Journal of Clinical Endocrinology and Metabolism
Volume 85 • Number 2 • February 1, 2000
Copyright © 2000 The Endocrine Society

 Dr.Sinan DOĞANTÜRK

ANKARA                                                                                                                     896

Original Studies

Estradiol Amplifies Interleukin-1-Induced Monocyte Chemotactic Protein-1 Expression by Ectopic Endometrial Cells of Women with Endometriosis ^*

 

ALI AKOUM ^{1 2}

CHRISTINE JOLICOEUR ¹

ANNIE BOUCHER <sup>1

1</sup> Laboratoire d'Endocrinologie de la Reproduction, Centre de Recherche, Pavillon Saint-Francois d'Assise, Centre Hospitalier Universitaire de Quebec, Universite Laval, Quebec, Canada G1L 3L5
² Chercheur-Boursier Senior of the Fonds de la Recherche en Sant&eacute; du Qu&eacute;bec.

Received August 2, 1999.
Revision received October 18, 1999.
Accepted October 22, 1999.

Address all correspondence and requests for reprints to: Ali Akoum, Ph.D., Laboratoire d'Endocrinologie de la Reproduction, Centre de Recherche, Hopital Saint-Francois d'Assise, 10 rue de l'Espinay, Local D0-711, Quebec, Canada G1L 3L5. E-mail: ali.akoum@ crsfa.ulaval.ca.

Copyright © 2000 by The Endocrine Society

 

ABSTRACT

Endometriosis, one of the most frequently occurring gynecological disorders, is estrogen dependent and is often associated with immunological changes. These include increased macrophage activation and infiltration into the endometriotic implants themselves as well as the peritoneal cavity where the implants often develop. Despite the critical role estrogens play in the development of endometriosis, the biochemical mechanisms of their action remain unclear. In the present study we report that estradiol (E₂ ) enhances endometriotic cell responsiveness to the proinflammatory cytokine interleukin-1beta by up-regulating interleukin-1-induced monocyte chemotactic protein-1 (MCP-1) expression at the level of both protein secretion and messenger ribonucleic acid (mRNA) synthesis, whereas progesterone had no significant effects. According to mRNA half-life experiments, E₂ action does not seem to be due to increased MCP-1 mRNA stability but, rather, to a higher level of transcription, as shown by run-on analysis. Interestingly, immunohistochemical analysis of MCP-1 expression in endometriotic tissue showed intense immunostaining in both epithelial glands and stroma regardless of the menstrual cycle phase, which is consistent with the cell culture data and indicates that MCP-1 expression is not subject to cyclic variation. The findings of the present study for the first time provide evidence that E₂ up-regulates, although in an indirect way, the expression of a potent chemotactic and activating factor by ectopic endometrial cells, which may occur locally in the inflammatory site and contribute to peritoneal macrophage recruitment and activation, and reveal a new means of E₂ action in the pathophysiology of endometriosis. ( J Clin Endocrinol Metab 85: 896-904, 2000)

 

ENDOMETRIOSIS is a common gynecological disorder, affecting from 5-15% of women of reproductive age ^[1] , and is characterized by the presence of endometrial-like tissue outside the uterus, mainly in the peritoneal cavity. The disease is frequently associated with abdominal pain, dysmenorrhea, dyspareunia, and infertility, causing profound physical and psychological distress ^[2] .

Endometriosis is known to be estrogen dependent. The disease usually arises at menarche and most often regresses spontaneously after menopause. However, it has been occasionally diagnosed in postmenopausal women with relatively high levels of estrogens ^{[3] [4] [5]} . In a primate model, surgically induced endometriosis persisted only in the presence of ovarian steroids ^[3] . Histological studies of endometriosis lesions during the menstrual cycle indicated that ectopic glands did not fully demonstrate the classic endocrine-mediated changes that manifest eutopic endometrium ^[6] . Nevertheless, endometriotic lesions express sex steroid receptors and can undergo cyclical changes similar, but not identical, to those of the intrauterine endometrium ^[7] . Moreover, current medical treatment of endometriosis is based on the suppression of ovarian function and the induction of hypoestrogenism ^{[8] [9] [10]} , which, although often resulting in temporary involution of the disease, have been shown to reduce symptomatology and to reduce endometriotic lesion volume ^{[10] [11]} .

According to numerous studies endometriosis is associated with immunological changes that were detected in the eutopic endometrium and the peripheral blood (reviewed in Refs. ^{[12] [13] [14]} ) but, in particular, were observed locally in the peritoneal cavity where the disease often develops. The peritoneal fluid of endometriosis patients was shown to contain elevated levels of autoantibodies ^{[4] [15]} and proinflammatory mediators ^{[16] [17] [18] [19] [20]} and to present increased infiltration by inflammatory cells ^[21] , especially activated macrophages ^{[20] [22]} . Macrophages are the predominant nucleated cells in the peritoneal fluid and represent the first line host response to inflammatory stimuli. However, in endometriosis, instead of favoring the elimination of misplaced endometrial cells, peritoneal macrophages may paradoxically contribute to their maintenance and growth by producing growth and angiogenic factors ^{[23] [24] [25]} and have a deleterious effect on the reproductive process through a direct cell-mediated damage or the release of embryotoxic substances ^{[16] [17] [26] [27]} .

Ectopic endometrial implants may be a major source of the peritoneal inflammation seen in patients with endometriosis. This may be triggered by in situ menstruation and breakthrough bleeding ^[28] , but also by numerous secretions, such as PGs ^[18] , complement components ^[19] , and proinflammatory cytokines ^{[29] [30] [31] [32]} , which may alter the peritoneal environment and contribute to endometriosis-associated pain and infertility ^{[16] [17] [20] [22] [27] [28] [33]} .

Using cell culture, we have recently been able to demonstrate

^* This research was supported by Grant MT-14638 (to A.A.) from the Medical Research Council of Canada.

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that eutopic endometrial cells of women with endometriosis have the intrinsic faculty to secrete increased amounts of monocyte chemotactic protein-1 (MCP-1) compared to those from normal women ^[34] and found that ectopic endometrial cells also abundantly secrete this factor ^[35] . Subsequently, we found that MCP-1 concentrations were elevated in the peritoneal fluid of patients with endometriosis, particularly in the mild but most active stages of the disease ^[36] . MCP-1, a chemokine with potent chemotactic and activation properties toward monocytes ^{[37] [38]} , could therefore play a key role in macrophage activation and their recruitment into the peritoneal cavity of patients with endometriosis and may also contribute to the infiltration of endometriotic implants by macrophages reported by many investigators ^[39] .

Despite the undeniable role of estrogens in the maintenance and progression of endometriosis, the biochemical mechanisms of their action in the disease and their possible link to the immunoinflammatory process occurring in women with endometriosis have been poorly investigated. In the present study we report that estradiol (E₂ ) enhances the responsiveness of ectopic endometrial cells to the proinflammatory cytokine interleukin-1beta (IL-1beta) by amplifying IL-1betainduced MCP-1 expression at the level of both messenger ribonucleic acid (mRNA) synthesis and protein secretion, and we demonstrate that this effect is exerted at the transcriptional level.

 

Materials and Methods

Source and handling of tissue

Tissue specimens used in this study ( Table 1 ) were obtained from women who signed a written informed consent for a research protocol approved by the Saint-Francois d'Assise Hospital ethics committee on human research. These women (mean age ± SD, 36 ± 5 yr; n = 13), consulting for infertility and/or pelvic pain, were found to have endometriosis during laparoscopy or laparotomy, had no endometrial hyperplasia or neoplasia, and had not received any antiinflammatory or hormonal medication during a period of at least 3 months before surgery. Endometriosis was staged during the operation according to the revised American Fertility Society classification system ^[40] . The cycle phase [proliferative (PF) or secretory (SR)] was determined according to the cycle history of patients and to the serum progesterone (P) levels measured. Endometriotic tissues were obtained from ovarian endometriomas (n = 6, 3 PF and 3 SR) and peritoneal foci (n = 7, 3 PF and 4 SR). Endometriotic biopsies were immediately placed at 4 C in sterile Hanks' Balanced Salt Solution containing 100 U/mL penicillin, 100

µg/mL streptomycin, and 0.25 µg/mL amphotericin; snap-frozen in liquid nitrogen with Tissue-Tek OCT compound (Miles, Inc., Elkhart, IN); and stored at -80 C until analyzed by immunohistochemistry or immediately used for cell culture experiments. Immunohistochemical analyses were performed on 12 of the 13 endometriotic tissue biopsies. However, for cell culture experiments, sufficient tissue was available from only 5 ovarian endometrioma cyst linings and 3 peritoneal endometriotic biopsies.

Immunohistochemistry

Serial 4- to 5-µm cryosections were first fixed in 4% formaldehyde solution (Fisher Scientific, Pittsburgh, PA) got 20 min at room temperature, then permeabilized with Triton X-100 (1% in phosphate-buffered saline for 20 min at room temperature) and treated with 0.3% H₂ O₂ in absolute methanol for 20 min at room temperature to eliminate endogenous peroxidase. Immunostaining was performed using a monoclonal mouse anti-MCP-1 antibody (10 µg/mL in phosphate-buffered saline containing 1% BSA; R&D Systems, Minneapolis, MN) and a Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA), diaminobenzidine (Sigma, St. Louis, MO) as the chromogen, and hematoxylin for counterstaining. The specificity of the immunoreactivity shown by the anti-MCP-1 antibody (primary antibody) was examined by preabsorption with an excess of MCP-1 (50 µg/mL) before incubation with endometriotic tissue sections. Sections incubated without the primary antibody or with mouse Ig of the same Ig class and concentration as the primary antibody were included as negative controls in all experiments. Endometriotic glands were identified using monoclonal antibodies specific to cytokeratins (AE1:AE3 Mix, ICN Biomedical, St. Laurent, Canada). Slides were viewed using a Leica Corp. microscope (model DMRB, Leica Corp.), and photomicrographs were taken with Kodak 100 ASA film (Eastman Kodak Co., Rochester, NY).

Tissue dissociation and cell culture

Endometriotic tissue was minced into small pieces and dissociated with collagenase as previously described ^[35] . Cells were pelleted by centrifugation (200 × g, 10 min); resuspended in DMEM-Ham's F-12 containing 10 µg/mL insulin, 5 µg/mL transferrin, and 10% FBS; and plated in 100-mm diameter culture dishes, then grown at 37 C in 5% carbon dioxide. In this study no attempt was made to separate epithelial cells from stromal fibroblast-like cells. These were identified morphologically in culture by light microscopy and immunocytochemically with specific monoclonal antibodies to cytokeratins and vimentin as previously described ^[35] . No leukocytes were detected in the endometriotic cells detached from culture dishes and assessed by flow cytometry (data not shown).

Culture stimulation and MCP-1 synthesis

Endometriotic cells grown to confluence were seeded at 20,000 cells/cm² in 25-cm² culture flasks in RPMI medium (Life Technologies, Inc., Gaithersburg, MD) containing 10% dextran-coated charcoal-treated FBS,

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10 µg/mL insulin, 5 µg/mL transferrin, and 1% antibiotics-antimycotics. For stimulation with IL-1beta, cells grown to confluence were incubated overnight with FBS-free medium before being exposed to different concentrations of IL-1beta (0-10 ng/mL; Genzyme Corp., Cambridge, MA) in a fresh FBS-free medium for varying periods of time (0-24 h). For treatment with ovarian steroids [P and 1,3,5^[10] -estratrien-3,17beta-diol 3-benzoate; Siigma], the culture medium was removed 2 days after cell passage and replaced with a fresh phenol red-free medium containing different concentrations of hormones. Cells were maintained in culture for 7-8 days (until confluence), and the medium was changed every 2 days. At confluence, cells were washed with serum-free RPMI enriched with 1% insulin-transferrin-selenium-linoleic acid (Becton Dickinson and Co., Mississauga, Canada), and incubation with hormones was continued in this medium for 42 h. Finally, cells were or were not exposed to IL-1beta, which was added to the culture medium to reach 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) as previously reported ^[36] , whereas cells were dissociated with trypsin/ethylenediamine tetraacetate (EDTA) and kept at -80 C until use for Northern blot analysis. To determine the combined effect of E₂ and P, cells were first treated with E₂ alone until confluence, then with P and E ₂ together before stimulation, or not, with IL-1beta. In some experiments, cycloheximide (Sigma) was added to the cell culture at the same time as IL-1beta, at a final concentration 100 µg/mL.

Northern blot analysis

Total RNA was extracted from cells with Trizol reagent according to the manufacturer's instructions (Life Technologies, Inc.). RNA was size fractionated by electrophoresis on 1% agarose gels containing 10% formaldehyde and transferred to a Hybond-N⁺ membrane (Amersham Pharmacia Biotech, Oakville, Canada). The membrane was then dehydrated at 37 C for 30 min; prehybridized with a hybridization buffer comprised of 5 × SSC (standard saline citrate)-5 × Denhardt's solution, 50 mmol/L NaH₂ PO₄ , 0.5% SDS, 200 µg/mL salmon sperm DNA and 50% formamide; hybridized with ³² P-labeled MCP-1 complementary DNA (cDNA; American Type Culture Collection, Manassas, VA) in the hybridization buffer; and washed with 1 × SSC, 0.2 × SSC, and 0.1% SDS, respectively, before being exposed to x-ray film (BioMax, Eastman Kodak Co.) at -80 C for about 18 h. Staining with ethidium bromide (Life Technologies, Inc.) and hybridization with 28S cDNA probe (American Type Culture Collection, Manassas, VA) were performed to ensure equal loading of RNA. Data were analyzed as ratios of the density of the hybridization signals of MCP-1 to 28S, as determined by computer-assisted densitometry (BioImage, Visage 110s, Genomic Solutions, Inc., Ann Arbor, MI).

mRNA stability and half-life experiments

Cells were treated with hormones, as described above, and incubated with IL-1beta (0.1 or 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 for RNA extraction and Northern blot analysis.

Nuclear run-on assay

Cell culture and treatment with hormones and IL-1beta 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% (vol/vol) Triton X-100, and homogenized in a Dounce homogenizer (Kontes Co., Vineland, NJ) on ice. 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 glycerol storage buffer [50 mmol/L HEPES (pH 8.0), 40% (vol/vol) glycerol, 5 mmol/L MgCl₂ , 0.1 mmol/L EDTA, and 2 mmol/L DTT]. For in vitro transcription, nuclei were resuspended in 200 µL reaction buffer containing 20 mmol/L HEPES (pH 8.0); 5 mmol/L MgCl₂ ; 90 mmol/L NH₄ Cl; 0.5 mmol/L MnCl₂ ; 16% (vol/vol) glycerol; 0.04 mmol/L EDTA; 2 mmol/L DTT; 0.4 mmol/L each of ATP, CTP, GTP (Life Technologies, Inc.); and 0.25 mCi [alpha-³² P]UTP (3000 Ci/mmol). The reaction was arrested by digestion with 100 µg/mL ribonuclease-free deoxyribonuclease I (Life Technologies, Inc.) and 100 µg/mL proteinase K (Life Technologies, Inc.) in the presence of 10 mmol/L CaCl₂ and 25 µg yeast transfer RNA (Roche, Mannheim, Germany) for 20 min at 37 C, followed by the addition of EDTA (15 mmol/L) and SDS (0.5%, wt/vol) and a further incubation for 20 min at 37 C. RNA was extracted twice with phenol/chloroform (1:1, vol/vol), precipitated overnight at -20 C with 100% ethanol (2:1, vol/vol) in the presence of 7.5 mol/L ammonium acetate (1:2, vol/vol), and collected by centrifugation at 12,000 rpm at 4 C for 15 min. The process of enzyme digestion, phenol/chloroform extraction, and ethanol precipitation was repeated; the RNA was precipitated again with ammonium acetate and ethanol and finally dissolved in 850 µL hybridization buffer containing 50 mmol/L PIPES (pH 7.0), 0.5 mol/L NaCl, 2 mmol/L EDTA, 0.4% (wt/vol) SDS, and 33% (vol/vol) formamide. The 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). Hybridization was carried out for 3 days at 42 C using 5-10 million cpm/mL hybridization buffer. Membranes were washed four times with 2 × SSC-0.1% SDS at 65 C for 30 min, incubated with 10 µg/mL ribonuclease A, and 100 µg/mL proteinase K for 30 min at 37 C, respectively, and washed twice again with 2 × SSC-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 analyzed using one-way ANOVA; and the Tukey test was used post-hoc for multiple comparisons. Differences were considered as statistically significant for P < 0.05.

 

Results

MCP-1 expression in endometriotic lesions

In the intrauterine endometrium, MCP-1 is expressed mostly in the glandular epithelium in the secretory phase of the menstrual cycle ^[41] . Surprisingly, this was not the case in ectopic endometrium. Intense MCP-1 immunoreactivity was localized in both endometriotic glands and stroma ( Fig. 1 ). A similar pattern of immunostaining was observed whether the tissue sections were from the inner lining of ovarian endometriomas ( Fig. 1A ) or from peritoneal endometriotic foci ( Fig. 1B ), albeit it was relatively less intense in the two white lesions included in the study. Furthermore, no apparent difference related to the menstrual cycle phase was noted. The epithelial glands of endometriotic lesions were identified because they reacted positively with mouse monoclonal antibodies specific to cytokeratins, whereas stromal cells did not show any positive immunostaining for these epithelial cell-specific markers (data not shown). Control experiments performed on serial sections of endometriotic tissue verified the specificity of the immunolocalization of MCP-1, as there was no noticeable immunostaining with normal mouse Igs (C and D), with the primary antibody after preabsorption with an excess of MCP-1, or when the primary antibody was omitted (data not shown).

Effects of IL-1beta and ovarian hormones on MCP-1 expression

Using an established protocol, endometriotic stromal and epithelial cells were cultured without any attempt of separation to preserve stromal/epithelial cell interactions and to evaluate the whole outcome of hormone treatment. According to previous investigations, differentiation and hormonal responsiveness of epithelial cells in vitro are dependent on an appropriate stromal environment ^{[42] [43]} .

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Figure 1. Immunohistochemical localization of MCP-1 in endometriosis lesions. Sections from the inner wall tissue of an ovarian endometrioma, endometriosis stage IV, proliferative phase, day 2 (patient 2, Table 1; A ), or from a peritoneal endometriotic implant, red vesicle, endometriosis stage II, secretory phase, day 20 (patient 6, Table 1; B ) were incubated with mouse monoclonal anti-MCP-1 antibody or with equivalent concentrations of normal mouse IgGs (C and D). Note the intense brown positive immunostaining in endometriotic glands and stroma (A and B) and the absence of staining in control sections (C and D).

IL-1 is one of the major proinflammatory cytokines found in elevated concentrations in the peritoneal fluid of patients with endometriosis ^{[16] [17]} . The protein is mainly secreted by activated macrophages, which are more numerous in the peritoneal fluid of patients and show a marked infiltration of endometriosis implants compared to eutopic endometrium ^[39] . In vitro experiments of the present study showed that without stimulation, cultured endometriotic cells did not express detectable amounts of MCP-1 mRNA by Northern blot. However, the addition of IL-1beta to the culture medium induced both MCP-1 mRNA and protein expression by endometriotic cells in a dose- and time-dependent manner. The dose-response data depicted in Fig. 2 showed detectable stimulation of MCP-1 mRNA and protein expression at as low as 0.01 ng/mL IL-1beta for a 6-h period of treatment and a maximal stimulation at 1 ng/mL. The levels of protein secretion increased 2 h after stimulation with 0.1 ng/mL IL-1beta and rose exponentially during the 24 h of treatment, whereas the highest level of mRNA was found after 6 h of stimulation and dropped afterward over time ( Fig. 3 ). The addition of cycloheximide, an inhibitor of protein synthesis, together with IL-1beta (0.1 ng/mL) completely abolished MCP-1 protein secretion, whereas a superinduction of the steady state levels of MCP-1 mRNA compared to those in cells exposed to IL-1beta alone was observed ( Fig. 2 ). Thus, IL-1beta exerts a direct effect on endometriotic cell steady state mRNA expression that does not necessarily require de novo protein synthesis.

As indicated earlier, ovarian function is crucial to the development and the progression of endometriosis. According to previous investigations, both stromal and epithelial cells from endometriotic tissues express estrogen and P receptors ^{[44] [45]} . To investigate whether ovarian hormones may be involved in the regulation of MCP-1 expression by endometriotic lesions, we used an established long term cell culture treatment protocol in which endometriotic cells were

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Figure 2. Dose-dependent expression of MCP-1 by IL-1beta-treated endometriotic cells. Confluent cultures were exposed to different concentrations of IL-1beta (0-10 ng/mL). Six hours later, cells and culture supernatants were recovered to evaluate MCP-1 mRNA by Northern blot and protein by ELISA. A, MCP-1 protein secretion (pg/mL). Values are the mean ± SEM of duplicate determinations in cultures from three different endometriotic lesions (patients 2, 7, and 8; Table 1 ). ^* and ^** , Significantly different from control ( P < 0.05 and P < 0.01, respectively); B, a representative autoradiogram showing MCP-1 mRNA steady state levels, and 28S ribosomal RNA demonstrating approximately equal RNA loading (patient 8, Table 1 ). , Effect of cycloheximide (100 µg/mL) on MCP-1 secretion (A) and mRNA steady state levels (B) in response to IL-1beta (0.1 ng/mL).

exposed first to estradiol at the start of culture, then to combined estradiol and progesterone after cell confluence. The ovarian steroid doses (10 ^-8 mol/L for E₂ and 10^-6 mol/L for P) used in this study were selected on the basis of previous reports showing that ovarian steroid concentrations in the peritoneal fluid are more elevated than those normally found in the peripheral blood ^{[23] [46]} . The results illustrated in Fig. 4 show that endometriotic cell treatment with E₂ and/or P had no significant effect on MCP-1 protein secretion ( Fig. 4A ) and mRNA steady state levels ( Fig. 4B ). Only MCP-1 protein secretion increased slightly in some cultures exposed to E ₂ alone or in combination with P ( Fig. 4A ).

In parallel experiments, we first treated endometriotic cells with ovarian hormones, and at the end of the culture, we exposed them to 0.1 ng/mL IL-1beta for 6 h. The suboptimal concentration of IL-1beta was used to detect any possible stimulatory or inhibitory effect of the reproductive steroids. As illustrated in Fig. 4, C and D , treatment with E₂ enhanced MCP-1 protein secretion and mRNA steady state levels by endometriotic cells in response to IL-1beta. However, treatment of endometriotic cells with P, either alone or in combination with E ₂ , had no significant effect on the IL-1beta-induced

Figure 3. Time course of MCP-1 expression by endometriotic cells stimulated with IL-1beta. Confluent cultures were treated with IL-1beta (0.1 ng/mL) or control medium for increasing periods of time. At each time point, the culture supernatant was recovered for MCP-1 protein secretion measurement by ELISA, and total cellular RNA was extracted to evaluate MCP-1 mRNA expression by Northern blot analysis. A, MCP-1 protein secretion (pg/mL). Values are the mean ± SEM of duplicate determinations in cultures from three different endometriotic lesions (patients 2, 7, and 8, Table 1 ). B, MCP-1 mRNA steady state levels. A representative autoradiogram showing hybridization of MCP-1 mRNA with ³² P-labeled MCP-1 cDNA probe (patient 7, Table 1 ). Hybridization with ³² P-labeled 28S cDNA probe demonstrated approximately equal RNA loading.

MCP-1 expression by these cells. Data from four independent experiments were analyzed. For MCP-1 secretion, the increase over the control value (cells stimulated only with IL-1beta), expressed as the mean ± SEM, was statistically significant for E₂ (65 ± 10%; P < 0.01) and E ₂ plus P (77 ± 8%; P < 0.01), whereas for P (-4 ± 3%) no significant effect was observed. MCP-1 concentrations in the conditioned medium ranged from 11.5-80.4 ng/mL in the control, and from 20.1-124.7, 21.4-136.5, and 10.3-75.4 ng/mL in the presence of E₂ , E₂ plus P, and P, respectively. For MCP-1 mRNA, steady state levels, expressed as the ratio of MCP-1 hybridization signal density to that of 28S, had increased by 201 ± 23% ( P < 0.01) after the addition of E₂ , by 223 ± 52% ( P < 0.01) after the addition of P to E₂ -treated cells, and by 16 ± 11% ( P > 0.05) after the addition of P alone. Similar results were obtained after simultaneous treatment with E₂ and P starting from the culture initiation or with P concentrations varying between 10^-8 -10^-6 mol/L (data not shown). A similar pattern of response was obtained whether cells were derived from ovarian cyst linings or from peritoneal endometriotic biopsies.

To determine whether the E₂ -mediated up-regulation of MCP-1 mRNA stteady state levels in endometriotic cells in

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Figure 4. Effect of E₂ (10^-8 mol/L) and P (10^-6 mol/L) on MCP-1 expression by endometriotic cells. Cells were treated with E₂ and/or P, as described in Materials and Methods without (A and B) or with (C and D) subsequent exposure to 0.1 ng/mL IL-1beta for 6 h. MCP-1 protein secretion in the culture medium was determined by ELISA and expressed as percentage of control (conditioned medium from cultures having not been submitted to any hormonal treatment). Values are the mean ± SEM of duplicate determinations in cultures from four different endometriotic biopsies (patients 4, 9, 10, and 11, Table 1 ). ^* and ^** , Significantly different from control ( P < 0.05 and P < 0.01, respectively). MCP-1 mRNA steady state levels in endometriotic cells were analyzed by Northern blot, and autoradiograms of representative experiments are shown (patient 10, Table 1 ). Concomitant hybridization of 28S ribosomal RNA demonstrated approximately equal RNA loading.

Figure 5. Effects of reproductive steroids on MCP-1 mRNA stability. Cells were pretreated with E₂ (10^-8 mol/L) and/or P (10^-6 mol/L) and stimulated with 0.1 ng/mL IL-1beta for 6 h. Actinomycin D (10 µg/mL) was added to stop de novo RNA synthesis, and cells were harvested after 0,2, and 4 h of incubation with actinomycin D for RNA extraction and Northern blot analysis. A, An autoradiogram showing a representative experiment (patient 11, Table 1 ). B, Kinetics of MCP-1 mRNA degradation. Data are expressed as the ratio of MCP-1 mRNA steady state level to that of 28S RNA as quantified by densitometric analysis of MCP-1 and 28S hybridization signals at each time point.

response to IL-1beta, occurred at the transcriptional and/or the posttranscriptional level, we evaluated MCP-1 mRNA stability and nuclear transcription in cells pretreated, or not, with E₂ (10^-8 mol/L) before stimulation with 0.1 ng/mL IL-1beta for 6 h, as previously described. As illustrated by Fig. 5 , which depicts a representative autoradiogram, treatment with E₂ had no detectable effect on MCP-1 mRNA kinetics of degradation after the 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

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exposed to E₂ before stimulation with IL-1beta was markedly higher (2.9 ± 0.7; mean ± SD of three independent experiments) than that in cells maintained without any previous hormonal treatment ( Fig. 6 ), suggesting a transcriptional regulation of MCP-1 expression by E₂ .

 

Discussion

Endometriosis, a disease occurring in women of reproductive age, is estrogen dependent. Estrogens are believed to be essential to the maintenance and growth of ectopic endometrium and may play a major role in the disease-associated biological changes and clinical manifestations. Among these, a chronic pelvic inflammatory reaction has been described with consistency in women suffering from endometriosis. However, despite these facts, the biochemical mechanisms of estrogen actions in endometriosis remain unclear. In the present study we report that E₂ enhances the responsiveness of ectopic endometrial cells to IL-1beta. Treatment by E₂ , which by itself had no significant effect on MCP-1 expression, amplified the stimulatory action of the proinflammatory cytokine at the level of both MCP-1 mRNA synthesis and protein secretion. These findings for the first time provide evidence that estrogens can indirectly up-regulate the synthesis and secretion of a potent monocyte chemotactic and activating factor for monocytes/macrophages by stromal and epithelial cells isolated from endometriosis lesions. They also put into prominence a synergistic stimulatory action between E₂ and the proinflammatory cytokine IL-1 exerted at the level of ectopic endometrial cells, which in endometriosis may, in fact, occur locally in the inflammatory site, and reveal a new mechanism of action of E₂ in the pathophysiology of this disease.

Little work has been performed to investigate the interactions between steroid hormones and the components of the immune system and their implications in the pathophysiology of endometriosis. E₂ and P have been shown to stimulate vascular endothelial growth factor (VEGF) production by

Figure 6. Nuclear run-on analysis of MCP-1 gene expression by endometriotic cells after pretreatment with ovarian hormones and stimulation by IL-1beta as described in Materials and Methods. DNA samples immobilized onto nylon membranes are as follows: lane 1, 28S cDNA; lane 2, MCP-1 cDNA; and lane 3, pUC18 plasmid DNA. Radioactive transcripts were from cells pretreated with E₂ (10^-8 mol/L) before stimulation with IL-1beta (A) and cells having not submitted to any hormonal pretreatment before exposure to IL-1beta (0.1 ng/mL; B). By densitometric analysis, the ratio of the hybridization signal of MCP-1 to that of 28S was 3-fold greater in A (E₂ ) than in B (control) This experiment was performed with endometriotic cells from patient 10 ( Table 1 ), and comparable results were obtained from two other independent experiments (patients 9 and 13).

nonactivated and activated peritoneal fluid macrophages, which are more frequently found in patients with endometriosis ^[23] . These steroids have also been shown to upregulate VEGF expression by uterine endometrial stromal cells of women with endometriosis ^[47] . However, in these studies the ovarian hormones appeared to exert a direct regulatory action. According to recent data, both eutopic endometrium of women with endometriosis and ectopic endometrial tissue may have the inherent capability to produce E₂ locally, as they were found to express P450 aromatase, which catalyses C₁₉ steroid conversion to estrogens ^[48] . This suggests that E₂ may contribute to the up-regulation of MCP-1 expression in the endometriotic tissue not only by an endocrine pathway, but also by a paracrine mechanism.

The findings of the present study are of additional interest as they extend our recently reported data revealing increased secretion of MCP-1 by eutopic endometrial cells of women with endometriosis ^[34] and elevated in situ expression of this factor in the eutopic endometrial tissue in the presence of the disease ^[41] . Our results illustrate the complexity of MCP-1 regulation in endometriosis and provide a new insight into the debate regarding biochemical and functional differences between endometrial cells implanted in ectopic locations and their eutopic counterparts. In the endometrium, MCP-1 expression was up-regulated in endometrial glands, particularly during the secretory phase of the menstrual cycle ^[41] . Immunohistochemical staining of MCP-1 in endometriosis tissues indicates that the chemokine is highly expressed in both the stromal and glandular compartments regardless of the menstrual cycle phase. Interestingly, cell culture experiments revealed that ectopic endometrial cells do respond to E₂ by increasing IL-1-induced MCP-1 expression as we observed previously for eutopic endometrial cells, but, in contrast, they show no noticeable responsiveness to P either alone or in combination with E₂ , as this normally occurs during the secretory phase of the menstrual cycle. On the one hand, these results suggest a dysregulation of MCP-1 gene in endometrial stromal cells implanted in ectopic locations. On the other hand, they may explain the absence of cyclic variation in MCP-1 expression in endometriosis lesions compared to intrauterine endometrium ^[41] and suggest a reduced sensitivity to P in ectopic endometrial cells, which probably could be ascribed to the decreased P receptors observed in endometriosis tissues ^[44] . Moreover, it is noteworthy that the lack of P-mediated regulation of MCP-1 expression by endometriotic cells and the particular cell responsiveness to E₂ displayed in our in vitro culture model were consistently observed regardless of whether cells originated from ovarian endometriomas or from pelvic peritoneal red vesicles. Further studies will be necessary to investigate the hormonal regulation of MCP-1 in cells from other types of endometriosis lesions.

The findings of the present study may have an interesting physiological significance, as estrogens are believed to exacerbate the progression and effects of endometriosis, and MCP-1 is known as a major mediator of monocyte activation and recruitment into inflammatory sites ^{[37] [38]} . Previous works from other laboratories and from our own have shown that MCP-1 concentrations and chemotactic activity for monocytes are elevated in the peritoneal fluid of women with

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endometriosis and varied according to the severity of the disease ^{[36] [49]} , supporting an important role for this factor in the local peritoneal inflammation related to endometriosis. MCP-1 could be produced by numerous cell types, including mesothelial cells ^[49] , activated macrophages themselves ^{[37] [38]} , and endometrial cells, which may spill into the peritoneal cavity by tubal reflux and show an inherent property to secrete increased levels of MCP-1 in endometriosis, as we have previously shown ^[41] . However, the high level of expression of MCP-1 that we observed in endometriosis implants in both epithelial glands and stroma and the ability of E₂ to amplify MCP-1 synthesis and secretion by endometriotic cells strongly suggest that these cells could induce macrophage activation and recruitment and contribute to the local inflammatory process taking place in the disease. Interestingly, increased infiltration of macrophages was observed in endometriotic implants compared to eutopic endometrium ^[39] , which makes plausible the involvement of MCP-1 in enhanced macrophage recruitment into the ectopic endometrial tissue. It should be pointed out that in the present study, ectopic endometrial cells were responsive to as little as 10 pg/mL IL-1beta. IL-1 is one of the major proinflammatory cytokines found in elevated concentrations in the peritoneal fluid of patients with endometriosis ^{[16] [17]} . The data reported by Taketani et al. ^[17] indicated that IL-1beta production is also increased in endometriotic tissue and may therefore account for the high expression of MCP-1 found in this tissue and the release of the chemokine in the peritoneal environment.

Other substances endowed with chemotactic activity for macrophages, such as complement component 3 (C3) ^[19] and the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted) ^[29] , have been found to be expressed by ectopic endometrial tissue. The hormonal modulation of C3 has not been documented, but added sex steroids had no apparent effect on RANTES expression ^[29] . This is in keeping with our data, as in our in vitro model, neither E₂ nor P by themselves demonstrated any significant effect on MCP-1 expression, but E₂ rather clearly presented an indirect action. Nevertheless, these observations taken together suggest that ectopic endometrial tissue can contribute to a feedforward cascade of events that activates the immune system and perpetuates the infiltration of inflammatory cells associated with endometriosis and may be involved in endometriosis-associated infertility. In fact, recent clinical data from our group revealed that laparoscopic resection or ablation of minimal and mild endometriosis enhanced fecundity in infertile women ^[33] .

The mechanisms by which E₂ enhances MCP-1 expression by ectopic endometrial cells in response to IL-1beta remain unclear. It is well documented that the induction of MCP-1 gene transcription by IL-1beta involves transcriptional factors, such as activating protein-1 and particularly nuclear factor-kappaB, which, according to a recent study, is essential for IL-1-induced MCP-1 gene transcriptional activity ^[50] . The MCP-1 gene promoter region sequence, although yet not complete, does not contain the palindromic estrogen-responsive elements necessary to E₂ /E ₂ receptor complex binding and activation of gene transcription. Even if such cis-acting elements were present, E₂ treatment without subsequent exposure to IL-1beta did not have any significant effect on MCP-1 mRNA steady state levels, making unlikely any direct mechanism involving E₂ receptor binding to the MCP-1 gene regulatory region. However, nuclear run-on analyses showed an enhancement at the transcriptional level of the IL-1beta-induced MCP-1 gene expression by ovarian hormones. This suggests a mechanism by which E₂ may activate a target gene whose products, in turn, may interact with IL-1beta-induced transcription signals.

In summary, the results of the present study show that E₂ up-regulates, although indirectly, the expression of a potent chemotactic and activating factor for monocytes by ectopic endometrial cells of women with endometriosis and that this chemokine is highly expressed by endometriotic lesions without noticeable cyclic variations. E₂ enhances the responsiveness of endometriotic cells to the proinflammatory cytokine IL-1beta by increasing IL-1beta-induced MCP-1 production and exerts its action at the transcriptional level. These findings may have an interesting significance in view of the biological properties of MCP-1, whose levels are elevated in the serum, peritoneal fluid, and eutopic endometrium of women with endometriosis, and the paramount role attributed to E₂ in the pathophysiology of endometriosis, which, in addition, is thought to be abnormally produced locally in eutopic and ectopic endometria. They also show for the first time a hormonal regulation of MCP-1 expression in ectopic endometrial cells and reveal a new method of interaction between the endocrine and the immune systems. Further investigations are needed to elucidate the mechanisms underlying the E₂ stimulatory action on MCP-1 expression and the effect of commonly used hormonal therapeutic agents or antiestrogens on that expression, which may have a great potential therapeutic interest.

Acknowledgments

We thank the group of investigation in gynecology (Drs, Jacques Bergeron, Andre Lemay, Rodolphe Maheux, Jacques Mailloux, and Marc Villeneuve) for patient evaluation and providing endometriotic biopsies, Monique Longpre and Johanne Pelletier for technical assistance, and Dr Lucile Turcot-Lemay for statistical analysis.

References

1. Goldman M, Cramer D. 1990 The epidemiology of endometriosis. In: Chadha D, Buttram V, eds. Current concepts in endometriosis. New York: Liss; 15-31.  

2. Strathy JH, Molgaard CA, Coulman CB. 1982 Endometriosis and infertility: a laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril. 38:667-672.  abstract

3. Di Zerga GS, Barger DL, Hodgen GD. 1980 Endometriosis: role of ovarian steroids in initiation, maintenance and suppression. Fertil Steril. 33:649-65.  abstract

4. Gleicher N, le-Roeiy A, Confino E, Friberg J. 1987 Is endometriosis an autoimmune disease? Obstet Gynecol. 70:115-122.  abstract

5. Meldrum DR. 1985 Management of endometriosis with gonadotropin-releasing hormone agonists. Fertil Steril. 44:581-582.  citation

6. Lyndrup J, Thorpe S, Glenthoj A, Obel E, Sele V. 1987 Altered progesterone/estrogen receptor ratios in endometriosis. Acta Obstet Gynecol Scand. 66:625-629.  abstract

7. Shaw RW. 1993 An atlas of endometriosis. New York: Parthenon Publishing Group; 23-25.  

8. Barbieri RL, Ryan KJ. 1981 Danazol: endocrine pharmacology and therapeutic applications. Am J Obstet Gynecol. 141:453-463.  abstract

9. Shaw RW. 1992 Treatment of endometriosis. Lancet. 340:1267-1271.  citation

10. Lemay A. 1993 La place de la suppression ovarienne dans le traitement de l'endometriose. Ref Gynecol Obstet. 1:243-250.  

11. Henzl M, Corson S, Moghissi K. 1988 Administration of nasal nafarelin as compared with oral danazol for endometriosis. A multicenter double-blind comparative clinical trial. N Engl J Med. 318:485-489.  abstract

904

12. Braun DP, Dmowski WP. 1998 Endometriosis: abnormal endometrium and Dysfunctional immune response. Curr Opin Obstet Gynecol. 10:365-369.  abstract

13. Giudice LC, Tazuke SI, Swiersz L. 1998 Status of current research on endometriosis. J Reprod Med. 43:252-262.  abstract

14. Vinatier D, Dufour P, Oosterlynck D. 1996 Immunological aspects of endometriosis. Hum Reprod Update 2371-384.  

15. Badawy SZA, Cuenca V, Frileich H, Stefanu C. 1990 Endometrial antibodies in serum and peritoneal fluid of infertile patients with and without endometriosis. Fertil. Steril. 53:930-932.  

16. Fakih H, Baggett B, Holtz G, Tsang KY, Lee JC, Williamson HO. 1987 Interleukin-1: a possible role in the infertility associated with endometriosis. Fertil Steril. 47:213-217.  abstract

17. Taketani Y, Kuo TM, Mizuno M. 1992 Comparison of cytokine levels and embryo toxicity in peritoneal fluid in infertile women with untreated or treated endometriosis. Am J Obstet Gynecol. 167:265-270.  abstract

18. Vernon MW, Beard JS, Graves K, Wilson EA. 1986 Classification of endometriotic implants by morphologic appearance and capacity to synthesize prostaglandins. Fertil Steril. 46:801-806.  abstract

19. Isaacson KB, Coutifaris C, Garcia CR, Lyttle CR. 1989 Production and secretion of complement component 3 by endometriotic tissue. J Clin Endocrinol Metab. 69:1003-1009.  abstract

20. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. 1983 Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol. 145:333-337.  abstract

21. Hill JA, Faris HMP, Schiff I, Anderson DJ. 1988 Characterization of leukocyte subpopulations in the peritoneal fluid of women with endometriosis. Fertil Steril. 50:216-222.  abstract

22. Haney AF, Muscato JJ, Weinberg JB. 1981 Peritoneal fluid cell populations in infertility patients. Fertil Steril. 35:696.  citation

23. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK. 1996 Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steriods. J Clin Invest. 98:482-489.  abstract

24. Halme J, White C, Kauma S, Estes J, Haskill S. 1988 Peritoneal macrophages from patientes with endometriosis release growth factor activity in vitro. J Clin Endocrinol Metab. 66:1044-1049.  abstract

25. Olive DL, Montoya I, Riehl RM, Schenken RS. 1991 Macrophage-conditioned media enhance endometrial stromal cell proliferation in vitro. Am J Obstet Gynecol. 164:953-958.  abstract

26. Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. 1996 Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril. 65:925-930.  abstract

27. Falcone T, Hemmings R. 1996 Review of peritoneal factors that affect fertility in patients with mild endometriosis. J SOGC. 18:121-130.  

28. Halme J. 1991 Role of peritoneal inflammation in endometriosis-associated infertility. Ann NY Acad Sci. 6:266-274.  

29. Hornung D, Ryan IP, Chao VA, Vigne JL, Shcriock ED, Taylor RN. 1997 Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab. 82:1621-1628.  full text

30. Akoum A, Lemay A, Rheault N, Paradis I, Maheux R. 1996 Secretion of interleukin-6 by endometriotic cells and regulation by inflammatory cytokines and sex steroids. Hum Reprod. 10:101-107.  

31. Tseng JF, Ryan IP, Milam TD, Murai JT, Scheriock ED, Landers DV, Taylor RN. 1996 Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic endometrial stromal cells from women with endometriosis. J Clin Endocrinol Metab. 81:1118-1122.  full text

32. Rier SE, Zarmakoupis PN, Hu X, Becker JL. 1995 Dysregulation of IL-6 responses in ectopic endometrial stromal cells: correlation with decreased soluble receptor levels in peritoneal fluid of women with endometriosis. J Clin Endocrinol Metab. 80:1431-1437.  full text

33. Marcoux S, Maheux R, Berube S, Canadian Collaborative Group on Endometriosis. 1997 Laparoscopic surgery in infertile women with minimal or mild endometriosis. N Engl J Med. 337:217-222.  abstract

34. Akoum A, Lemay A, Brunet C, Hebert J, Groupe d'Investigation en Gynecologie. 1995 Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis. Fertil Steril. 63:322-328.  abstract

35. Akoum A, Lemay A, Brunet C, Hebert J, Groupe d'Investigation en Gynecologie. 1995 Cytokine-induced secretion of monocyte chemotactic protein-1 by human endometriotic cells in culture. Am J Obstet Gynecol. 172:594-600.  full text

36. Akoum A, Lemay A, McColl S, Turcot-Lemay L, Maheux R. 1996 Elevated concentration and biological activity of monocyte chemotactic protein-1 in the peritoneal fluid of patients with endometriosis. Fertil Steril. 66:17-23.  abstract

37. Schall TJ. 1991 Biology of the RANTES/SIS cytokine family. Cytokine. 3:165-183.  citation

38. Leonard EJ, Yoshimura T. 1990 Human monocyte chemoattractant protein-1 (MCP-1). Immunol Today. 11:97-101.  abstract

39. Witz CA, Schenken RS. 1997 Pathogenesis of endometriosis. Semin Reprod Endocrinol. 15:199-208.  abstract

40. American Fertility Society. 1985 Revised American Fertility Society classification of endometriosis. Fertil Steril. 43:351-352.  citation

41. Jolicoeur C, Boutouil M, Drouin R, Paradis I, Lemay A, Akoum A. 1998 Increased expression of monocyte chemotactic protein-1 in the endometrium of women with endometriosis. Am J Pathol. 152:125-133.  abstract

42. Metzger DA, Szpak CA, Haney AF. 1993 Histologic features associated with hormonal responsiveness of ectopic endometrium. Fertil Steril. 59:83-88.  abstract

43. Cunha GR, Bigsby RM, Cooke PS, Sugimura Y. 1985 Stromal-epithelial interactions in adult organs. Cell Differ. 17:137-148.  citation

44. Bergqvist A, Ferno M. 1993 Oestrogen and progesterone receptors in endometriotic tissue and endometrium: comparison of different cycle phases and ages. Hum Reprod. 8:2211-2217.  abstract

45. Nisolle M, Casanas-Roux F, Wyns C, de Menten Y, Mathieu PE, Donnez J. 1994 Immunohistochemical analysis of estrogen and progesterone receptors in endometrium and peritoneal endometriosis: a new quantitative method. Fertil Steril. 62:751-759.  abstract

46. De Leon FD, Vijayakumar R, Brown M, Rao CHV, Yussman MA, Schultz G. 1986 Peritoneal fluid volume, estrogen, progesterone, prostaglandin, and epidermal growth factor concentrations in patients with and without endometriosis. Obstet Gynecol. 68:189-194.  abstract

47. Shifren JL, Tseng JF, Zaloudek CJ, et al. 1996 Ovarian steriod regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab. 81:3112-3118.  full text

48. Noble LS, Simpson ER, Johns A, Bulun SE. 1996 Aromatase expression in endometriosis. J Clin Endocrinol Metab. 81:174-179.  full text

49. Arici A, Oral E, Attar E, Tazuke SI, Olive DL. 1997 Monocyte chemotactic protein-1 concentration in peritoneal fluid of women with endometriosis and its modulation of expression in mesothelial cells. Fertil Steril. 67:1065-1072.  abstract

50. Ueda A, Okuda K, Ohno S, et al. 1994 NF-kappaB and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol. 153:2052-2063.  abstract

 

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