Molecular Human Reproduction, Vol. 6, No. 7, 618-626,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Ovarian hormones modulate monocyte chemotactic protein-1 expression in endometrial cells of women with endometriosis
1 Department of Obstetrics and Gynecology and 2 Department of Medicine, Faculty of Medicine, Laval University, Quebec, Canada
Abstract
Endometriosis, a frequent oestrogen-dependent disease believed to result from an aberrant proliferation of endometrial tissue outside the uterine cavity, is associated with an increased expression of monocyte chemotactic protein-1 (MCP-1) in the intrauterine endometrium. This makes it plausible that migrating endometrial cells are intrinsically able to initiate monocyte chemoattraction and activation, a phenomenon which has been consistently observed in the peritoneal cavity of patients and recently in their eutopic endometrium. To elucidate the mechanisms involved in the regulation of MCP-1 expression in eutopic endometrial cells, we studied the effects of ovarian hormones and found that oestradiol (10–9 and 10–8 mol/l) markedly increased MCP-1 mRNA steady-state levels and protein secretion by endometrial cells in response to interleukin-1ß (IL-1ß) (0.1 ng/ml). The IL-1ß-induced MCP-1 expression was even higher following pretreatment of cells with both oestradiol (10–9 mol/l) and progesterone (5x10–8 mol/l). This did not seem to be due to increased MCP-1 mRNA stability, but rather to a higher level of gene transcription. Our results provide evidence that ovarian steroids regulate, indirectly, the synthesis and the secretion of a potent chemotactic and activating factor for monocytes/macrophages by endometrial cells of women with endometriosis and reveal a new mechanism for oestradiol action.
endometriosis/endometrium/oestradiol/MCP-1
Introduction
Endometriosis, the proliferation of endometrial-like tissue outside the uterus, is a common gynaecological disease causing pelvic pain and infertility in ~10% of the female population (Strathy et al., 1982
). Endometriosis is an oestrogen-dependent disorder (Dizerega et al., 1980), associated with an immuno-inflammatory process observed both locally in the peritoneal cavity where endometriotic lesions are commonly found (Oral et al., 1996; McMaster et al., 1998) and eutopically, in the intrauterine endometrium (Ota et al., 1996; Tseng et al., 1996; Jolicoeur et al., 1998). It primarily affects women of reproductive age and occasionally is diagnosed in post-menopausal women with relatively high oestrogen concentrations (Meldrum, 1985; Gleicher et al., 1987). Oestradiol is thought to be the hormonal factor that plays a paramount role in the maintenance of endometriosis. The current medical treatment of the disease, which is actually based on the suppression of ovarian steroidogenesis and the creation of a hypo-oestrogenic milieu less favourable to the growth of endometriotic tissue (Barbieri and Ryan, 1981; Shaw, 1992; Lemay, 1993), has been shown to be associated with reduced peritoneal inflammation (Haney and Weinberg, 1988; Taketani et al., 1992; Leiva et al., 1993) and decreased antibody titres in vivo in surgically induced endometriosis (Homm et al., 1989) and in treated patients (El-Roeiy et al., 1988).
One of the most consistent biological changes reported and observed in women with endometriosis is that of macrophage activation and recruitment into the peritoneal cavity (Haney et al., 1981; Halme et al., 1983; Braun et al., 1994). Other studies revealed an increased activation of peripheral blood monocytes (Dmowski et al., 1994) and a greater number of macrophages in the endometrium of patients (Ota et al., 1996). Activated monocytes/macrophages secrete angiogenic (McLaren et al., 1996) and growth (Halme et al., 1988; Olive et al., 1991) factors which may promote ectopic development of endometrial cells. They also secrete numerous pro-inflammatory and embryotoxic substances (Braun et al., 1996; Falcone and Hemmings, 1996; Rana et al., 1996) that may contribute to endometriosis-associated infertility. We have previously found that both the peritoneal fluid and the peripheral blood of women with endometriosis contain increased amounts of monocyte chemotactic protein-1 (MCP-1) (Akoum et al., 1996a,b), a chemokine of which the major biological property known to date is that of monocyte chemoattraction and activation (Leonard and Yoshimura, 1990; Schall, 1991). MCP-1 may play an important role in monocyte/macrophage activation and recruitment into the peritoneal cavity of patients with endometriosis as well as in macrophage infiltration of eutopic endometrial tissue. This chemotactic factor may be secreted by mesothelial cells (Arici et al., 1997) or by many other cell types including endothelial cells and activated monocytes/macrophages (Leonard and Yoshimura, 1990; Schall, 1991). We have, however, found that ectopic endometrial cells abundantly secrete MCP-1 (Akoum et al., 1995) and that eutopic endometrial cells of women with endometriosis have the intrinsic faculty to secrete increased amounts of this factor in vitro (Akoum et al., 1995). We also observed an in-vivo up-regulation of MCP-1 expression in women with endometriosis that occurs, at the level of both protein and mRNA synthesis, in the intrauterine endometrium (Jolicoeur et al., 1998). This appeared throughout the menstrual cycle but became more pronounced during the secretory phase, indicating a possible hormonal influence. Hence, the objective of the present study was to investigate whether ovarian hormones may be involved in the control of MCP-1 expression by endometrial cells in endometriosis.
Materials and methods
Sources and handling of tissue
Endometrial biopsies used in this study were obtained from 16 women with endometriosis (Table I). These women have signed an informed consent for a research protocol approved by the Saint-Franciois d'Assise Hospital Ethics Committee on Human Research. They consulted for infertility and/or pelvic pain and were found to have endometriosis at the time of laparoscopy. No endometrial hyperplasia or neoplasia was present in the subjects and none had received any anti-inflammatory or hormonal medication during a period of at least 3 months before laparoscopy. Endometrial biopsies were obtained by aspiration with the use of a Pipelle (Unimar Inc., Prodimed, NeuillyEnTchelle, France). They were immediately placed at 4°C in sterile Hanks' balanced salt solution (HBSS) containing 100 U/ml penicillin, 100 µg/ml streptamycin, and 0.25 µg/ml amphotericin B (Gibco BRL, Burlington, Ontario, Canada) and transported to the laboratory.
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Tissue dissociation and cell culture
Endometrial tissue was minced into small pieces, then incubated with collagenase (Sigma Chemical Co., St Louis, MO, USA) to dissociate the epithelial glands from stromal fibroblast-like cells. Further separation was done by differential sedimentation and adhesion as previously reported (Akoum et al., 1995). Epithelial glands were dissociated with 0.05% trypsin/0.53 mmol/l EDTA (Gibco BRL) for 10 min at 37°C, 5% CO2, then cultured in 56 cm2 tissue culture dishes in Dulbecco's modified Eagle's medium-F12 (DMEM-F12) (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL), 10 µg/ml insulin (Sigma), 5 µg/ml transferrin (Sigma), 1% antibiotics–antimycotics (Gibco BRL). At confluence, the purity of primary endometrial epithelial cell cultures was verified morphologically by light microscopy, and immunocytochemically on parallel coverslip cultures as previously described (Akoum et al., 1995b
). No CD-45 positive leukocytes were detected in culture, and less than 1% contamination by stromal vimentin-positive or endothelial Factor VIII-positive cells was generally observed (data not shown).
Culture stimulation and MCP-1 synthesis
Epithelial cells from primary cultures were grown to confluence and sub-cultured (one passage) with a split ratio of 1:2 in 56 cm2 Petri dishes in Roswell Park Memorial Institute (RPMI) medium (Gibco BRL) containing 10% dextran-coated charcoal-treated FBS (FBS-DC), 10 µg/ml insulin, 5 µg/ml transferrin and 1% antibiotics–antimycotics. For stimulation by interleukin (IL)-1ß, cells were grown to confluence, rinsed three times with FBS-free medium, then incubated overnight with the latter before being exposed to different concentrations of IL-1ß (0–10 ng/ml) (Genzyme, Cambridge, MA, USA) in fresh FBS-free medium for varying periods of time (0–24 h). For treatment with ovarian steroids [progesterone: 4-pregnen-3, 20-dione; oestradiol: 1,3,5, (10)-oestratrien-3, 17ß-diol 3-benzoate] (Sigma), the culture medium was removed 2 days following cell passage and replaced with fresh medium containing different concentrations of hormones. Cells were maintained in culture for 7 to 8 days (until confluence) and the medium was changed every 2 days. At confluence, cells were washed three times with serum-free RPMI enriched with 1% ITS+ (insulin–transferrin–selenium–linoleic acid) (Becton–Dickinson Inc., Mississauga, Ontario, Canada) and incubation with hormones continued in this medium for 42 h. Finally cells were or were not exposed to IL-1ß, which was added in a fresh medium at a final concentration of 0.1 ng/ml. Six hours later, the culture supernatant was collected and kept in small aliquots at –80°C until use in the MCP-1 assay by enzyme-linked immunosorbent assay (ELISA), which has a detection limit of ~50 pg/ml (Akoum et al., 1996b). Cells were dissociated with trypsin/EDTA and kept at –80°C until use for Northern blot analysis. To determine the combined effect of oestradiol and progesterone, cells were first treated with oestradiol alone until confluence, then with progesterone and oestradiol together before stimulation or not with IL-1ß. In some experiments, cycloheximide (Sigma) was added to the cell culture at the same time of IL-1ß, at a final concentration of 100 µg/ml.
Northern blot analysis
Total RNA was extracted from cells with TRIzol reagent according to the manufacturer's instructions (Gibco BRL). RNA was size-fractionated by electrophoresis on 1% agarose gels containing 10% formaldehyde and transferred to a Hybond-N+ membrane (Amersham, Oakville, Ontario, Canada). The membrane was then dehydrated at 37°C for 30 min, prehybridized with a hybridization buffer comprised of 5xsodium saline citrate (SSC), 5xDenhardt's solution, 50 mmol/l NaH2PO4, 0.5% sodium dodecyl sulphate (SDS), 200 µg/ml salmon sperm DNA and 50% formamide, hybridized with 32P-labelled MCP-1 cDNA (ATCC, Rockville, MA, USA) in the hybridization buffer and washed with 1xSSC, 0.2xSSC and 0.1% SDS respectively, before being exposed to X-ray film (BioMax, Eastman Kodak, Rochester, NY, USA) at –80°C for ~18 h. Staining with ethidium bromide (Gibco BRL) and hybridization with a 28S cDNA probe (ATCC) were performed to ensure equal loading of RNA. Data were analysed as ratios of the density of the hybridization signals of MCP-1 to 28S rRNA, as determined by computer-assisted densitometry (BioImage, Visage 110s; Genomic Solutions Inc., Ann Arbor, MI, USA).
mRNA stability and half-life experiment
Cells were treated with hormones, as described earlier, and incubated with IL-1ß (0.1 ng/ml) for 6 h. Transcription was then stopped with actinomycin D (10 µg/ml) and cells were harvested after different times of incubation with actinomycin D and used for RNA extraction and Northern blot analysis.
Nuclear run-on assay
Cell culture and treatment with hormones and IL-1ß were performed as mentioned above. At the end of the treatment, cells were scraped in a lysis buffer containing 0.25 mol/l sucrose, 10 mmol/l HEPES pH 8.0, 10 mmol/l MgCl2, 2 mmol/l dithiothreitol (DTT) and 0.1% (v/v) Triton X-100, and homogenized on ice in a Dounce Homogenizer. Nuclei were isolated by centrifugation at 600 g for 5 min at 4°C, washed twice by homogenization in fresh buffer, collected by centrifugation and stored in 80 µl of glycerol storage buffer [50 mmol/l HEPES pH 8,0, 40% (v/v) glycerol, 5 mmol/l MgCl2, 0.1 mmol/l EDTA and mmol/l DTT]. For in-vitro transcription, nuclei were resuspended in 200 µl of reaction buffer containing 20 mmol/l HEPES pH 8.0, 5 mmol/l MgCl2, 90 mmol/l NH4Cl, 0.5 mmol/l MnCl2, 16% (v/v) glycerol, 0.04 mmol/l EDTA, 2 mmol/l DTT, 0.4 mmol/l each of ATP, CTP, GTP (Gibco BRL), and 0.25 mCi of [
-32P]UTP (3000 Ci/mmol). The reaction was arrested by digestion with 100 µg/ml of RNase-free DNase I (Gibco BRL) and 100 µg/ml of proteinase K (Gibco BRL) in the presence of 10 mmol/l CaCl2 and 25 µg of yeast tRNA (Boehringer Mannheim, GmBH, Germany) for 20 min at 37°C, followed by the addition of EDTA (15 mmol/l) and SDS [0.5% (w/v)] and incubated for a further 20 min at 37°C. RNA was extracted twice with phenol/chloroform (1:1, v/v), precipitated overnight at –20°C with 100% ethanol (2:1, v/v) in the presence of 7.5 mol/l ammonium acetate (1:2, v/v) and collected by centrifugation at 12 000 r.p.m. at 4°C for 15 min. Enzyme digestion, phenol/chloroform extraction, and ethanol precipitation were repeated, RNA was again precipitated with ammonium acetate and ethanol, and finally dissolved in 850 µl of hybridization buffer containing 50 mmol/l PIPES pH 7.0, 0.5 mol/l NaCl, 2 mmol/l EDTA, 0.4% (w/v) SDS and 33% (v/v) formamide. Radioactive RNA was used to probe alkali-denatured plasmid DNA (5 µg) or insert DNA (1 µg) immobilized on nylon membranes using a slot blot apparatus (Hoefer, San Francisco, CA, USA). Hybridization was carried out for 3 days at 42°C using 5–10x106 c.p.m./ml of hybridization buffer. Membranes were washed four times with 2xSSC, 0.1% SDS at 65°C for 30 min, incubated with 10 µg/ml RNase A and 100 µg/ml proteinase K for 30 min at 37°C respectively, and washed twice again with 2xSSC, 0.1% SDS at 65°C for 30 min before being exposed to X-ray films (BioMax) at –80°C.
Statistical analysis
All experiments were repeated at least three times. Data were analysed using one-way analysis of variance (ANOVA), and the Tukey test was used post-hoc for multiple comparisons. P < 0.05 was considered statistically significant.
Results
The present study showed that IL-1ß stimulated both mRNA expression and protein secretion of MCP-1 in a dose- and time-dependent manner. Dose–response data depicted in Figure 1 revealed detectable stimulation of MCP-1 protein and mRNA expression at as low as 0.01 ng/ml IL-1ß for a 6 h period of treatment and a maximal stimulation at 1 ng/ml. MCP-1 protein concentrations and mRNA steady-state levels increased 2 h following stimulation with 0.1 ng/ml of IL-1ß and rose gradually during the 24 h of treatment (Figure 2). The addition of cycloheximide, a protein synthesis inhibitor, together with IL-1ß (0.1 ng/ml) completely abolished MCP-1 protein secretion while a super-induction of the steady-state levels of MCP-1 mRNA as compared to cells exposed to IL-1ß alone was observed (Figure 1). Thus, IL-1ß exerts a direct effect on endometrial epithelial cell steady-state mRNA expression that does not depend on de-novo protein synthesis.
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Effect of cycloheximide (100 µg/ml) on MCP-1 secretion (A) and mRNA steady-state levels (B) in response to IL-1ß (0.1 ng/ml).
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Treatment of endometrial epithelial cells with oestradiol (10–10 to 10–8 mol/l) or with progesterone (5x10–9 to 5x10–7 mol/l), had no significant effect on MCP-1 expression. MCP-1 protein secretion (Figure 3A
) and mRNA steady-state levels (Figure 3B) were either not modified or increased slightly in some cultures exposed to 10–9 or 10–8 mol/l oestradiol.
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In parallel experiments, we first treated endometrial cells with ovarian hormones, and, at the end of the culture, we exposed them to 0.1 ng/ml IL-1ß for 6 h. As illustrated in Figure 3C and D
, pretreatment with oestradiol (10–9 and 10–8 mol/l) enhanced both MCP-1 protein and mRNA steady-state levels by endometrial cells in response to IL-1ß. However, pretreatment with progesterone (5x10–9 to 5x10–7 mol/l) had no significant effect on the IL-1ß-induced MCP-1 expression by these cells.
To evaluate the combined effect of oestradiol and progesterone on MCP-1 expression, cells were treated with oestradiol alone until confluence, then with progesterone and oestradiol together before stimulation or not with IL-1ß. The results presented in Figure 4A and B show that without subsequent exposure to IL-1ß, simultaneous treatment of cells with oestradiol and progesterone, both used at physiological concentrations of 10–9 and 5x10–8 mol/l respectively, did not significantly affect MCP-1 expression as compared to control. However, a marked increase in MCP-1 protein and mRNA steady-state levels following treatment with oestradiol and stimulation with IL-1ß was observed (Figure 4C, D). Moreover, the combination of oestradiol and progesterone caused even greater, although not statistically significant, increase in MCP-1 expression as compared to oestradiol alone (Figure 4C, D). This, curiously, was perceptible only when progesterone was added after confluence to oestradiol-treated cells (Figure 4D). In addition, cell responsiveness to ovarian hormones did not appear to depend on the cycle phase where endometrial tissues were collected. Data from four independent experiments were analysed. MCP-1 mRNA steady-state levels, expressed as the ratio of MCP-1 hybridization signal density to that of 28S, increased by 174 ± 65% over control (cells stimulated only with IL-1ß) (P < 0.01) in oestradiol-treated cells, 212 ± 52% (P < 0.01) after the addition of progesterone to oestradiol-treated cells and by 19 ± 14% (P > 0.05) in cells treated with progesterone alone. For MCP-1 secretion, the increase over control was generally less obvious than that of mRNA steady-state levels, but statistically significant for oestradiol (55 ± 9%) (P < 0.01) and for oestradiol + progesterone (73 ± 10%) (P < 0.01), while for progesterone (8 ± 5%) no significant effect was noted.
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To determine whether the up-regulation of MCP-1 mRNA steady-state levels induced by the ovarian hormones in endometrial cells in response to IL-1ß was exerted at the transcriptional or post-transcriptional level, or both, we evaluated MCP-1 mRNA stability and nuclear transcription in cells pretreated and not with oestradiol (10–9 mol/l) and progesterone (5x10–8 mol/l) prior to stimulation with 0.1 ng/ml IL-1ß for 6 h as previously described. As illustrated in Figure 5
, treatment with ovarian hormones had no detectable effect on MCP-1 mRNA kinetics of degradation following arrest of de-novo RNA transcription by actinomycin D (10 µg/ml). However, as shown by nuclear run-on analysis, the level of MCP-1 transcription in cells exposed to ovarian hormones prior to stimulation with IL-1ß was markedly higher (2.9 ± 0.7; mean ± SD of three independent experiments) than that in cells maintained without previous hormonal treatment (Figure 6), suggesting transcriptional regulation of MCP-1 expression by ovarian hormones.
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Discussion
In the present study we report that oestradiol enhances the responsiveness of endometrial epithelial cells to IL-1ß in women with endometriosis. The oestradiol treatment, which by itself had no significant effect on MCP-1 expression, amplified IL-1ß stimulatory action, providing first-time evidence that oestradiol can indirectly up-regulate the synthesis and the secretion of a potent monocyte chemoattractant and activating factor for monocytes/macrophages by endometrial epithelial cells of women with endometriosis. These findings also reveal a synergistic stimulatory action between oestradiol and the pro-inflammatory cytokine IL-1ß exerted at the level of eutopic endometrial cell in endometriosis, and demonstrate a new mode of action for this ovarian steroid.
Treatment with progesterone did not result in any detectable effects on MCP-1 expression by endometrial cells, with or without subsequent stimulation with IL-1ß. The addition of progesterone to oestradiol-treated cultures prior to stimulation with IL-1ß engendered nevertheless a further, although not statistically significant, increase in MCP-1 expression as compared to oestradiol alone. These in-vitro data are consistent with our previous in-vivo observations of a cycle-dependent expression of MCP-1 in the endometrial glands of women with endometriosis, which was present throughout the menstrual cycle of patients, but more apparent during the secretory phase (Jolicoeur et al., 1998).
To date, the interactions between steroid hormones and the components of the immune system, and their implications in the pathophysiology of endometriosis have been poorly investigated. Oestradiol and progesterone have been shown to stimulate vascular endothelial growth factor (VEGF) production by non-activated and activated peritoneal macrophages which are more frequently found in patients with endometriosis (McLaren et al., 1996). They also have been shown to up-regulate VEGF expression by endometrial stromal cells of women with endometriosis (Shifren et al., 1996). In these studies, the ovarian hormones appeared, however, to exert direct regulatory action. According to recent data, oestradiol could be unusually produced in the endometrium of endometriosis patients, as P450 aromatase, which catalyses the conversion of C19 steroids to oestrogens, was found to be abnormally expressed in this tissue during the course of the disease (Noble et al., 1996). This suggests that oestradiol may contribute to the up-regulation of MCP-1 expression in the endometrial tissue of women with endometriosis not only by an endocrine pathway, but also by a paracrine mechanism. These findings may have an interesting significance, as the main biological property of MCP-1 is that of monocyte activation and recruitment into the site of inflammation (Leonard and Yoshimura, 1990; Schall, 1991). The intrinsic property of endometrial cells to secrete increased levels of MCP-1 in endometriosis (Akoum et al., 1995), and the ability of oestradiol to augment their responsiveness to the pro-inflammatory cytokine IL-1ß by amplifying MCP-1 synthesis and secretion, strongly suggest that these cells, which may be spilled into the peritoneal cavity, have the potential to induce macrophage activation and recruitment and to initiate the local inflammatory process taking place in the disease. Interestingly, increased infiltration of monocytes has been observed in the eutopic endometrium of women with endometriosis (Ota et al., 1996), making plausible an involvement of MCP-1 in the enhanced monocyte recruitment into the endometrial tissue.
It is known that synthesis of multiple cytokines that may participate in the normal physiology of the endometrial immunological system takes place in the endometrium (Tabibzadeh, 1991). The pattern of leukocyte infiltration in this tissue suggests that hormonal factors are involved in regulating cytokine expression and leukocyte traffic (Tabibzadeh, 1991). Interestingly, it has been reported that IL-8 expression by endometrial stromal cells is up-regulated by progesterone (Arici et al., 1996). RANTES, another chemokine that is a chemoattractant and activator of lymphocytes and monocytes, was also found to be expressed in normal human endometrium and in a manner dependent on the menstrual cycle phase (Hornung et al., 1997). The present study provides evidence supporting a synergistic relationship between ovarian steroids and a pro-inflammatory cytokine in the regulation of MCP-1 synthesis and secretion by endometrial epithelial cells in endometriosis. It remains to be seen whether a similar regulatory mechanism operates for the endometrial cells of normal women.
The findings of the present study are of additional interest as they extend our recently reported data regarding ectopic endometrial cell responsiveness to ovarian hormones (Akoum et al., 2000). Like eutopic endometrial cells, ectopic cells responded to oestradiol by increasing IL-1-induced MCP-1 expression, but, in contrast, they showed no noticeable responsiveness to progesterone either alone, or in combination with oestradiol. These results are consistent with the absence of cyclic variation in MCP-1 expression in endometriosis lesions (Akoum et al., 2000) as compared to intrauterine endometrium (Jolicoeur et al., 1998), and suggest a reduced sensitivity to progesterone in ectopic endometrial cells, which probably could be ascribed to the decreased progesterone receptors observed in endometriosis tissues (Bergqvist and Ferno, 1993). This illustrates the complexity of MCP-1 regulation in endometriosis, and provides a new insight into the debate regarding biochemical and functional differences between intrauterine endometrial cells and their ectopic counterparts.
The mechanisms by which oestradiol, alone or associated with progesterone, enhance MCP-1 expression by endometrial cells in response to IL-1ß remain unclear. It is well documented that the induction of MCP-1 gene transcription by IL-1ß involves transcriptional factors such as AP1, and particularly NF
B, which according to a recent study is essential for IL-1-induced MCP-1 gene transcriptional activity (Ueda et al., 1994). A putative oestrogen-responsive element (ERE), where an oestradiol/oestradiol receptor complex could bind and trigger gene transcription, has yet to be reported within the MCP-1 gene promoter sequence. Even if such a cis-acting element existed, oestradiol treatment without subsequent exposure to IL-1ß showed no significant effect on MCP-1 mRNA synthesis. This makes unlikely any direct mechanism involving oestradiol receptor binding to the MCP-1 gene regulatory region. On the other hand, nuclear run-on analyses showed that ovarian hormones enhanced IL-1ß-induced transcription of the MCP-1. This suggests a mechanism by which oestradiol, alone or combined with progesterone, may activate a target gene whose products in turn may interact with IL-1ß-induced transcription signals.
In summary, the results of the present study show that oestradiol up-regulates, although indirectly, the expression of a potent chemotactic and activating factor for monocytes by endometrial epithelial cells of women with endometriosis. Oestradiol enhanced the responsiveness of these cells to the pro-inflammatory cytokine IL-1ß by amplifying IL-1ß-induced MCP-1 production. These findings could be of considerable significance in view of the biological properties of MCP-1 whose levels are elevated in the serum, the peritoneal fluid and the eutopic endometrium of women with endometriosis, and in view of the paramount role attributed to oestradiol in the pathophysiology of endometriosis. In addition, we demonstrate, for the first time, a hormonal regulation of MCP-1 expression in the endometrium of women with endometriosis and reveal a novel interaction between the endocrine and the immune system. Further investigations are needed to elucidate the mechanisms underlying oestradiol stimulatory action on MCP-1 expression and the effect of anti-oestrogens on that expression, which may be of great therapeutic interest and potential.
Acknowledgments
The authors wish to thank the group of investigation in gynecology (Drs Jacques Bergeron, Simon Carrier, Jean-Yves Fontaine, Céline Huot, Johanne Hurtubise and Rodolphe Maheux) for patient evaluation and providing endometrial biopsies, Monique Longpré and Johanne Pelletier for technical assistance, Dr Lucile Turcot-Lemay for statistical analysis, Christine Jolicoeur for technical assistance and Dr Mahéra Al-Akoum for the critical review of the manuscript. Ali Akoum is a `Chercheur-Boursier Senior' of the `Fonds de la Recherche en Santé du Québec (FRSQ)'. This research was supported by grant MT-14638 to AA from the Medical Research Council of Canada.
Notes
3 To whom correspondence should be addressed at: Laboratoire d'Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-Franciois d'Assise, 10 rue de l'Espinay, Local D0-711, Québec, Canada, G1L 3L5. E-mail: ali.akoum{at}crsfa.ulaval.ca 
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Submitted on December 6, 1999; accepted on May 4, 2000.
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