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BOR - Papers in Press, published online ahead of print March 12, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.065219
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BIOLOGY OF REPRODUCTION 79, 142–153 (2008)
DOI: 10.1095/biolreprod.107.065219
© 2008 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract; 

Interleukin 1 Signaling Is Regulated by Leukemia Inhibitory Factor (LIF) and Is Aberrant in Lif–/– Mouse Uterus1

A.A Fouladi-Nashta 3 4 5, L Mohamet 4 5, J.K Heath 6, and S.J Kimber 2 5

Faculty of Life Sciences,5 The University of Manchester, Core Technology Facility, Manchester M13 9MNT, United Kingdom School of Biosciences,6 University of Birmingham, Birmingham B15 2TT, United Kingdom

ABSTRACT

This study addresses the regulation of the interleukin 1 (IL1) system in the murine uterine luminal epithelium (LE) and stroma by leukemia inhibitory factor (LIF). Using RT-PCR we compared expression of Il1a, Il1b, Il1rn, Il1r1, and Il1r2 during the pre- and peri-implantation periods of pregnancy in wild-type (WT) and LIF-null LE and stroma. In WT LE, Il1a transcripts were down-regulated on Day 4 of pregnancy (D4), with renewed expression by the evening of D4 (D4 pm). In Lif –/– LE there was a gradual decrease in expression on D2, and expression became undetectable by D6. Il1b and Il1r1 expression were similar in WT and null mice, but Il1rn expression was almost completely lost during the peri-implantation period in Lif –/– LE. In the stroma, Il1a was sharply down-regulated on D4 and reappeared on D4 pm but was only expressed from D3 to D5 in the null mice. Stromal Il1r1 and Il1r2 were also misregulated. Il1rn showed constitutive expression in null stroma in contrast to the loss of expression on D4 in the WT mouse. In Lif-deficient mice, immunostaining indicated a reduction of endometrial IL1A at the time of implantation and of IL1B in stroma. LE-stromal coculture revealed that LIF stimulated the apical secretion of both IL1A and PTGES2 by LE cells without affecting basal secretion of IL1A and with only a small effect on basal PTGES2 secretion. We conclude that Il1a and Il1rn in LE and Il1a, Il1rn, and Il1r1 in stroma are regulated by LIF, which stimulates apical secretion of IL1A by LE.

implantation, interleukin 1, leukemia inhibitory factor, prostaglandin, uterus

INTRODUCTION

Embryo implantation involves a complex and dynamic interaction between the trophoblast, the uterine epithelium, and the stroma that must occur within a specific temporal window during which the uterine endometrium is receptive to the embryo. Although it is well established that this "window of implantation" is primarily controlled by the steroid hormones estrogen and progesterone (P4) [1, 2], recent evidence has shown that a plethora of other molecules, including growth factors and cytokines, mediate and modulate the actions of these steroid hormones [3, 4]. Uterine leukemia inhibitory factor (LIF) is expressed in two transient peaks during early pregnancy. First, on Day 1 of pregnancy (D1; vaginal plug is observed), LIF expression is stimulated by ovulatory estrogen in both luminal and glandular epithelium (LE and GE, respectively). Second, on D4, nidatory estrogen stimulates expression of both Lif mRNA and protein in the GE [5–7]. This second peak of LIF expression is essential for successful embryo implantation into the uterus in the evening on D4 (D4 pm) [8]. The cellular target of LIF in the uterus during pregnancy appears to be the LE, and Lif receptor (Lifr) transcripts and protein have been found to be present predominantly in the LE from D3 to D5 [9, 10]. It has been known for some time that uteri of Lif-deficient mice are unable to support embryo implantation [6]. However, Lif –/– blastocysts can undergo implantation when transferred into pseudopregnant recipients and develop to term, demonstrating that the implantation defect is maternal. Rescue of implantation can be achieved by exogenous delivery of LIF on D4 in the homozygous mutants [6, 8, 11]. The importance of LIF for successful embryo implantation in the mouse may be of general significance to all mammals and other species. Indeed, increased levels of LIF during pregnancy have been shown to be conserved in several species including humans and rhesus monkeys [12–15], whereas low levels of Lif have been correlated with infertility in women [16–19].

Furthermore, the uteri of Lif-deficient mice do not undergo decidualization, a process that involves the differentiation of the uterine stroma and that is essential to support the implanting embryo [6–8]. Decidualization is triggered by a number of molecules and is first discerned by an increase in vascular permeability at the site of implantation [1, 20]. Among the best candidates for roles in the initiation of decidualization are prostaglandins (PGs), which increase at the time of implantation. PTGES2 is a central PG involved in the initiation of uterine vascular permeability [21–23]. PGs are produced by both uterine epithelial and stromal cells, and their synthesis is induced by interleukin 1 (IL1), also produced by the uterine epithelium, as well as by other cell types, including macrophages [24]. The IL1 system is composed of two agonists, IL1A and IL1B; one antagonist, IL1RN; and two membrane-bound receptors, IL1 receptor type one (IL1R1) and type two (IL1R2) [25, 26]. Endogenous control of secreted IL1 activity is achieved by regulation of IL1 synthesis and processing and by release from intracellular and membrane-bound stores [26]. This control of IL1 bioavailability is further regulated by a unique receptor antagonist (IL1RN), which binds with high affinity to IL1 receptors, thus preventing access by IL1 ligands and inhibiting signaling [27]. In mouse, IL1R1 protein is reported to be induced in uterine LE cells during the preimplantation period and subsequent blockade of IL1 signaling by injection of IL1RN during early pregnancy, thereby preventing attachment of the blastocyst to the LE [28, 29].

Epithelial derived IL1A has been previously reported to up-regulate the synthesis of PTGES2 and PGF2{alpha} in mouse and rat uterine stromal cells [30, 31], and other studies in vitro have shown that IL1A increases levels of mRNA for Ptgs2 (a rat-limiting enzyme for PG synthesis) in rat uterine stromal cells [32]. Evidence from in vivo studies has demonstrated that mRNA and protein levels of PTGS2 are reduced in the uterine stroma of Lif-deficient mice at the implantation site [7, 33]. We have shown, however, that LIF does not directly promote the synthesis of PTGES2 by uterine stromal cells in vitro, suggesting that PTGES2 is not a direct target of LIF here [34]. In human endometrial epithelial cells, IL1B up-regulates LIFR, and this effect is abrogated by inhibition of IL1R1 [35]. This suggests that in human and murine endometrium it is likely that feedback loops exist between LIF and IL1 in uterine epithelial cells. Together with the reduction of PTGS2 expression at the implantation site in Lif –/– females, these findings support a signaling cascade involving LIF induction of IL1 in the LE that triggers the onset of the decidual response via PGs. Therefore, using a coculture system, we have investigated the effects of LIF on IL1A production and gene expression in cultured mouse uterine LE and stromal cells in a physiologically relevant model. We have also shown that IL1 and its associated molecules are precisely regulated in LE and stroma during early pregnancy in vivo. Moreover, the temporal sequence of changes in Il1-related gene expression (specifically Il1a and Il1rn) during uterine LE development for implantation is seriously altered in Lif –/– mice, indicating that a close relationship exists between LIF and IL1A in the regulation of endometrial cells as demonstrated in vitro.

MATERIALS AND METHODS

Animals

All mice were maintained under conditions in accordance with the U.K. Home Office as in Fouladi-Nashta et al. [7], and procedures were in accordance with our U.K. Home Office license. MF1 (wild-type [WT] outbred) female mice (Harlan Olac Ltd., Bicester, U.K.) between 7 and 9 wk of age were placed with MF1 males overnight for mating, and pregnancy was confirmed by the presence of a vaginal plug (D1). MF1 female mice used for in vitro culture were induced to ovulate by an i.p. injection of a single dose of 5 IU eCG (Intervet, Milton Keynes, U.K.), followed by a single injection of 5 IU hCG (Intervet) 48 h later. Mating was confirmed by the observation of a vaginal plug the following morning. Mice were killed by cervical dislocation on D2 (48 h following hCG), and uterine tissues were processed as below. The Lif –/– MF1 founder mice were provided by Dr. Andrew Sharkey (University of Cambridge, Cambridge, U.K.) from an original colony generated at the Institute for Stem Cell Research (University of Edinburgh, Edinburgh, U.K.) [36]. Since Lif –/– females are infertile, propagation of Lif –/– mice was achieved by breeding from null males and heterozygote females as previously described [7, 37]. Genotyping for identification of Lif –/– mice was carried out by PCR on DNA samples from progeny following weaning as previously reported by us [7, 37]. Animals were killed by cervical dislocation on the required day of pregnancy, and uterine tissue was processed as detailed below. Uteri were harvested in the morning between 0900 and 1000 h on all days of pregnancy and also in the evening on D4 (D4 pm) between 2100 and 2200 h.

Reagents

All reagents were purchased from Sigma (Dorset, U.K.) unless otherwise indicated. Primary antibodies were used as follows: Goat anti-mouse IL1A (2 µg/ml; R&D systems, Oxfordshire, U.K.); rabbit anti-mouse IL1B (1 µg/ml; Santa Cruz Biotechnology, Heidelberg, Germany); monoclonal 11–5F against desmoplakin (1:10; courtesy of Prof. D. Garrod, University of Manchester, Manchester, U.K.); rabbit anti-mouse TJP1 (1 µg/ml; Zymed, Cambridge, U.K.); rat anti-mouse f4/80 (Serotec, Oxford, U.K.); and fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat, -rat, or -rabbit IgG secondary antibodies (4 µg/ml; Jackson Immunoresearch Laboratories, PA) or alternatively an Alexa 488-conjugated donkey anti-goat IgG (10 µg/ml; Molecular Probes, Invitrogen, Paisley, U.K.) or a biotinylated goat anti-rabbit IgG (7.5 µg/ml; Vector Laboratories, Peterborough, U.K.). Texas red-X phalloidin was used at 1:50 (Molecular Probes). Normal goat serum (NGS) was used at a 1:20 dilution to minimize nonspecific binding. Mouse IL1A used as the standard in ELISA was purchased from Chemicon (Hampshire, U.K.). For use in culture, LIF was obtained courtesy of Dr. A. Vernallis (Aston University, Birmingham, U.K.), and its activity was calibrated by the proliferation response of BAF cells (gift from Dr. A. Vernallis). The LIF inhibitor (hLIF-05), an LIFR antagonist, was used at 10 times the concentration of supplemented LIF [34, 38, 39].

Uterine Epithelial Cell Layer Dissociation for RNA Extraction

Uterine horns were dissected from WT or Lif-null females on D2–D6, and the LE cell "tube" was dissociated from the stroma and gently squeezed out according to [40]. The uterine horns were then slit longitudinally, and stromal cells were scraped from LE-depleted horns using a cell scraper (BD Biosciences, Oxfordshire, U.K.). The samples were centrifuged at 3000 x g for 3 min. Total RNA was isolated from the cells using the RNeasy Kit (Qiagen, West Sussex, U.K.) according to the manufacturer's instructions. Briefly, the tissue was lysed by drawing 10 times through a 21-gauge needle (BD Biosciences) in either 350 µl (epithelial extracts) or 600 µl (stromal extracts) of guanidine isothiocyanate and 0.1% (v/v) β mercaptoethanol. To ensure complete homogenization of the tissue, the samples were added to a Qiashredder column (Qiagen) following the manufacturer's instructions. RNA preparations were quantified by absorbance at 260 nm (A260) using a Nanodrop spectrophotometer (Labtech Intl., E. Sussex, U.K.) or Genequant (Amersham Bioscience, Amersham, U.K.) spectrophotometer. Purity was calculated from the A260/A280 ratio.

Isolation of Total RNA from Cultured Uterine Epithelial and Stromal Cells

The stromal cells were detached from the wells using a cell scraper (Corning), and the cell suspensions were centrifuged at 1000 x g for 5 min. The supernatant culture medium was removed, and the pellet was stored in liquid nitrogen. The LE cells attached to the membranes were transferred directly to the lysis buffer. RNA was isolated from all samples using RNeasy mini kit (Qiagen) as above.

Reverse Transcription-Polymerase Chain Reaction

Relative changes in Il1a, Il1b, Il1rn, Il1r1, and Il1r2 mRNA were examined in uterine LE and stromal isolates from D2 to D6 in WT and Lif-null females using RT-PCR. Samples from a minimum of three independent animals were used in each case. Changes in PCR products obtained for Il1 were normalized by comparison with an endogenous housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), expression of which has been shown to be consistent in the uterus [40]. Briefly, 2 µg total RNA from each sample was reverse transcribed using Superscript II first strand cDNA synthesis (Invitrogen, Paisley, U.K.) following the manufacturer's instructions, with omission of reverse transcriptase run in parallel in all reactions. PCRs were assembled to a final volume of 25 µl containing 0.5 µl of cDNA template, 10 pmol (final concentration) primers, and Red Taq PCR Buffer reaction mix (Sigma). No template and a reverse transcriptase negative control were assembled in parallel. Optimal annealing temperatures and cycle number are shown in Table 1. Cycle conditions were as follows: initial denaturation at 94°C for 1 min, then a cycle of 30 sec at 94°C, an annealing cycle for 30 sec at a temperature determined as optimum, and extended at 72°C for 30 sec. PCR products were resolved on a 2% (w/v) agarose gel, and the results were visualized under UV transillumination (GRI, Essex, U.K.). PCRs were also taken to saturation (40 cycles) to determine if transcripts were weakly expressed or absent. The PCR products were verified by automated capillary gel electrophoresis by Manchester Sequencing Services using an ABI Prism 377 sequencer (Applied Biosystems, Cheshire, U.K.), and products were confirmed by a BLAST search.


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  		TABLE 1 Primer sequences and characteristics.

Immunolocalization of IL1A/IL1B

Uterine horns were fixed in either 4% paraformaldehyde for 4 h at room temperature or in Carnoys fixative for 30 min at room temperature and dehydrated through an ethanol series before being embedded in paraffin wax and sectioned. Deparaffinized sections were either processed for antigen retrieval by microwave treatment (750 W) with TEG buffer (1.2 g/L Tris and 0.190 g/L EGTA, in distilled water; pH 9; IL1A) as previously described [7] or, following exposure to 0.3% (v/v) hydrogen peroxide in methanol for 12 min, subjected to antigen retrieval with 0.01 M citrate buffer (pH 6.0) for 6 min (IL1B). After cooling, nonspecific binding was blocked in 10% (v/v) NGS and 0.1% (w/v) BSA in PBS (blocking solution).

For immunoperoxidase staining (IL1B), endogenous biotin was blocked using an avidin-biotin blocking kit as per manufacturer's instructions (Vector Laboratories). The primary rabbit anti-IL1B or irrelevant control antibodies were diluted 1:50 in blocking solution and incubated overnight at 4°C. Following washing, the sections were incubated in the appropriate biotinylated secondary antibody for 45 min at room temperature. ABC reagent (Vector Laboratories) was applied to the sections for 30 min, and positive immunoreactivity was detected using a diaminobenzidine peroxidase substrate kit (Vector Laboratories). Nuclei were counterstained with Harris hematoxylin, and sections were mounted in a permanent mountant (CellPath, Newtown, Powys). To determine macrophage and IL1A immunoreactivity, uterine tissue from D4 was placed into aluminum foil containers of cryo-embedding compound OCT (Raymond A Lamb Laboratories, Sussex, U.K.). The samples were then flash-frozen in liquid nitrogen and stored at –80°C. Serial sections (7 µm) were taken using a cryostat (Leica UK Ltd., Milton Keynes, U.K.) and fixed for 10 min in ice-cold acetone at –20°C. The sections were rehydrated in 0.1% (w/v) BSA and 0.1% (v/v) Tween 20 in PBS. A 1:20 dilution of NGS was used to block nonspecific binding. The diluted primary antibody (1:50 for both IL1A and f4/80) was added to each section and left overnight at 4°C. Following washing, the sections were incubated with the appropriate FITC-conjugated secondary antibody for 45 min at room temperature. The sections were mounted in Vectashield with 1.5 µg/ml 4',6'-diamidino-2-phenylindole (Vector Laboratories) and stored in the dark at 4°C. For all experiments, relevant isotypes were used as negative controls and carried out in parallel. A secondary antibody-only control was also used to check for nonspecific secondary antibody binding.

Isolation and Culture of Uterine Luminal Epithelial and Stromal Cells

Briefly, fat-trimmed uteri were cut longitudinally to expose the lumen. They were placed in trypsin dissociation solution (0.5% Type II bovine trypsin and 0.165% pancreatin in Hanks Balanced Salt Solution [HBSS; Invitrogen]) for 1 h at 4°C followed by 1 h at room temperature. The medium was removed from the uteri, discarded, and replaced with ice-cold DNase medium (1 µg/ml DNAse [Type II from bovine pancreas], 10 mM MgCl2, and 0.1% heat-inactivated fetal calf serum [HIFCS; Invitrogen] in HBSS) before vortexing for 10 sec at medium speed. The supernatant cell suspension was transferred to a 50-ml Falcon tube on ice. The whole process was repeated, and the supernatants were pooled for isolation of LE cells. The remaining uteri were washed with HBSS and used for isolation and culture of stromal cells as described below.

Isolation and Culture of Uterine LE Cells

Preparation and culture of epithelial cells was as developed by Blissett and Kimber [41] is modified from [42]. The epithelial cell suspension was centrifuged at 200 x g for 5 min at 4°C. The supernatant was removed, and the cell pellet was resuspended in 10 ml ice-cold DNAse medium for 1 min before recentrifugation. This procedure was repeated three times. DNAse medium was replaced with HBSS, and the Falcon tube was placed at a 45° angle (15 min on ice) to allow LE cell plaques to separate under gravity. The supernatant was removed, and the epithelial cells were resuspended in 10 ml ice cold HBSS. The process was repeated for a total of four gravitational separations before adjusting cell density to 8.0 x 105 cells per milliliter in LE culture medium (1:1 Ham F12:Dulbecco modified Eagle medium [DMEM; Gibco BRL Life Technologies Ltd., Paisley, U.K.] containing 0.1% BSA [Fraction V Albumin, ICN], 100 mg/ml penicillin streptomycin [Invitrogen], 2.5% NuSerum [Collaborative Research Inc., Bedford, U.K.], 2.5% HIFCS, 15 mM Hepes buffer, and 200 mM L-glutamine). LE cells were cultured on Cellagen membranes (ICN-Flow, Thame, U.K.) as previously described [43, 44]. Cellagen discs were preincubated with culture medium. After preincubation, media in the apical and basal compartments were replaced with 250 µl cell suspension and 450 µl LE culture medium, respectively (Fig. 1). Cells grown on these membranes are cuboidal and show a semipolarized phenotype, intermediate between the highly polarized LE morphology seen in vivo from D1 to D3 and the flattened morphology seen for cells grown on plastic. The transepithelial resistance (TER) of the cultures was measured using a Millicell-ERS TER meter (Millipore, Watford, U.K.). All cultures used in these experiments had a TER above 400 cm2.


Figure 01
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  		FIG. 1 Schematic presentation of coculture system. Mouse uterine LE cells were cultured on the suspended Cellagen membrane, and stroma cells were cultured on the base of the wells in a 24-well culture dish.

Isolation and Culture of Uterine Stromal Cells

Uterine stromal cells were isolated and cultured as previously described [34]. Upon removal of LE from the uterine tissue (see above), 10 glass beads were added to the remaining LE-denuded endometrium extract, together with stromal trypsin dissociation solution (0.05% trypsin and 0.02% EDTA [BDH] in HBSS). The tubes containing cell extracts were incubated for 20 min at 37°C and vortexed at medium speed for 10 sec every 10 min. This process was repeated by incubation at room temperature. The content of the tube was passed through a 70-µm gauze filter (Falcon), and the enzymatic digestion was stopped (2% Soybean trypsin inhibitor in HBSS) after filtration. The cell suspension was then centrifuged at 400 x g for 10 min at 4°C. The pellet was washed in stromal cell culture medium (1:1 mixture of DMEM and Ham F12 medium [Invitrogen] supplemented with 1.2 g/L of sodium bicarbonate, 100 IU/ml penicillin streptomycin (Invitrogen), and 2% HIFCS) and centrifuged for 10 min at 4°C. The pellet was resuspended in culture medium, and live cells were assessed by trypan blue exclusion using a Neubauer hemocytometer. We have already shown that cells stained with epithelium-specific antibody marker (H001) were less than 2% of cells [34], and leukocytes were <1% by 48 h under these conditions.

Isolated stromal cells were cultured in 24-well dishes (Nunc) at 1.5 x 105 cells per milliliter in 5% CO2 in air at 37°C. Evaluation was undertaken on a minimum of three cultures in each case. For coculture, uterine stromal cells were cultured in the basal compartment, and LE cells were introduced on to the inserts at the time of stromal seeding. Media in both compartments were changed at 48 h and 96 h. Culture media from both compartments were collected and stored at –80°C for IL1A and PTGES analysis in triplicate. All experiments were repeated on a minimum of three separate occasions.

ELISA for IL1A

IL1A secretion into the culture media by uterine stromal and LE cells was measured using a mouse IL1A ELISA module set (BMS611MST; Medsystems Diagnostic GmbH, Vienna, Austria) according to the manufacturer's instructions. Briefly, Microwell plates (Maxisorb) were coated with rabbit anti-mouse IL1A (3 µg/ml) overnight at 4°C. Nonspecific binding was blocked with 250 µl of assay buffer (5 mg/ml BSA, 0.05% Tween 20 in PBS) for 2 h at room temperature. Serial dilutions of mIL1 standard protein in PBS were added in duplicate to the standard wells (for construction of a standard curve). Wells were then incubated with biotin-conjugate (1 in 10 000) for 2 h at room temperature. They were washed three times in wash buffer (0.05% Tween 20 in PBS), Streptavidin-horseradish peroxidase was added, and they were incubated for 1 h at room temperature. After washing, TMP substrate solution (1:2 mixture of H2O2 and tetramethylbenzidine) was added and shaken for 20 min in the dark. The enzyme reaction was stopped by 100 µ1 4N sulphuric acid, and the color intensity was read on a microplate reader at 450 nm to calculate IL1A concentrations.

PGE Radioimmunoassay

The concentration of PTGES2 was measured in the culture media as in [34] using Sigma radioimmunoassay (RIA) and standards (0–100 pg/ml) prepared in RIA buffer (0.01 M PBS [pH 7.4] containing 0.1% BSA and 0.1% sodium azide). One hundred microliters of sample or standards and 500 µl of antibody working solution were added to 1.5-ml Eppendorf tubes, vortexed, and incubated for 3 min at 4°C, and then 3H PGE (Amersham), diluted in RIA buffer to give 6000 cpm in 700 µl, was added. The tubes were vortexed and incubated for 1 h at 4°C, and 200 µl cold dextran-coated charcoal suspension (0.1% dextran, 1% activated charcoal [100–400 mesh] in RIA buffer) was added. After shaking, the tubes were centrifuged at 800 x g for 15 min at 4°C, and the supernatants were transferred into scintillation vials with 4 ml of scintillation cocktail (Optiphase Hisafe 2, Wallac). Radioactivity was measured with a β counter (Wallac-M1214), and the sample concentration was extrapolated from the standard curve. The values were considered reliable only in the logit interval of ±2.2 when the unlabeled molecules displace between 10% and 90% of maximum radioactivity bound [45].

Immunofluorescence Staining of Junctional Proteins in Cultured LE Cells

Cellagen discs were removed from culture wells, and the membranes (carrying LE cells) were detached from the supports and cut in two pieces. One half of each membrane was used for isolation of total RNA, and the other half was fixed and deposited on a coverslip for immunofluorescence staining of junctional proteins including Z0–1, desmoplakin, as in [7]. Primary antibodies and controls were as above. The coverslips were incubated for 2 h at room temperature with an appropriate affinity-purified FITC-conjugated secondary antibody (green) containing 10 µg/ml phalloidin (red), washed, and incubated for 5 min in 5 µg/ml bizbenzimide (Hoescht 33342, blue staining) before mounting in hydrophilic mounting media containing anti-fading reagent Gelvatol (0.1% gelvatol in 0.1M Tris buffer 33% glycerol).

RT-PCR for the Il1a in Cultured Cells

A one-step RT-PCR kit (Qiagen) was used according to the manufacturer's instructions for room temperature and amplification of a 220-bp product. One microgram of RNA was used for reverse transcription and PCR over 30 cycles with an annealing temperature of 60°C and 5 min extension. For experiments where Il1a mRNA transcripts were compared between different groups, the tubes were removed from the cycler (Eppendorf) every two cycles after the 18th cycle (amplification cycles in the linear range). Extension was then continued in another machine. The cycle number at which Actb was first detected was used to normalize for cDNA quantities.

Statistical Analysis

Data are presented as mean ± SEM. Statistical analysis was performed with the SPSS 13.0 program to carry out a two-way ANOVA using the General Linear Models procedure. Effects in the linear model consisted of batch effects and the effects of time and LIF treatments. A post hoc test was then used to analyze the difference between control and treatments. A Tukey test was also used to reveal the differences between each treatment.

RESULTS

Il1 Family Members Are Regulated at the Transcript Level in Peri-Implantation Uterus

Characterization of Il1a, Il1b, Il1rn, Il1r1, and Il1r2 mRNA expression on D2–D6 in WT and Lif-null females was performed by RT-PCR (Fig. 2) on RNA extracted separately from uterine stromal and LE isolates. Transcript patterns shown are representative of three separate animals at each stage and genotype.


Figure 02
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  		FIG. 2 Gene expression analysis of the Il1-associated proteins in the peri- implantation uterus of WT and Lif-null mice. Messenger RNAs were prepared separately from LE and stroma of WT and Lif-null uteri on the required days of pregnancy. RT-PCRs were performed for (A) Il1a, (B) Il1b, (C) Il1rn, (D) Il1r1, and (E) I1r2. F) Gapdh. Negative controls consisting of no template (water only) and no reverse transcriptase (-RT) were assembled in parallel in each experiment (final two columns).

Both ligands, Il1a and Il1b, showed temporal regulation in the uteri of WT mice during early pregnancy (Fig. 2, A and B). Specifically, transcript bands were observed on D2 in both LE and stromal isolates, and the intensity of the bands appeared to then decrease such that on the morning of D4 (0900 h) no transcripts could be detected for Il1a (even when PCRs were taken to saturation), although a very faint band was seen for Il1b in LE and stroma. However, by D4 pm (2200 h), which follows elevated levels of estrogen and LIF, mRNAs for Il1a and Il1b in both LE and stromal isolates were again detected as seen on D2. Although Il1a mRNA was continually expressed up until D6 in both the stroma and LE, Il1b mRNA was undetectable on D6 in both the LE and stroma, suggesting only transient re-expression on D4 pm and D5. Moreover, the pattern of disappearance of Il1a on the morning of D4 in WT uteri was not paralleled in the uteri of Lif-deficient mice on D2–D6. Il1a mRNA levels appeared to decline progressively from D2 onwards in the LE, whereas stromal expression of Il1a transcripts was only detected on D3 and D5 in Lif-null mice. Interestingly, in null females, the pattern of Il1b expression in the LE was parallel to that seen in WT mice, but stromal expression of Il1b was markedly different. Obvious stromal Il1b mRNA signal was detected on D2, D3, D4, and D6 but was undetectable on D4 pm and D5, when it was readily detectable in WT stroma.

Transcriptional expression of Il1r1 was similar to that seen for Il1b in WT mice where a reduction in detectable transcripts was identified on D4 in the stroma and LE (Fig. 2D). On D5 stromal transcript levels declined, and on D6 no transcripts could be detected in the LE and little in the stroma. Il1r2 transcripts were consistently detected in the LE from D2 onwards (Fig. 2E). Strong signal for Il1r2 mRNA was seen on D4 pm and D5, with lowest levels being on D4. By D5 no stromal expression of Il1r2 mRNA could be detected. Patterns of gene expression seen in Lif-null uteri for Il1r1 and Il1r2 in the LE were similar to those in WT uteri. However, stromal expression of Il1r1 mRNA appeared to be delayed relative to WT, with strong signal on D3, D4, and D6, but barely detectable signals on D2, D4 pm, and D5. In the null uterus, Il1r2 transcripts were only detected in the stroma on D3 and D5.

Il1rn transcripts were consistently expressed throughout D2–D6 in both LE and stromal isolates from WT mice, with only a transient but marked reduction on D4 in the stromal isolate (Fig. 2C). In LE of Lif nulls, Il1rn mRNA could be reliably detected only on D2 and D6. In the stroma, however, Il1rn transcripts were consistently expressed on D2–D6, with no loss of expression on D4 as in the WT stroma.

IL1A Protein Expression Is Reduced in the Lif-Null Uterus at Implantation

Transcript analysis revealed that Il1a was regulated differently during early pregnancy in the uteri of WT and Lif-null animals. To investigate whether similar changes occurred in protein expression, immunohistochemistry was performed on uterine sections from both WT and Lif-null mice (three females for each genotype) on D3–D6 using an antibody to IL1A (Fig. 3). Immunoreactive IL1A was not restricted to the site of embryo attachment/invasion in either WT or lif-null uterus, so sections were stained at and adjacent to the implantation site in WT mice and the presumptive implantation sites in Lif-null uteri. In WT mice, the protein profile was similar to that seen for mRNA. On D3, IL1A protein was identified in LE cells, and staining of a higher intensity was observed in the stroma. The IL1A-positive cells in the stroma (particularly on D3) were interspersed with nonstained cells, appeared to be larger in size than adjacent stromal cells, and may be macrophages. Attempts at double staining for IL1A and macrophage markers were hampered by the different antigen-antibody requirements. However, staining on sequential frozen uterine sections suggested both macrophages and IL1A protein are in the same areas, with distinct expression for IL1A compared to that of macrophage distribution (Fig. 3). By D4 only very weak staining was observed in the stroma, but by D4 pm, IL1A was detected in the LE and decidualizing stromal cells. Intense punctate staining could also be seen in the uterus on D5, particularly in the decidualized stroma and the embryo itself. On D6, IL1A was still detectable in the primary decidual zone around the embryo and in the outer decidual cells at the mesometrial pole of the uterus. In contrast, overall levels of immunoreactive IL1A appeared greatly reduced in the uteri of Lif-null mice compared to WT mice from D4 onwards. Thus, on both D3 and D4, IL1A protein was present in the LE, stroma, and glands, but by D4 pm IL1A staining was barely detectable, with only small sporadic patches of IL1A-positive stromal cells visible on D5. By D6 no IL1A was apparent in either the LE or stroma.


Figure 03
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  		FIG. 3 Immunolocalization of IL1A during early pregnancy. Immunofluorescence detection of IL1A in paraffin-embedded uterine sections on D3–D6 in WT and Lif-null females. Note that the overall expression of IL1A is reduced in the Lif-null uterus compared to WT. Strong signals were also apparent in the embryo on D5 in WT mice. A, B) Frozen sections of D4 mice uterus stained with f4/80 antibody (Pan Macrophage Marker). Both macrophages and IL1A protein are in the same areas, with distinct expression for IL1A to that of macrophage distribution. LE, Luminal epithelium; S, stroma; E, embryo; G, glands. Bars = 100 µm.

IL1B Protein Is Only Transiently Expressed on the Evening of D4 in Lif-Null Uteri

The cellular expression of IL1B was also investigated by immunohistochemistry in WT and Lif-null mice on D4 and D5 (Fig. 4). These days were chosen based upon the RT-PCR analysis, showing that changes in expression of Il1b transcripts were greatest around the time of implantation. On D4, faint IL1B immunoreactivity was observed in the cells of the LE and GE in WT mice. By D4 pm, intense staining of IL1B was observed in the LE, GE, and stromal cells, and a similar pattern of expression was detected on D5, but the staining was of a lower intensity. In contrast, in Lif-null mice, immunoreactive IL1B was predominantly observed in the cells of the LE and GE on D4 pm, although some faint staining was also evident in the subluminal stroma. Immunoreactive IL1B was not detected on D4 or D5 in Lif-null uteri.


Figure 04
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  		FIG. 4 Immunolocalization of IL1B in the peri-implantation uterus from WT and Lif-null mice. Detection of IL1B was performed on paraffin-embedded uterine sections from WT and Lif-null mice on D4 and D5 by immunohistochemistry. Cytoplasmic staining of IL1B (brown) can be seen in the LE, stroma, and uterine glands of the WT mouse. In contrast, IL1B immunoreactivity was only evident in the uteri of Lif-null mice on D4 pm (2000–2200 h). All nuclei were counterstained with hematoxylin (blue). LE, Luminal epithelium; S, stroma; G, glands. Bars = 100 µm.

Establishment of Coculture System

Since our data suggested that the changing expression of IL1 and associated molecules is disrupted in the Lif-null uterus, we investigated the effect of LIF on stromal and LE cells in vitro. For this purpose we used our coculture system, in which LE cells are grown on suspended membranes. LE cells proliferated and formed a pavement-like epithelium on Cellagen membranes. They became confluent after 4 days of culture, at which time the TER plateaued at or above 400 {Omega} cm2, indicative of a tight junctional network. The LE cells were immunostained for the tight junctional protein TJP1 and the desmosomal protein desmoplakin, together with cytoplasmic staining for actin, and examination by confocal microscopy demonstrated intact junctional complexes with neighboring cells (Fig. 5).


Figure 05
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  		FIG. 5 Confocal microscopic analysis of junctional proteins in the cultured LE cells. LE cells cultured on Cellagen membranes were stained for tight junction proteins TJP1 and desmoplakin protein (11.5F; green) and also the nuclear stain Hoescht (blue) and Texas red-conjugated phalloidin (red). A single optical section is shown. Note high number of binuclear LE cells. Bar = 20 µm.

Influence of LIF on Production of PTGES2 and IL1A by LE and Stromal Cells In Vitro

For coculture experiments, uterine LE cells from D2 were cultured on Cellagen membranes with stromal cells in the culture well as described in Materials and Methods (Fig. 1). Preliminary experiments using increasing concentrations of LIF showed that 50 ng/ml LIF had a stimulatory effect on release of IL1A by LE cells into the apical compartment, an effect that was prevented when the LIF inhibitor (LIF05) was added to the medium (Fig. 6). Subsequent experiments were carried out using this LIF concentration. LIF and/or the inhibitor were added to the culture media in both compartments, and the medium was collected at 24 h and then every 48 h up until 120 h and used for measurements of IL1A and PTGES2. LIF significantly (P < 0.01) increased secretion of both IL1A and PTGES2 in the apical medium from the LE compartment of the coculture system at both 72 and 120 h, and IL1A was also increased at 24 h (Figs. 7A and 8A). By 120 h in culture, IL1A concentration increased more than twice in LIF cultures compared to inhibitor or LIF plus inhibitor cultures, and the PTGES2 concentration in cultures with LIF had tripled compared to all other groups. When the effect of LIF on the concentration of IL1A and PTGES2 was analyzed in the lower chamber (adjacent to stromal cells), no significant difference was found during the entire culture (Figs. 7B and 8B). In addition, LIF had no significant effect on IL1A production by LE cells cultured on membranes without cocultured stromal cells (Fig. 9, A and B). However, LIF increased PTGES2 concentration only in the apical compartment of LE cells (Fig. 9C).


Figure 06
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  		FIG. 6 Effects of LIF on production of IL1A by murine endometrial LE cells cocultured with stromal cells. In preliminary experiments, LE cells were cultured on Cellagen membranes in the absence (control) or presence of increasing concentrations of LIF with stromal cells on the floor of the wells. LIF inhibitor was also supplemented in a tenfold excess to the concentration of LIF. Concentration of IL1A in culture media increased in a dose-dependent manner. The data represent average ± SEM in three separate replicates. Treatments marked with different numbers of asterisks are significantly different from each other: * vs. **, P < 0.05. LIF + Inhibitor, 50 ng/ml LIF + 500 ng/ml inhibitor.


Figure 07
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  		FIG. 7 Effects of LIF on production of IL1A by cocultured murine endometrial cells. LE cells were cultured on Cellagen membranes placed in the wells of 24-well plates, and the stromal cells were cultured on the bottom of the culture wells. A) Concentration of IL1A in the LIF-treated LE culture media (apical compartment) increased significantly with time in culture. LIF + Inhibitor = 50 ng/ml LIF + 500 ng/ml inhibitor. B) No significant differences were observed in the concentration of IL1A in stromal culture media (basal) between treatments. The data represent average ± SEM in three separate replicates. At each time point, treatments marked with different numbers of asterisks are significantly different from each other. * vs. **, P < 0.05; * vs. ***, P < 0.01; ** vs. ***, P < 0.05; **** vs. ***, P < 0.05; **** vs. **, P < 0.01; **** vs. *, P < 0.01. LIF + Inh = 50 ng/ml LIF + 500 ng/ml inhibitor.


Figure 08
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  		FIG. 8 Effects of LIF on production of PTGES2 by cocultured murine endometrial cells. LE cells were cultured on Cellagen membrane and the stromal cells were cultured on the bottom of the culture wells. A) LIF increased concentration of PTGES2 in the LE culture media (apical) with time in culture. B) No differences were observed in the concentration of PTGES2 in stroma culture media (basal) between treatments after 24 and 72 h of culture, but there was a significant increase compared to other groups at 120 h. The data represent average ± SEM in three separate experiments. At each time point treatments marked with different numbers of asterisks are significantly different from each other. * vs.***, P < 0.05; **vs. ***, P < 0.05; * vs. ****, P < 0.01; ** (both control and LIF + Inhibitor treatments at 120h) vs. ****, P < 0.05.


Figure 09
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  		FIG. 9 Effects of LIF on production of IL1A and PTGES2 by murine endometrial LE cells cultured alone. LE cells were cultured on the insert Cellagen membrane in the absence (control) or presence of 50 ng/ml LIF. No differences were observed in the concentrations of IL1A between apical (A) and basal (B) culture media or in the presence or absence of LIF. Similarly, no differences were observed in the concentration of PTGES2 in the basal media (D). However, LIF increased PTGES2 concentrations in the apical compartment at 72 and 120 h (C) (*P < 0.05). Data represents average ± SEM in three separate experiments in each case.

Effects of LIF on Expression of mRNA for Il1a

In order to assess effects of LIF on mRNA for Il1a, LE cells and stromal cells were cultured in the coculture system above, and RNA was extracted. Preliminary observations confirmed presence of Il1a mRNA in all cell types (data not presented). Semiquantitative RT-PCR showed no differences between treatments after any of 2, 4, or 6 (data not shown) days of culture (Fig. 10, A and B).


Figure 10
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  		FIG. 10 Effects of LIF on expression of mRNA for Il1a in murine endometrial cells. Total RNA was extracted from either of freshly collected and cultured stromal and LE cells. A) Semiquantitative analysis of mRNA for Il1a in LE and stroma cells after 6 days of culture in the absence (control) or presence of LIF or LIF inhibitor. No differences were observed in the intensity of bands for Il1a mRNA among treatments. B) Semiquantitative analysis of changes in the expression of mRNA for Il1a in LE cells in relation to time of culture.

DISCUSSION

LIF expression on D4 is critical for successful implantation [5, 8], and in its absence the expression of a variety of molecules is affected in both the LE and stroma [7, 8, 11, 33, 46] (reviewed in [10]). Uteri of Lif-deficient mice are unable to support embryo implantation or to mount a decidual response [6]. However, the precise mechanisms by which LIF exerts its effects on implantation are still largely undefined. The identification of LIF-regulated molecules in the endometrium around the time of implantation will provide insights into the mechanisms that underlie uterine receptivity and decidualization.

LIF is known to influence cells of the immune system [47, 48] in addition to cells in the reproductive tract. The cytokine IL1 and its associated molecules also play well-established roles in inflammatory processes in the body [49]. Indeed, the changes associated with implantation have been compared to an inflammatory reaction [50, 51]. Moreover, several studies have suggested that IL1 expression is regulated by the local effects of estrogen and P4, and estrogen also regulates Lif [8, 48].

We found that components of the Il1 system are spatially and temporally regulated during the pre- and peri-implantation period. Abundant transcripts for Il1a, Il1b, Il1rn, and Il1r2 were detected in the LE from both WT and Lif-null uteri on D2, suggesting that at least at the transcript level, the ability of the Lif-deficient mouse to induce a pro-inflammatory response in the early stages of pregnancy is not compromised. However, we have shown previously that the proportions and distribution of leukocytes, particularly macrophages and NK cells, are already disrupted in Lif-null uteri by D3 [37].

Increased transcript signals for Il1a, Il1b, Il1rn and Il1r1, and Il1r2 were observed in WT mice at the time of implantation on D4 pm in both LE and stroma. These findings agree with previous studies where Il1a and Il1b mRNAs and their bioavailability were shown to peak between D4 and D5 [52]. Wood et al. [53] also reported that mRNA levels of uterine Il1a and Il1b decreased, from peak levels on D1 and D2 to very low levels on D3, but expression increased in the peri-implantation period. We have further shown that Il1rn mRNA is consistently expressed up until D4 pm in the LE but barely detectable on D5, whereas stromal expression was uninterrupted, except for a transient loss of transcript on D4. A similar pattern was observed for stromal Il1r2 transcripts. That expression of Il1rn in the LE is continuous during the preimplantation phase suggests that IL1RN in the LE may play a role in inhibiting IL1 receptor activation up until just after implantation has been initiated. In line with this, experiments have suggested that IL1 signaling is crucial to embryo implantation in vivo, as functional blockade of IL1R1 by repeated i.p. IL1RN administration from D3 to D9 resulted in reduced implantation rates [28]. This inhibition of embryo implantation was attributed to a down-regulation of integrins {alpha}4 and β3 on the LE by IL1RN [54]. However, Abbondanzo et al. [55] found that administration of IL1RN to C57BL/6 x 129Sv hybrids had no effect on implantation. This difference may have a methodological source, but it casts uncertainty on the absolute requirement for IL1 signaling for implantation. Our data suggest that IL1 signaling is likely to be functional in the later stages of implantation.

Since activation of the IL1R2 by any IL1 ligand does not elicit a biological response, it is proposed to function by limiting the bioavailability of IL1A and IL1B by acting as a decoy receptor, reducing the amount of free IL1 [56, 57]. Examination of Il1r2 mRNA levels in the WT peri-implantation uterus revealed that stromal transcript expression of Il1r2 was low, with detection only on D3, D4 pm, and D5, and only D3 and D5 in the null stroma. In contrast, LE extracts on D2–D6 consistently gave bands both in WT and null animals. Therefore, transcriptional regulation of stromal Il1r2 during the peri-implantation period may play a role in regulating the bioavailability of IL1A and IL1B, but it is little affected by LIF.

The spatio-temporal expression of several components of the IL1 system in the Lif-null uterus was altered from that of WT uteri from D3 onwards. Transcripts for Il1a in the LE of Lif-null uteri were low except for a strong band on D2, in contrast to WT LE, where signaling for Il1a was high on D4 pm following the nidatory burst of LIF expression. This suggests that LIF does either directly or indirectly regulate Il1a over the peri-implantation period. Stromal expression of Il1a mRNA in the nulls was restricted to D3 and D5, suggesting major misregulation in transcriptional timing.

Interestingly, PCR signal for Il1rn in the Lif-null LE similarly declined from D2 onwards, with barely detectable signal by D4–D5. The low levels of Il1rn in the uteri of Lif-null compared to WT mice could contribute to the implantation defect. The premature reduction in antagonism of IL1A and IL1B may result in overstimulation of the IL1 signaling cascade, potentially altering the inflammatory response [20]. Huang et al. [58] postulated that an appropriate ratio of IL1 to IL1RN is crucial during embryo implantation. Furthermore, work from our laboratory has shown that both macrophage number and distribution (a primary source of IL1) were altered in the uteri of Lif-deficient mice from D3 compared to WT [37]. It has also been confirmed in the present study that IL1 is synthesized in LE and probably GE, as well as stromal cells, and that leukocytes are by no means the only source of IL1 ligands in the preimplantation uterus. Surprisingly, there were no significant differences in the expression of Il1b transcripts in the uteri of WT and Lif-null mice, indicating that the strong Il1b bands seen on D4 pm are not a direct result of LIF production on D4.

No significant alterations were observed in the gene expression of Il1r1 or Il1r2 in the uterine LE of WT and Lif-null mice during early pregnancy in the LE, suggesting these receptors are not regulated by LIF. However, null stromal Il1r1 was expressed in a pattern at variance with that of WT, with loss of transcripts 12–24 h after the known peak of Lif expression in WT animals. Lack of proper regulation of epithelial derived IL1A in the Lif-null uterus may lead secondarily to lack of IL1B in the glands observed by immunocytochemistry on D5. This may reflect a failure of normal LE to stromal signaling. Indeed, it is likely that the stromal misexpression of several Il1 components in the uteri of Lif-null mice compared to WT may be partly attributed to the lack of secondary signaling between unstimulated LE and the stroma in the null uteri, particularly on D4 or D5.

On D3 in both WT and Lif-null uteri, immunoreactive IL1A was detected in the LE, GE, and some stromal cells. IL1A-positive cells within the stroma appeared larger than other stromal cells and may represent IL1A-producing immune cells such as macrophages, but it was not possible to confirm this definitively due to the differences in fixation requirement of the various antibodies. Previous studies by our laboratory have shown that there are increasing numbers of macrophages at this time that are known to be major producers of IL1 [37]. Intense staining of IL1A was observed in the decidua on D5 and D6 but markedly reduced in Lif-null uteri from D4 onwards. Since these animals lack decidualization and IL1 is known to induce decidualizing molecules [30, 31], the lack of IL1A here is consistent with it being a mediator of LIF-induced decidualization. In vitro studies in murine endometrial stromal cells have also shown that the extracellular matrix glycoprotein tenascin C (TNC) expression is up-regulated by Il1a [7, 59]. Similarly, TNC has also been shown to be absent from the site of implantation in the uteri of Lif-null mice during the implantation period [7]. However, while evidence suggests a fundamental role for IL1 signaling in embryo implantation, gene deletion experiments in mice have revealed that there are no overt reproductive phenotypes in mice lacking either Il1r1 or Il1b [60–62]. Various members of the IL1 system have been identified in the uterus, including the novel ligands IL1F5 and IL1F7 [63], but to date they have no identifiable function. Perhaps the lack of overt phenotypes in the gene-deleted mice can be explained by compensatory effects of these and other novel ligands or receptors.

In order to obtain functional evidence for the interrelationship of IL1 and LIF, we cocultured LE with stromal cells to examine the influence of LIF in a system which more closely mimics the physiological relationship of these two cell layers than does separate cultures. A surprising result here was that the predominant secretion of IL1 induced by LIF in our in vitro culture system is directed towards the lumen, a location removed from potential tissue targets. However, it is possible that this molecule is involved in leukocyte recruitment to the point of possible pathogen entry from luminal fluids. This would seem logical since the LE barrier is breached during implantation, with potential risk of infection.

LIF stimulated IL1A secretion by LE cells in a dose-dependent manner, an effect that was abrogated by an established LIF inhibitor. It did not stimulate IL1A secretion by the basal stromal cells. Expression of Il1a mRNA in cultured LE (by semiquantitative PCR) did not appear to be affected by adding LIF to medium or by LIF inhibition. Therefore, stimulation of secretion of this cytokine into the culture medium by LIF is likely to occur posttranscriptionally at the level of protein synthesis or of the secretory apparatus. These would be consistent with an effect of the inhibitor by 24 h. Stimulation of IL1 secretion by LIF is in keeping with the increased protein seen by immunohistochemistry. Thus, this is a level of LIF regulation of IL1A that is in addition to the regulation of LE Il1a transcription in vivo, inferred from the rapid loss of PCR signal after D2 in Lif-null LE. The lack of direct transcript regulation in culture suggests that, in vivo, regulation may be indirect and/or on mRNA stability. Alternatively, direct transcript stimulation may be prevented under our coculture conditions.

LIF also induced release of PTGES2 by LE cells in coculture, an effect which was also observed in culture of LE alone. In the coculture system, LE and stromal cells are able to establish a dialogue, resulting in modulation of cytokine production [64], thus making this model more physiologically relevant. Jacobs and Carson [30] reported that IL1 induces PTGES2 secretion by uterine stromal cells in vitro. This effect is mediated through up-regulation of Ptgs2 mRNA in stromal cells [32, 65]. Moreover, uterine secretion of IL1A by epithelial cells increases PTGS2 enzyme activity [66–68]. We have previously reported defects in PTGS2 protein expression and decidualization of uterine stroma cells in Lif-null mice [7]. We have also shown that LIF does not stimulate PTGES2 by stromal cells cultured without LE [34]. However, since LIF does not directly promote the secretion of PTGES2 by uterine stromal cells in vitro, PTGES2 is not likely to be a direct target of LIF in the stroma [34]. LIF may exert its effect mainly though up-regulation of intermediate messengers in the LE that in turn regulate decidualization. It is unlikely that IL1A from LE acts directly on stromal cells per se since our results suggest it is secreted mainly apically. However, IL1A from GE and/or other cells in the stroma may regulate stromal PTGS2 in vivo and thus contribute to decidualization and angiogenesis. Furthermore, autocrine activation of IL1R1 signaling pathways in the LE is likely. This may contribute to LE receptivity and susceptibility to embryonic signals that in turn directly or indirectly initiate the decidual response. It should not be forgotten that the embryo itself expresses IL1A [69–70 and this study], which could also interact locally with LE. Moreover, IL1 is known to affect the phenotype of invasive trophoblast in human [71–73] and mouse [74], so one target of apically secreted IL1 could be the trophoblast as well as a possible immunomodulatory one, mentioned above.

In LE cells, LIF may induce PTGES2 production through an autocrine influence of IL1 via the IL1 receptor. This may also activate basally released modulators of stromal cells to contribute to the decidual response following signaling from the embryo. In addition, LIF modulates IL1 signaling by regulating IL1RN in the LE to dampen both the autocrine effect and any paracrine influence on the blastocyst. Our results confirm the extreme complexity of the interacting network of secreted molecules that regulate implantation-related events.

ACKNOWLEDGMENTS

We thank Dr. Anne Vernallis, University of Aston, for the gift of hLIF-05. We also acknowledge Mr. Tony Bagley for assistance on confocal microscopy.

FOOTNOTES

1Supported by a grant from the Biological and Biotechnological Research Council (BBSRC) United Kingdom to S.J.K. and a BBSRC graduate studentship to L.M. Back

Correspondence: 2FAX: 161 275 3915; e-mail: sue.kimber{at}manchester.ac.uk

4These authors contributed equally to this work. Back

3Current address: The Royal Veterinary College, Hawkshead Lane, Hatfield AL9 7TA, United Kingdom. Back

Received: 23 August 2007.

First decision: 13 September 2007.

Accepted: 3 March 2008.

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