells in culture. Subsequent examination established that IL-11 secretion was enhanced merely by chopping and culture, but that this induced secretion could be minimized by careful manipulation of culture conditions. It has previously been shown that simply removing murine tissues and incubating them in tissue culture medium rapidly induces secretion of many cytokines including IL-11 (Metcalf et al., 1995). Sheer stress also increases IL-11 secretion by other cell types (Sakai et al., 1999). Furthermore, human decidual explants produce substantially lower levels of growth factors and cytokines than decidual cells cultured under the same conditions (Lonsdale et al., 1996). In agreement with these findings, a study by Cork showed that high levels of IR-IL-11 were secreted by endometrial stromal cells that had been cultured in serum (Cork et al., 2001). Caution must therefore be applied in the interpretation of in-vitro cytokine secretion data and care must be taken to verify such data with in-vivo studies where possible. Our preliminary studies emphasized the need to optimize culture conditions to test the function of IL-11 during the in-vitro decidualization of human endometrial stromal cells. The addition of TSL to the medium assisted in maintaining low levels of IL-11 secretion throughout the decidualization culture and enabled manipulation of the IL-11 available to the cells.
The process of decidualization is a continuum, controlled by a highly synchronized activation of specific genes (Tabibzadeh and Babaknia, 1995) resulting in co-ordinated expression of specific new products which appear sequentially as the process proceeds (Bryant-Greenwood et al., 1993; Tang et al., 1994). The level of PRL secretion by stromal cells correlates with the degree of decidualization (Maslar and Riddick, 1979). In the present study, the secretion of two biochemical markers of decidualization, PRL and IGFBP-1, was measured and morphological alterations were assessed in the in-vitro model. Biologically active IL-11 was produced during progesterone-induced decidualization in our model system, as observed in vivo (Dimitriadis et al., 2000), and IL-11R mRNA was expressed throughout the decidualization culture. This reflects studies in the mouse where no difference is seen in IL-11R mRNA expression between non-pregnant and pregnant uterus (Robb et al., 1998), and in the human endometrium where IL-11R and gp130 mRNA expression did not change throughout the menstrual cycle (Dimitriadis et al., 2000).
Therefore, it is unlikely that IL-11R and gp130 mRNA levels are regulated by progesterone. Similarly, IL-11 secretion by human endometrial stromal cells does not appear to be regulated by progesterone. In other cell types, IL-11 secretion is regulated by factors such as transforming growth factor (TGF)ß1 (Taki et al., 1999) and PGE2 (Mino et al., 1998).
Importantly, IL-11 both increased and accelerated the secretion of PRL and IGFBP-1 by decidualizing cells, suggesting a role in the progression of decidualization. Furthermore, the production of PRL was altered earlier in the decidualization culture period compared with IGFBP-1, suggesting differences in the mechanisms of regulation of PRL and IGFBP-1 by IL-11 and possible changes in its function as decidualization progresses.
Neutralizing endogenous IL-11 production by AB reduced secretion of both PRL and IGFBP-1 while subsequent removal of the AB and addition of IL-11 rapidly restored secretion of both hormones to maximal levels. Interestingly, in the presence of AB the levels of secretion were not reduced to the very low levels seen in non-decidualized cultures. Explanations include that IL-11 is not absolutely required for decidualization or that the secreted IL-11 may not have been fully neutralized at the concentration of AB used. The rapid secretion of the decidual markers following reintroduction of IL-11 probably reflects partial decidualization in the presence of AB. In the IL-11R null mouse, decidualization of the uterine stromal cells did occur in the primary decidual, zone in the antimesometrial section of the uterus, although it was markedly reduced. The severe disruption of the process resulted in an absence of decidualization in the mesometrial zone resulting in the inability of the embryo to remain implanted (Robb et al., 1998). This suggests that, in the mouse, initiation of decidualization occurs but the process cannot continue in the absence of IL-11R signalling. In the human, there is no evidence for subtypes of decidual cells as in the mouse. However, decidualization occurs progressively in the endometrial stroma, initially in cells close to the spiral arterioles. Furthermore, both expression of IL-11 mRNA and the presence of IR-PRL in intact endometrium occur in subsets of stromal cells during the late secretory phase (Dimitriadis et al., 2000).
We have also demonstrated that the functional differentiation of human endometrial stromal cells was accompanied by morphological differentiation, and that this involved IL-11. The loss of distinct cell borders corresponds with the degree of decidualization (Frank et al., 1994) and addition of IL-11 to cells throughout the decidualization culture in the present study resulted in a loss of distinct cell borders which was not seen in the decidualizing cells treated only with E2 and progesterone. The increased and accelerated response in decidualization following treatment of the cells with IL-11 was not accompanied by a change in cell number, suggesting that the function of IL-11 during decidualization is unlikely to involve endometrial stromal cell proliferation.
In conclusion, this study has clearly demonstrated that IL-11 is involved in the functional differentiation that occurs during progesterone-mediated decidualization of human endometrial stromal cells. The results suggest that the role of IL-11 in decidualization possibly involves autocrine or paracrine mechanisms. The interactions of IL-11 with other cytokines produced by endometrial stromal cells such as IL-6 (Tabibzadeh and Babaknia, 1995; Zoumakis et al. 2000), TNF (Hunt et al., 1992) and the TGFßs (Chegini et al., 1994) and ligands coupled to the cAMP pathway, such as PGE2 (Frank et al., 1994) still need to be examined. The contribution of IL-11 to decidualization may prove to be critical in the establishment of pregnancy. In humans, failure of implantation and placental development are clinically important. Of particular relevance is the high rate of early spontaneous abortion (one-third of all pregnancies) (Wilcox et al., 1988), the failure of IVF embryos to implant, and the important consequences of impaired fetal health resulting in subsequent disorders in adult life. Strategies for overcoming unexplained infertility and early abortion and to improve well-being in threatened pregnancies during the first trimester are essential. The identification of IL-11 as an important player in the decidualization process provides a potential target for manipulation.
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We are grateful to Professor Gabor Kovacs and his patients for provision of endometrial tissue, and to Anne Clare and Heather Widjaja for collection of tissue. We thank Rachel Mansfield, Professor Glenn Begley and Professor Don Metcalf for assistance in establishing the Ba/F3/IL-11R
/gp130 bioassay, and Wendy Carter, Helene Martin and Kathy Davern together with members of the WEHI monoclonal laboratory for development of the mouse anti-human IL-11R antibodies. We are grateful to Professor Tom Kennedy for critical reading of the manuscript, Sue Panckridge for assistance with the illustrations and Samantha Park in preparing the manuscript. Support for this Subproject (CIG-99-27) was provided by the CICCR Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School. The views expressed by the authors do not necessarily reflect the views of CONRAD or CICCR. E.D. is supported by CONRAD. L.A.S. and L.R. were supported by the National Health and Medical Research Council of Australia and L.R. by the Sylvia and Charles Viertal Charitable Foundation, Australia. |
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3 To whom correspondence should be addressed. E-mail: evdokia.dimitriadis{at}med.monash.edu.au
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Submitted on August 20, 2001; resubmitted on December 10, 2001; accepted on March 17, 2002.
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