Molecular Human Reproduction, Vol. 9, No. 3, 151-158,
March 2003
© 2003 European Society of Human Reproduction and Embryology
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Leptin regulation of the interleukin-1 system in human endometrial cells
Submitted on October 2, 2002; accepted on December 13, 2002
1 Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, 2 Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, 3 Department of Physiology, Tufts University School of Medicine, Boston, MA 02111 and 4 Analytical Biotechnology Inc., 64 Grove Street, Watertown, MA 02472, USA
5 To whom correspondence should be addressed. e-mail: gonzalezr{at}bbri.org
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ABSTRACT |
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We have previously shown that (i) leptin and leptin receptor (Ob-R) are expressed in the human endometrium, and (ii) leptin secretion is regulated in blastocyst and endometrial epithelial cell (EEC) co-cultures. Interleukin-1ß (IL-1ß) up-regulates leptin and Ob-R, and both cytokines up-regulate ß3 integrin expression in EEC. In the present investigation we examined the effect of leptin on the expression of the IL-1 system in EEC and endometrial stromal cells (ESC) cultured in a medium containing insulin, leptin or IL-1ß (0–3 nmol/l). Leptin stimulated IL-1 antagonist (IL-1Ra), IL-1ß secretion and expression of IL-1 receptor type I (IL-1R tI) in both cell types. IL-1ß and IL-1Ra secretion were down-regulated by IL-1R tI blockade using specific antibodies. Interestingly, leptin partially neutralized this effect. The blockade of Ob-R neutralized the effects of both leptin and IL-1ß on expression of the IL-1ß system and ß3 integrin and on phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that leptin regulates the IL-1 system and that the blockade of functional Ob-R impairs leptin and IL-1ß functions at the endometrial level. Leptin could be an important molecule for implantation and a molecular mediator for actions of the IL-1 system. The fact that leptin, in the absence of IL-1, can trigger the expression of markers of endometrial receptivity and of the invasive trophoblast phenotype (as does IL-1), suggest that leptin could substitute for these IL-1 functions during the implantation process.
Key words: human endometrial cells/implantation/interleukin-1/leptin/leptin receptor
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Leptin, a 16 kDa polypeptide, known primarily for its effects on food intake, also appears to play a critical role in reproductive function and is linked to the inflammatory response (Gonzalez et al., 2000a; Moschos et al., 2002).
Recent published data has provided compelling evidence that leptin could play key roles in the development of the preimplantation embryo and in the implantation process. Leptin promotes preimplantation embryo development (Kawamura et al., 2002) and leptin (Gonzalez et al., 2000b; Wu et al., 2002) and leptin receptor (Ob-R) are expressed by endometrium (Alfer et al., 2000; Gonzalez et al., 2000b; Kitawaki et al., 2000; Wu et al., 2002). Moreover, human blastocysts co-cultured with endometrial epithelial cells (EEC) modulate leptin secretion (Gonzalez et al., 2000b).
A major consequence of leptin binding to Ob-R, the product of the db gene, is activation of JAK-2, which leads to phosphorylation of Stat3 (signal transducer and activator of transcription 3) (p-Stat3), that in turn activates a number of downstream signalling pathways (Takahashi et al., 1997). Ob-R expression in human endometrium fluctuates during the menstrual cycle (Kitawaki et al., 2000). The down-regulation of Ob-R expression at the time of implantation could play a role in subfertility (Alfer et al., 2002). Soluble leptin receptor (sOb-R) may also negatively regulate biological effects of leptin (Lewandowski et al., 1999; Huang et al., 2001a; Laimer et al., 2002).
Although leptin is regulated in several tissues by interleukin-1 (IL-1), tumour necrosis factor-
(TNF-) and transforming growth factor-ß (TGF-ß) (Sarraf et al., 1997), leptin may itself induce the synthesis of inflammatory cytokines in vivo and in vitro (Loffreda et al., 1998). Interestingly, leptin mRNA and protein expression are increased in ectopic endometriotic lesions (Wu et al., 2002).
The IL-1 system, composed of ligand (IL-1ß), receptor type I (IL-1R tI) and receptor antagonist (IL-1Ra), is produced by both preimplantation embryos and endometrium. It has been proposed to be an important factor in embryo-maternal molecular cross-talk during implantation (Sheth et al., 1991; Simon et al., 1993). The factor(s) responsible for embryonic up-regulation of IL-1 secretion when preimplantation embryos and EEC are co-cultured together has not yet been identified (De los Santos et al., 1996).
IL-1ß (Simon et al., 1997) and leptin (Gonzalez and Leavis, 2001) up-regulate ß3 integrin expression (a molecular marker of endometrial receptivity) by EEC. However, leptin exerts a significantly greater effect on ß3 integrin up-regulation than IL-1ß at similar concentrations and IL-1ß stimulates leptin secretion and Ob-R expression by EEC (Gonzalez and Leavis, 2001).
Even though evidence has accumulated that supports roles for IL-1ß during implantation (Sheth et al., 1991; Simon et al., 1993, 1994, 1997, 1998; De los Santos et al., 1996), gene-targeting studies with mice deficient in IL-1ß, IL-1R tI and IL-1Ra have not been able to demonstrate that these molecules are indispensable for reproduction (Abbondanzo et al., 1996; Stewart and Cullinan, 1997).
In contrast, leptin or Ob-R-deficient mice cannot reproduce. Exogenous leptin injected into starved mice restores fertility (Ahima et al., 1996). Leptin-deficient mice (ob/ob) are sterile, but fertility can be restored by exogenous leptin (Chehab et al., 1996). In addition, embryos implanted in STAT3 deficient mice rapidly degenerate (Takeda et al., 1997). Finally, pregnancies from leptin-treated ob/ob females failed when treatment with a minimal dose of leptin was stopped at 0.5 or 3.5 days post-coitum, indicating that leptin is essential for normal preimplantation and/or implantation processes (Malik et al., 2001).
We have previously proposed that leptin produced and secreted locally by preimplantation embryos and EEC could act in an autocrine or paracrine manner to regulate biological functions that may mediate endometrial receptivity (Gonzalez et al., 2000a,b). In addition, we have also hypothesized that the actions of IL-1ß and leptin could be related at the endometrial level during the time of implantation (Gonzalez et al., 2000a; Gonzalez and Leavis, 2001).
In this study we investigated (i) leptin regulation of the expression of the IL-1 system in endometrial cells and (ii) whether blocking of the functional Ob-R could impair leptin and IL-1ß functions at the endometrial level.
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Reagents and antibodies
All chemicals and cultured media were obtained from Sigma Chemical Co., St Louis, MO, USA and from GIBCO BRL Products, Gaithersburg, MD, USA. Antibody for phosphorylated STAT3 (p-Stat3 B-7) was obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. IL-1R tI monoclonal antibody, goat polyclonal anti the extracellular domain of Ob-R, human recombinant leptin and IL-1ß were provided by R&D Systems Inc., MN, USA. Anti-ß3 integrin subunit antibody was purchased from Becton Dickinson, San Diego, CA, USA and anti-vimentin, anti-cytokeratin and anti-CD45 antibodies were from Dako Corporation, Carpinteria, CA, USA. Non-specific mouse and goat IgGs (Santa Cruz Biotechnology) were used as negative controls.
Endometrial tissues
Human endometrial tissue was obtained from women undergoing hysterectomy. Informed consent was obtained from each patient and ethical approval was obtained from the ethical committee of St Mary’s Hospital, CT, USA. Samples used in the study were of non-malignant aetiologies. Tissues were digested with proteases for endometrial cell isolation as described elsewhere (Gonzalez et al., 1999). Briefly, endometrial biopsies were minced and treated with collagenase I (0.1%)–DNAse I, 0.005% for 1 h at 37°C. After gland sedimentation, endometrial stromal cells (ESC) were isolated from supernatants. EEC were purified of ESC and macrophage contaminants by repeated incubation at 37°C in a Falcon flask. Stromal and epithelial cell dispersions were counted in a haemocytometer and cell viability was assessed by optical microscopy using the Trypan Blue exclusion method. The mean cell viability was 
90%. To assess homogeneity of cell preparations several specific monoclonal antibodies were used in cell smears (Gonzalez et al., 1999), i.e. anti-vimentin (Vm; ESC+), cytokeratin (Ck; EEC+) and CD45 (leukocyte +). Homogeneity of cell preparations was 
98%.
General experimental design
To study the effects of leptin on the regulation of the IL-1 system in endometrial cells (ESC and EEC) we performed the followings experiments: (i) Flow cytometric (FC) analysis to investigate if the anti-Ob-R polyclonal antibody can bind to and block the receptor function in ESC. Furthermore, this antibody was used to assess the specificity of leptin effects on the cell cultures. (ii) The effects of leptin and anti-Ob-R antibody on the secretion of IL-1ß and IL-1Ra by cell cultures were determined by ELISA, and the IL-1Ra/IL-1ß ratio was calculated. (iii) The effects of IL-1ß on the secretion of IL-1Ra and expression of IL-1R tI by cell cultures were also evaluated. A monoclonal antibody (anti-IL-1R tI) was used to block the receptor and assess the specificity of the IL-1ß effects. (iv) We also investigated whether leptin affects the expression of IL-1R tI in both cell types and whether it could overcome the blocking effects of the anti-IL-1R antibody on IL-1ß and IL-1Ra secretion. (v) To assess the functionality of the Ob-R after leptin or anti-Ob-R treatments, electrophoresis on SDS–PAGE and Western blot analysis of p-Stat3 and IL-1R tI were performed. (vi) The effects of leptin, IL-1ß and blocking of Ob-R on the expression of ß3 integrin by EEC were evaluated by immunocytochemistry using a specific monoclonal antibody. The staining intensities were used to semi-quantitatively calculate the expression of ß3 integrin by the HSCORE approach (Lessey et al., 1992). ß3 integrin expression was also determined by Western blot analysis. (vii) Finally, to test whether leptin is involved in IL-1 up-regulation of ß3 integrin by EEC, we blocked the leptin receptor with anti-Ob-R antibodies.
Cell cultures
Homogeneous preparations of endometrial cells (300 000 cells/well) were cultured for 5–7 days in fetal bovine serum (FBS)-enriched medium. The medium used was DMEM-MCDB105 plus 10% FBS, 5 µg/ml insulin, 1% amphopthericin B, 100 µg/ml streptomycin and 100 U/ml penicillin until confluent layers were obtained. To eliminate any cytokine effect from the FBS supplement, the cells were washed twice with PBS–2% BSA and cultured for an additional 2 days in the same medium containing 2% BSA but without FBS. Cells were washed again and cultured in the same FBS-free medium but containing leptin, IL-1ß (0–3 nmol/l) and/or Ob-R antibody (1–20 µg/ml) or IL-1R tI antibody (20 µg/ml) for 24 h. Conditioned media were collected, lyophilized and stored at –80°C for ELISA determinations. Duplicate wells were run for each treatment with leptin and IL-1ß at the concentrations described above and the experiments repeated at least three times with different endometrial preparations. Controls were the same cellular preparations cultured in the absence of cytokines and antibodies.
Western blot analysis
Following cytokine and antibody treatments, endometrial cell cultures were washed once with ice-cold PBS and disrupted by homogenization in RIPA buffer [20 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 2 mmol/l EDTA, 50 mmol/l sodium pyrophosphate, 2 nmol/l sodium orthovanadate, 2 nmol/l phenylmethyl sulphonyl fluoride, 1% Nonidet P-40, 10% glycerol, 100 µmol/l antipain hydrochloride, 0.1 mg/ml trypsin inhibitor and protease cocktail inhibitor 1:50 (Sigma)] on ice. Cellular lysates were centrifuged for 10 min at 24 000 g at 4°C. Protein concentrations were determined using the Bradford protein assay (BioRad Laboratories Inc., Hercules, CA, USA). The supernatants were combined with the appropriate amount of SDS sample buffer, 10 µg of proteins were loaded per lane and electrophoresis was performed at 50 V for 1.5 h (BioRad, electrophoresis apparatus) in 10% SDS–PAGE gel. Electroblotting onto nitrocellulose membranes of 0.2 µm was performed at 22 V for 4 h. Membranes were blocked with 5% blocking reagent from the ECL system (Amersham Pharmacia Biotech) in Tris–glycine (TBS)–0.1% Tween-20 solution overnight at room temperature and washed three times with TBS plus 0.1% Tween-20. The membranes were subsequently incubated at room temperature for 1 h with 1 µg/ml of IL-1R tI, 1 µg/ml of ß3 integrin or 0.5 µg/ml of p-Stat3 mouse monoclonal antibodies in TBS-0.1% Tween-20. This was followed by incubation with anti-mouse horse radish peroxidase-conjugated secondary antibodies (ECL system, Amersham) for 30 min at room temperature. Positive specific antigen–antibody reactions were analysed with the ECL-chemiluminescent assay (Amersham).
Determination of IL-1ß and IL-1Ra secretion by endometrial cell cultures
Lyophilized conditioned media (n = 3 per treatment) from ESC and EEC cultures were resuspended in 0.5–1 ml of deionized water and IL-1ß and IL-1Ra concentrations were determined by ELISA (Quantikine® HS, R&D Systems). Cytokine concentrations as determined by ELISA were within the dynamic range of the standard curve and were divided by the lyophilization concentration factor and expressed in pg/ml. Standards, controls and samples were assayed in duplicate. The intra- and interassay coefficients of variations were 5.8 and 8.3% for IL-1ß; 4.4 and 6.6% for IL-1Ra. According to the manufacturer, the performance characteristics of ELISA were as follows: 100% specificity, sensitivity 0.1 and 14 pg/ml for human IL-1ß and IL-1Ra, respectively.
Immunocytochemistry and HSCORE determinations
EEC were cultured as described above in duplicate in 8-well glass culture plates designed for immunocytochemical studies (Nalgene Nunc International, Naperville, IL, USA). After cytokine and antibody treatments the expression of ß3 integrin by EEC was assessed by immunocytochemistry by incubation for 1 h at room temperature with the ß3 integrin antibody (diluted 1:80 in PBS–2% BSA) and further with a streptavidin-biotin-horse radish peroxidase system (Dako, LSAB kit system). Negative controls were incubated with non-specific mouse IgG. EEC were stained by amine-ethyl-carbazol (AEC) enzymatic product and counter-stained with haematoxylin (Dako).
ß3 integrin positive cells were recorded in randomly chosen fields and a total of 300 cells were counted in each of three fields. Treatments were performed in triplicate. Staining intensity was assigned using semi-quantitative HSCORE (Lessey et al., 1992). The HSCORE was calculated using the following equation: HSCORE = 
pi(i + 1), where ‘i’ is the intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong, respectively) and ‘pi’ is the percentage of stained EEC for each intensity.
Flow cytometric measurements
To determine the binding of Ob-R antibody to living cells, ESC monolayers were incubated for 2 h at 37°C with goat Ob-R antibody at 1, 5 and 20 µg/ml in DMEM-MCDB105–2% BSA. After washing three times with PBS, cells were further incubated for 45 min with an anti-goat FITC-conjugated antibody (Dako). After washing as before, the cells were scraped from the plates. Non-specific goat IgGs were used as controls. FC measurements were performed in a Coulter Epics XL flow cytometer using System II software for data acquisition and analysis (Coulter Corporation, FL, USA).
Statistical analysis
A one-way ANOVA test with Dunnett error protection and confidence interval of 95% was used from the Analyse-it for Microsoft Excel (Leeds, UK, htpp://www.analyse-it.com) for data analysis. Data are expressed as mean ± SEM. Values for P < 0.05 were considered statistically significant.
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Results |
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Blocking the leptin receptor with Ob-R antibody
To study leptin effects on ESC and EEC cultures we first addressed the possibility that Ob-R antibody could block leptin receptor function. FC results of ESC cultures in medium containing Ob-R antibody demonstrated that this antibody specifically binds to live cells (Figure 1A). The binding of Ob-R antibody to ESC showed a dose–response effect that reached a plateau for maximal binding between 5 and 20 µg/ml (Figure 1B). Western blot analysis demonstrated that leptin increased the p-Stat3 in ESC cultures and that this effect was down-regulated by the addition of Ob-R antibody (Figure 1C).
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The Ob-R antibody used in this study recognizes the N-terminus of long and functional Ob-R, and it is recommended by the manufacturer for Western blot analysis and immunocytochemistry analysis of Ob-R expression. However, this polyclonal antibody binds to and blocks Ob-R function in endometrial cell cultures. This new feature of Ob-R antibody was used to study the in vitro blocking of functional leptin receptor in the present investigation.
Leptin regulation of IL-1ß and IL-1Ra secretion and IL-1R tI expression by ESC
Lyophilization of conditioned culture media and reconstitution in a small volume allowed the effective determination of IL-1ß and IL-1Ra secretion by endometrial cell cultures even after the cells were cultured for a 2 day period in medium free of steroids and growth factors.
Leptin in the absence of steroid hormones substantially up-regulated all components of the IL-1ß system (ligand, receptor and receptor antagonist) in ESC cultures. Basal secretion of IL-1ß (Figure 2A) and IL-1Ra (Figure 2B) in control ESC cultures was significantly up-regulated by leptin treatment at doses between 0.06 and 3 nmol/l. Leptin did not show a dose–response effect on IL-1ß and IL-1Ra up-regulation. However, a maximal effect of leptin on IL-1ß stimulation (>2-fold) and IL-1Ra stimulation (>3-fold) was found at a concentration of 0.6 nmol/l.
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Interestingly, ESC cultured in medium containing both leptin and Ob-R antibody down-regulated their IL-1ß and IL-1Ra secretions which demonstrates that the effect of leptin was abolished by Ob-R blockade (Figure 2A and B).
Western blot analysis demonstrated that IL-1R tI expression was decreased in ESC cultures under basal conditions. Interestingly, leptin up-regulates the expression of IL-1R tI by ESC in a dose-dependent manner at concentrations between 0.06 and 3 nmol/l (Figure 2C). The addition of Ob-R antibody (1 µg/ml) decreased leptin-induced (0.06 nmol/l) up-regulation of IL-1R tI by ESC. However, when leptin concentrations were increased (0.6 or 3 nmol/l), the effects of Ob-R antibody on IL-1R tI expression were abolished (Figure 2C). Therefore, we also looked at the effect of higher Ob-R antibody concentrations. Leptin up-regulation of IL-R tI by ESC was neutralized by Ob-R antibody at concentrations between 5 and 20 µg/ml (Figure 2D).
IL-1Ra/IL-1ß ratio in leptin-stimulated ESC
Leptin significantly increased (P < 0.05) the IL-1Ra/IL-1ß secretion ratio in ESC cultures. Although leptin up-regulated both ligand and antagonist receptor secretion by ESC, the overall effect of leptin stimulation was higher on IL-1Ra (Figure 3). Following similar profiles to those found for individual cytokine secretion, the IL-1Ra/IL-1ß ratio was higher in ESC cultures stimulated by leptin at 0.6 nmol/l.
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Interestingly, the blockade of IL-1R tI with specific antibodies decreased the IL-1Ra/Il-1ß ratio in ESC, but leptin also partially inhibited the antibody effect (Figure 3). This effect of leptin could be due to leptin up-regulation of IL-1R tI expression by ESC.
Leptin regulation of IL-1ß and IL-1Ra secretion and IL-R tI expression by EEC
Similarly to the effects of leptin on the IL-1ß system in ESC, leptin also up-regulated IL-1ß (Figure 4A) and IL-1Ra (Figure 4B) secretion by EEC cultures. However, IL-1ß secretion was only significantly increased at the highest leptin concentration (3 nmol/l) tested (Figure 4A). In contrast, IL-1Ra secretion was significantly stimulated by leptin (0.6 and 3 nmol/l) in a dose–response manner (Figure 4B). As for ESC, the addition of Ob-R antibody to EEC cultures stimulated with leptin abolished leptin up-regulatory effects on IL-1ß (Figure 4A) and IL-1Ra (Figure 4B) secretion.
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The overall effect of leptin was a significant increase of the IL-1Ra/IL-1ß ratio in EEC cultures. However, the IL-1Ra/IL-1ß ratio showed a plateau when EEC were stimulated with leptin at 0.6 or 3 nmol/l concentrations (Figure 4C).
Leptin also up-regulated the expression of IL-1R tI by EEC. However, the addition of Ob-R antibody (1 µg/ml) to EEC cultures stimulated with leptin (0.6 nmol/l) was not enough to block the up-regulatory effects of leptin on IL-1R tI expression (Figure 4D).
IL-1ß also up-regulated the expression of IL-1R tI by EEC. The addition of Ob-R antibody at 1 µg/ml did not affect the IL-1ß increase of IL-1R tI expression. However, higher Ob-R antibody concentrations effectively abolished leptin and IL-1ß stimulatory effects on IL-1R tI expression (data not shown). Leptin and IL-1ß stimulated IL-1R tI expression by EEC in a synergistic manner (Figure 4D).
The role of functional leptin receptor in leptin and IL-1ß up-regulation of ß3 integrin
Immunocytochemical results from ß3 integrin in EEC cultures demonstrated that basal expression (Figure 5A, a) of this integrin was significantly (P < 0.05) increased by leptin (Figure 5A, b) or IL-1ß (Figure 5A, d). However, the stimulatory effect of leptin and IL-1ß was abolished by the addition of Ob-R antibody (Figure 5A, c and e, respectively). HSCORE determinations of ß3 integrin expression by EEC indicated that leptin and IL-1ß significantly up-regulate ß3 integrin in EEC cultures. Blockade of Ob-R with a specific antibody effectively decreased the stimulatory effects of leptin and IL-1ß integrin expression (Figure 5B). Western blot analysis of ß3 integrin expression in leptin or IL-1ß-stimulated EEC corroborated the immunocytochemical results (Figure 5C). In contrast to leptin, the IL-1ß stimulatory effect on ß3 integrin expression was totally blocked by Ob-R antibody (Figure 5C). These findings suggest that leptin and IL-1ß up-regulation of ß3 integrin expression by EEC requires the presence of functional Ob-R. These results also suggest that at least for ß3 integrin up-regulation leptin could act as a mediator molecule for actions of IL-1ß.
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Discussion |
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We have previously suggested that leptin could be a regulator of implantation (Gonzalez et al., 2000a,b). It may improve endometrial receptivity by up-regulation of ß3 integrin expression (Gonzalez and Leavis, 2001) and enhance the trophoblast invasive phenotype (Gonzalez et al., 2001a).
The results from the present investigation suggest a role for leptin in endometrial receptivity via its relationships with IL-1ß. Our results demonstrate that leptin, acting through its functional receptor expressed by ESC and EEC, triggers the secretion of IL-1ß, IL-1Ra and up-regulates the expression of IL-1R tI at the protein level.
Although leptin stimulates the secretion of IL-1ß and its natural antagonist in both cell types, there were differences between ESC and EEC responses to leptin stimulation. Basal secretion of IL-1ß and IL-1Ra were higher in ESC than EEC cultured in a FBS-free medium containing insulin. In comparison, leptin at a lower dose (0.06 nmol/l) stimulates IL-1ß secretion by ESC while a 50-fold higher dose (3 nmol/l) of leptin was necessary to up-regulate IL-1ß secretion by EEC. Similarly, leptin up-regulates IL-1Ra secretion in both cell types, but again leptin at 0.06 nmol/l was enough to up-regulate IL-1Ra secretion by ESC. In contrast, a 10-fold higher leptin dose (0.6 nmol/l) was required to up-regulate IL-1Ra secretion by EEC. This secretion was increased by leptin in a dose–response manner.
These results suggest that ESC are more sensitive to leptin up-regulation of the IL-1 system than EEC. The biological significance of these findings needs to be investigated.
In this study we have only evaluated the regulatory effects of leptin on the IL-1 system at the post-transcriptional level. Therefore, further studies are needed to assess if leptin also up-regulates gene expression of the IL-1 system in endometrial cells. However, it is well known that these two processes are not always simultaneously regulated (Dutt and Lee, 2000). Low correlation coefficients have been obtained between mRNA and protein concentrations in human tissues, suggesting that post-transcriptional regulation of gene expression is a frequent phenomenon in higher organisms (Anderson and Seilhamer, 1997). Similar results have been reported from studies in microorganisms, showing that the correlation between mRNA and protein levels is insufficient to predict protein expression levels from quantitative mRNA data (Gygi et al., 1999).
The up-regulatory effect of leptin on the IL-1 system in endometrial cell cultures found in the present study occurs together with a significant increase of p-Stat3 production that is abolished by the blockade of Ob-R. Interestingly, leptin in the db/db mice, which lack functional Ob-R, increased IL-1 Ra mRNA levels in the brain. This leptin effect on IL-1Ra may be mediated through Stat3 independent mechanisms (Hosoi et al., 2002).
Although the Ob-R antibody used in this investigation recognizes the N-terminus of the Ob-R extracellular domain in Western blot and immunohistochemical analyses, it also appears to bind to live cells in a dose-dependent manner. Moreover, the culture of ESC and EEC with 1–20 µg/ml Ob-R antibody neutralized leptin-induced enhancement of p-Stat3. This new feature of Ob-R antibody was useful to study the effects of Ob-R blockade on the expression of the IL-1ß system in endometrial cells.
Taking into account that (i) IL-1ß can up-regulate leptin secretion and the expression of Ob-R in EEC (Gonzalez and Leavis, 2001), (ii) leptin up-regulates the IL-1 system in ESC and EEC, and (iii) both cytokines showed synergistic actions for up-regulation of IL-1R tI, it can be anticipated that these molecules should have very complex relationships at the endometrial level for promoting cellular changes during the peri-implantation and implantation processes. The leptin-cytokine network in the endometrium may be more complex, insofar as the biological actions of TGF-ß1, IL-1, IL-6 and leptin appear to be closely related. Leptin levels are thought to be regulated by these cytokines in many tissues as well as by both pituitary and ovarian hormones and insulin (Granowitz, 1997; Janik et al., 1997; Sarraf et al., 1997; Zumbach et al., 1997; Gonzalez et al., 2000a).
In vitro IL-1ß up-regulates the expression of ß3 integrin by EEC (Simon et al., 1997); ß3 integrin is an adhesion molecule and a marker of endometrial receptivity, which is likely to increase the possibility of successful implantation (Lessey et al., 1992; Gonzalez et al., 1999, 2001b). The observations that IL-1ß (De los Santos et al., 1996) and leptin (Gonzalez et al., 2000b) secretion are themselves regulated in embryos co-cultured with EEC, led us to propose that the actions of IL-1ß and leptin could be related at the time of implantation. Leptin produced and secreted locally by preimplantation embryos and EEC could act in an autocrine or paracrine manner to regulate biological functions that may mediate the implantation process (Gonzalez et al., 2000a,b).
Leptin seems to be a more potent stimulator of ß3 integrin expression by human EEC than IL-1ß at similar concentrations. Interestingly, in EEC cultures, IL-1ß up-regulates secretion of leptin and expression of both leptin and Ob-R. No IL-1ß effect was found on leptin or Ob-R in ESC cultures (Gonzalez and Leavis, 2001).
IL-1ß up-regulates the synthesis of IL-1ß and IL-1Ra mRNA, down-regulates IL-1R tI mRNA (Huang et al., 2001b) and negatively affects proliferation and decidualization of ESC (Kariya et al., 1991; Frank et al., 1995). IL-1 also stimulates production of TNF- by EEC that in turn leads to the inhibition of ESC decidualization (Laird et al., 1996). Thus, it seems that IL-1 could play a dual role in human implantation by promoting expression of markers of endometrial receptivity in EEC and by disrupting blastocyst implantation through the inhibition of ESC decidualization.
Leptin exhibits regulatory effects on the IL-1 system in other cell types, i.e. IL-1Ra in human monocytes (Gabay et al., 2001) and IL-1ß mRNA in mouse glial cells (Hosoi et al., 2000). The principal biological function of IL-1Ra is to regulate IL-1ß action (Hannum et al., 1990). An appropriate ratio of IL-1ß to IL-1Ra might favour the process of embryo implantation (Tanaka et al., 2000; Huang et al., 2001b). Conflicting data have been reported on IL-1Ra disruption of blastocyst implantation capabilities (Simon et al., 1994; Abbondanzo et al., 1996; Stewart and Cullinan, 1997). Results from the present investigation show that leptin can overcome the IL-1R tI blockade by significant up-regulation of the IL-1 system. These results raise the possibility that leptin regulation of IL-1 system expression at the endometrial level can diminish to some extent the consequences of IL-1R tI blockade by IL-1Ra.
In addition, we found that leptin and IL-1ß up-regulation of ß3 integrin by EEC was negatively affected by the addition of Ob-R antibody. These data reinforce the possibility that IL-1ß effects on endometrial receptivity may involve the action of the leptin system.
Leptin could also be involved in endometrial inflammatory disorders. Leptin up-regulation of the IL-1 system found in the present investigation and IL-1ß up-regulation of leptin and its receptor in EEC (Gonzalez and Leavis, 2001) could also be related to the up-regulation of leptin (protein and mRNA) found in endometriotic lesions (Wu et al., 2002) where IL-1, IL-6 and TNF- secretion are significantly higher (Bergqvist et al., 2001). Moreover, leptin stimulates its own mRNA expression in ectopic but not in eutopic ESC, indicating the different biochemical natures of these cells (Wu et al., 2002).
Although endometriosis is the most common gynaecological disorder, relatively little is known regarding its aetiology (Oral et al., 1996; Vinatier et al., 1996). One might wonder whether the abnormal production of leptin by endometrial cells could be a biochemical cause of endometriosis. It is also probable that over-secretion of leptin by endometriotic cells significantly contributes to the progression of endometriosis by up-regulation of the secretion of inflammatory and angiogenic cytokines, and matrix metalloproteinases. Consequently, we hypothesize that the blockade of Ob-R expressed by ectopic endometrial cells might be a new strategy for the treatment of endometriosis.
On the other hand, a negative correlation between IL-1 and endometrial cancer differentiation has been reported. The over-expression of IL-1 in less differentiated tumours might contribute to their invasiveness and malignancy (Singer et al., 2002). Interestingly, leptin seems to be related to the incidence of endometrial cancer. Leptin levels are positively associated with endometrial cancer. However, it remains to be assessed whether leptin increase, as a consequence of obesity, plays a role in endometrial carcinogenesis or whether it is a simple correlate of obesity (Petridou et al., 2002).
In conclusion, the present data suggest that leptin regulates the IL-1 system in human endometrium. Leptin, acting in an autocrine/paracrine manner, may be an endometrial molecular mediator of the actions of IL-1ß on ß3 integrin up-regulation. These findings reinforce our hypothesis for a role of leptin in human embryo implantation and its relationships with IL-1 actions.
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Acknowledgement |
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This study was funded in part by Analytical Biotechnology Inc.
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