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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 12 5785-5792
Copyright © 2002 by The Endocrine Society

Original Article

Estradiol and Interleukin-1ß Exert a Synergistic Stimulatory Effect on the Expression of the Chemokine Regulated upon Activation, Normal T Cell Expressed, and Secreted in Endometriotic Cells

Ali Akoum, André Lemay and Rodolphe Maheux

Unité d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, Centre Hospitalier Universitaire de Québec, Faculté de Médecine, Université Laval, Québec, Canada G1L 3L5

Address all correspondence and requests for reprints to: Ali Akoum, Ph.D., Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, 10 rue de l’Espinay, Local D0–711, Québec, Canada G1L 3L5. E-mail: ali.akoum{at}crsfa.ulaval.ca.

Abstract

Endometriosis, commonly associated with intraperitoneal inflammation, is estrogen dependent. Possible links between the immunoinflammatory and endocrine changes observed in endometriotic women have been poorly understood. In this study, we report that estradiol (E2) and IL-1ß exert a synergistic stimulatory action on RANTES (regulated upon activation, normal T cell expressed, and secreted) expression by endometriotic cells. Treatment of endometriotic cells with IL-1ß had a dose-dependent effect on RANTES protein secretion and mRNA steady state levels, whereas cell treatment with E2 or progesterone had no detectable effect. Interestingly, treatment of endometriotic cells with E2 before stimulation with IL-1ß resulted in a further increase in RANTES protein secretion and mRNA steady state levels, compared with IL-1ß alone, whereas treatment with progesterone did not significantly affect cell responsiveness to IL-1ß. Assessment of RANTES mRNA half-life revealed that cell pretreatment with E2 enhanced RANTES mRNA stability and increased gene transcription as shown by run-on analysis. Immunohistochemical analysis of RANTES in endometriotic tissue showed immunostaining to be predominant in the stroma with no noticeable differences in tissues from the proliferative and secretory phase of the menstrual cycle. This appears to be consistent with the cell culture data and indicates that RANTES expression in endometriotic tissue is not subject to cyclic variation. These findings reveal a new regulatory mechanism by which IL-1ß produced by activated macrophages can in synergy with ovarian and locally produced E2 lead to enhanced macrophage and T-lymphocyte recruitment, thereby exacerbating the local immunoinflammatory process. Furthermore, the findings provide a further evidence for a close relationship between the endocrine and immunological changes observed in endometriosis.

TUBAL REFLUX LEADING to the presence of endometrial tissue in the peritoneal cavity is common to almost all menstruating women (1). In some of them, however, and according to Sampson’s theory (2), endometrial tissue adheres to the peritoneal lining and develops into endometriotic lesions.

Besides genetic, hormonal, and environmental factors reported to be involved in the development of endometriosis, immunological factors may account for the susceptibility of endometrial tissue to escape immunosurveillance and implant in ectopic locations (3). Endometriosis is often associated with immunoinflammatory changes observed in the peritoneal fluid, which bathes the organs of the pelvic cavity, as well as in endometriotic implants (4, 5). One of the hallmarks of such a local inflammatory reaction is the increased number of activated peritoneal leukocytes, particularly macrophages (6, 7, 8, 9). Moreover, these cells show evidence of activation in endometriosis patients and were also found to infiltrate endometriotic tissue (5, 7, 8, 9, 10, 11). Paradoxically, instead of eliminating misplaced endometrial tissue, activated macrophages appear to be involved in endometrial tissue adhesion, implantation, and growth. In fact, peritoneal macrophages from women with endometriosis seem to resist apoptosis (12) and have a reduced capacity to eliminate misplaced endometrial tissue (13). In contrast, macrophages from women with endometriosis were shown to secrete growth factors for endometrial cells (14, 15, 16) and promote angiogenesis directly by secreting angiogenic factors (17) and indirectly through the ability of macrophage-derived cytokines such as TNF{alpha} (18, 19) and IL-1 to induce angiogenic phenotype in endometriotic cells (20). Local inflammatory reaction is also thought to be a possible cause of endometriosis-associated pain and infertility (8, 21), and increased numbers of activated macrophages have been found in the peritoneal fluid of infertile women with endometriosis (7, 8, 9).

Increased recruitment of macrophages into the peritoneal cavity can at least in part be due to cyclic stimulation by refluxed endometrial tissue (22). However, data from our and other laboratories showed that chemokines produced by endometriotic tissue may contribute to a feed-forward cascade of events that accentuate the recruitment of leukocyte into the peritoneal cavity of endometriosis patients (23, 24, 25, 26, 27).

Regulated upon activation, normal T cell expressed, and secreted (RANTES) is a chemokine reported to have a potent ability to chemoattract monocytes and T cells (28). Elevated concentrations of this mediator have been detected in the peritoneal fluid of endometriotic women, and RANTES levels correlated with the stage of disease (29). This suggests that RANTES may be involved in leukocyte recruitment into the endometriosis implantation sites. Although expression of RANTES was first thought to be limited to activated T cells, it is now known to be secreted by a variety of tissue types including macrophages and endometrial cells and has been reported to be expressed by endometriotic tissue (26, 30). Furthermore, recent studies indicate that RANTES expression by endometriotic cells is up-regulated by IL-1ß, a proinflammatory cytokine produced mainly by monocytes/macrophages (31). Our present study corroborated these data and further revealed a synergism between estradiol (E2) and IL-1, resulting in an increased RANTES secretion by endometriotic cells. Interestingly, E2 appeared to enhance IL-1-mediated RANTES expression both at the transcriptional and posttranscriptional levels. This may be of interest in view of the critical role of E2 in the development and maintenance of endometriosis (32, 33, 34) and its abnormal production locally in ectopic endometrial tissue (35).

Materials and Methods

Source and handling of tissue

Tissue specimens used in this study were obtained from healthy ovulating women who signed a written informed consent for a research project approved by the Saint François d’Assise Hospital Ethics Committee on Human Research. Endometriosis was diagnosed during laparoscopy and staged according to the revised American Fertility Society classification system (36). Women recruited into the study 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. The cycle phase (proliferative or secretory) was determined according to the patients’ cycle history and serum progesterone (P). The mean age was 35.0 ± 7.5 yr (Table 1Go).


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  		Table 1. Clinical characteristics of patients, sources of endometriotic tissue, laparoscopic findings, and type of experiment performed on each biopsy

 
Endometriotic biopsies were immediately placed at 4 C in sterile Hanks’ balanced salt solution (HBSS) containing 1% antibiotics-antimycotics (Invitrogen, Burlington, Ontario, Canada), snap frozen in liquid nitrogen with Tissue-Tek OCT compound (Miles Inc., Elkhart, IN), and stored at -80 C until analyzed by immunohistochemistry or were directly used for cell culture experiments. Some endometriotic specimens were fixed in Tissufix no. 2 solution (Laboratoire Gilles Chaput Inc., Montreal, Quebec, Canada) for 24 h. They were then placed in a Tissue-Tek VIP apparatus (Miles Scientific, Etobicoke, Ontario, Canada) for another 12 h before being embedded in paraffin (TissuePrep, Fisher Scientific, Fair Lawn, NJ) for pathological investigations.

Immunohistochemistry

Immunohistochemistry was performed according to a previously described procedure (25), which was modified for RANTES. Briefly, immunostaining was performed on 4- to 5-µm tissue sections using a monoclonal mouse antihuman RANTES antibody (15 µg/ml in PBS containing 1% BSA; R&D Systems, Minneapolis, MN), a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA), and diaminobenzidine (Sigma, St. Louis, MO) as chromogen. Hematoxylin was used for counterstaining. Sections incubated without the primary antibody or with normal mouse IgGs used at the same concentration as the primary antibody were included as negative controls in all experiments. Slides were viewed using a microscope (mikroskopie und systeme GmbH, model DMRB), and photomicrographs were taken with 100 ASA film (Kodak, Rochester, NY).

Tissue dissociation and cell culture

Endometriotic cells were prepared and cultured according to a well-established and routinely used protocol (23). Briefly, endometriotic tissue was washed with cold HBSS, minced into small species, and digested with 1 mg/ml collagenase in HBSS for 1 h at 37 C. After a Ficoll-Paque gradient (Amersham Biosciences Corp., Baie d’Urfé, Quebec, Canada) to eliminate leukocytes, cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum (FBS), 1.7 µM insulin, 6.2 µM transferrin, and 1% antibiotics-antimycotics (Invitrogen). Cultures using this protocol are free of leukocytes and contain predominantly stromal fibroblastlike cells. Epithelial and stromal cells were not separated because cell responsiveness to sex steroid hormones appeared to depend on stromal-epithelial cell interaction (37, 38).

Culture stimulation and RANTES synthesis

Cells were grown in 25-cm2 culture flasks in DMEM medium containing 10% dextran-coated charcoal-treated FBS. At confluence, the complete medium was discarded and replaced overnight with FBS-free medium, and cells were cultured for additional periods of time (0–24 h) with fresh FBS-free medium containing different concentrations of IL-1ß (0–600 pM). In some experiments, cultures were stimulated with IL-1ß (6 pM) in the presence of 355 µM cycloheximide (Sigma), an inhibitor of protein synthesis.

The effect of ovarian steroids [P: 4-pregnen-3,20-dione; E2: 1,3,5,(10)-estratrien-3, 17ß-diol-3-benzoate] (Sigma) was evaluated using an established long-term cell culture treatment protocol in which hormones were added to endometriotic cells at the onset of culture (25) and maintained for 7–8 d (until confluence). At confluence, cells were washed with serum-free medium, and incubation with hormones [E2, P or E2 + P] further continued in this medium for 42 h before stimulation or not with IL-1ß. Ovarian steroid doses (10-8 M for E2 and 10-6 M for P) were chosen on the basis of previous studies showing that ovarian steroid concentrations in the peritoneal fluid are higher than those normally found in the peripheral blood (17, 39).

RANTES protein secretion was evaluated in the culture medium using ELISA, whereas RANTES mRNA steady state level in endometriotic cells was evaluated by Northern blot.

RANTES ELISA

RANTES concentrations were measured using an ELISA procedure developed in the laboratory. Briefly, this technique uses a capture mouse monoclonal antihuman-RANTES antibody (500 ng/well in 96-well plates) (R&D Systems), a goat polyclonal antihuman-RANTES antibody for detection (0.5 µg/ml in PBS/0.5% BSA) (R&D Systems), alkaline phosphatase-conjugated goat antirabbit IgGs peroxidase-conjugated AffiniPure rabbit antigoat IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) (1:4000 dilution in PBS/0.5% BSA) (Chemicon International Inc., Temecula, CA), and 3,3', 5,5',-tetramethylbenzidine-peroxidase (Bio-Rad Laboratories, Inc. Ltd., Mississauga, Ontario, Canada) as substrate. The OD was read at 450 nm, and RANTES concentrations were extrapolated from a standard curve using recombinant human RANTES (gift from Dr. Shaun McColl, Department of Molecular Biosciences, University of Adelaide, Adelaide, Australia). The sensitivity limit of the assay was 13 pM, with intraassay and interassay coefficients of variation less than 5%.

Northern blot analysis

Total RNA was extracted from cells with TRIzol reagent according to the manufacturer’s instructions (Invitrogen). 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 composed of 5x saline sodium citrate (SSC), 5x Denhardt’s solution, 50 mM NaH2PO4, 0.5% SDS, 200 µg/ml salmon sperm DNA, and 50% formamide, hybridized with 32P-labeled RANTES cDNA (also a gift from Dr. Shaun McColl) in the hybridization buffer and washed with 1x SSC, 0.2x 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 (Invitrogen) 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 RANTES 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 earlier, and incubated with IL-1ß (6 pM) for 6 h. Transcription was then stopped with actinomycin D (8 µM), and cells were harvested after different times (0–4 h) of incubation with actinomycin D 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 and then homogenized using a Dounce homogenizer in a lysis buffer containing 0.25 M sucrose, 10 mM HEPES (pH 8.0), 10 mM MgCl2, 2 mM dithiothreitol (DTT), and 0.1% (vol/vol) Triton X100. Nuclei were pelleted and washed twice in fresh buffer by centrifugation at 600 x g for 5 min at 4 C, and stored in 80 µl glycerol storage buffer [50 mM HEPES (pH 8.0), 40% (vol/vol) glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 2 mM DTT]. For transcription assays, nuclei were incubated in 200 µl reaction buffer containing 20 mM HEPES (pH 8.0); 5 mM MgCl2; 90 mM NH4Cl; 0.5 mM MnCl2; 16% (vol/vol) glycerol; 0.04 mM EDTA; 2 mM DTT; 0.4 mM each of ATP, CTP, and GTP (Invitrogen); and 0.25 mCi of [{alpha}-32P]UTP (3000 Ci/mmol); the reaction was stopped by the addition of 100 µg/ml RNase-free Dnase I (Invitrogen). After treatment with proteinase K (100 µg/ml) (Invitrogen) in the presence of 10 mM CaCl2 and 25 µg yeast tRNA (Roche Diagnostics, Laval, Quebec) 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 M ammonium acetate (1:2, vol/vol), pelleted by centrifugation at 12,000 rpm at 4 C for 15 min, and dissolved in 850 µl hybridization buffer containing 50 mM PIPES (pH 7.0), 0.5 M NaCl, 2 mM 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 d at 42 C using 5 million to 10 million cpm/ml hybridization buffer. Membranes were washed four times with 2x SSC, 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, washed twice again with 2x SSC, 0.1% SDS at 65 C for 30 min, and finally exposed to x-ray films (BioMax, Kodak) at -80 C.

Statistical analysis

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 values less than 0.05.

Results

RANTES expression in endometriotic lesions

RANTES immunoreactivity was found throughout endometriotic tissue but was predominant in stromal cells (Fig. 1AGo). Control experiments performed on serial sections of endometriotic tissue confirmed the specificity of RANTES immunoreactivity because there was no noticeable immunostaining with normal mouse IgGs (Fig. 1BGo), with the primary antibody following preabsorption with an excess of RANTES or when the primary antibody was omitted (data not shown). Similar pattern of staining was observed whether endometriotic tissues were from the inner lining of ovarian endometriomas or from peritoneal lesions. Furthermore, no apparent difference related to the menstrual cycle phase was noted.



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  		Figure 1. Immunohistochemical analysis of RANTES expression in endometriotic tissue (biopsy of a black endometriotic lesion from a 32-yr-old woman with stage IV endometriosis). Note positive brown immunostaining throughout endometriotic tissue, particularly in stromal cells, in the presence of a mouse monoclonal anti-RANTES antibody (A) and the absence of immunostaining in the presence of mouse IgGs used instead of the primary antibody (B) (negative control). Scale bar, 30 µm.

 
Effect of IL-1ß and ovarian hormones on RANTES expression

The absence of apparent variations in RANTES immunostaining in relation with the menstrual cycle phase, prompted us to examine endometriotic cell responsiveness to ovarian hormones and assess whether these latter may have any regulatory effect on RANTES expression. Thus, endometriotic cell cultures were exposed to ovarian hormones at concentrations similar to those found in the peritoneal fluid of endometriosis patients (17, 39). As shown in Fig. 2Go, unstimulated endometriotic cells had no detectable expression of RANTES mRNA, as assessed by Northern blot, and did not release any amount of RANTES protein in the culture medium detectable by ELISA. In addition, endometriotic cell treatment with E2, P, or a combination of these hormones had no detectable effect on RANTES mRNA expression and resulted in a slight increase in RANTES secretion.



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  		Figure 2. Effect of E2 (10-8M) and P (10-6 M) on RANTES expression by endometriotic cells. Cells were continuously incubated with E2 and/or P from the culture onset, as described in Materials and Methods. A, RANTES protein secretion in the culture medium as determined by ELISA (pM). B, RANTES mRNA steady state levels and ribosomal 28S RNA in endometriotic cells as analyzed by Northern blot. M, Medium only (control).

 
In view of these data, we used IL-1ß as stimulus to induce the synthesis of RANTES by endometriotic cells and assessed whether ovarian hormones may regulate the IL-1-induced RANTES expression. IL-1, one of the major proinflammatory cytokines found in elevated concentrations in the peritoneal fluid of patients with endometriosis (40, 41), has been reported to induce RANTES production by endometriotic cells (31). IL-1 is mainly secreted by activated macrophages (42), which have been shown to be actively recruited into the peritoneal cavity of patients and infiltrate endometriotic lesions (5, 6, 7, 8, 9, 10, 11).

Thus, endometriotic cells were first incubated with increasing concentrations of IL-1ß for different time periods to determine optimal stimulation conditions. Our data showed that IL-1ß stimulated RANTES mRNA steady state levels and protein secretion in a dose- and time-dependent manner. Detectable levels of RANTES mRNA and protein were observed following stimulation with 0.6 pM IL-1ß, and maximal stimulation occurred in the presence of 60 pM for a 6-h period of treatment (Fig. 3Go). The protein secretion increased 6 h after stimulation with 6 pM IL-1ß and rose gradually during the 24 h of treatment, whereas mRNA levels peaked after 6 h of stimulation and declined afterward over time (Fig. 4Go). Incubation of endometriotic cells with cycloheximide (355 µM) to inhibit protein synthesis completely abolished RANTES protein secretion in response to IL-1ß (6 pM) but led to a marked increase in RANTES mRNA steady state levels (Fig. 3Go). Thus, IL-1ß exerts a direct effect on endometriotic cell steady state mRNA expression that does not necessarily require de novo protein synthesis.



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  		Figure 3. Dose effect of IL-1ß on RANTES expression by endometriotic cells. Cells grown to confluence were incubated for 6 h with increasing concentrations of IL-1ß (0–600 pM). At the end of the incubation period, cells and culture media were recovered to evaluate RANTES protein secretion by ELISA and mRNA steady state levels by Northern blot. A, RANTES protein secretion (pM). B, RANTES mRNA as observed by autoradiography following hybridization of RANTES mRNA with 32P-labeled RANTES cDNA probe. The 28S ribosomal RNA demonstrated approximately equal RNA loading. Cycloheximide (CHX; 355 µM), added together with IL-1ß (6 pM), inhibited RANTES protein secretion (A) and markedly increased mRNA steady state levels (B).

 


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  		Figure 4. Time course of IL-1ß-induced RANTES secretion and mRNA synthesis by endometriotic cells. Confluent cultures were treated with IL-1ß (6 pM) or control medium for different time periods. The culture supernatant was recovered at each time point to measure RANTES protein secretion by ELISA (pM), and total cellular RNA was extracted to evaluate RANTES mRNA expression by Northern blot. A, RANTES protein secretion. B, RANTES mRNA steady state levels and 28S ribosomal RNA.

 
Endometriotic cells were then exposed to E2, P, or to E2 and P in combination before being exposed to 6 pM IL-1ß for 6 h. A 6-h period of treatment and a concentration of 6 pM IL-1ß were therefore used in the subsequent experiments to detect any possible effect of ovarian hormones. Data depicted in Fig. 5Go show that pretreatment with E2 increased RANTES mRNA steady state levels in endometriotic cells in response to IL-1ß and stimulated as well RANTES protein secretion in the culture medium. However, pretreatment with P, either alone or in combination with E2, had no significant effect on RANTES mRNA or protein secretion. Results from three independent experiments were analyzed. For RANTES secretion, the increase over control (cells stimulated only with IL-1ß) was statistically significant for E2 (56.8% ± 11.3%, P < 0.001) and E2 + P (47.4% ± 4.8%, P < 0.01), but P effect (-20.9% ± 9.0%) was not statistically significant. For RANTES mRNA, steady state levels expressed as the ratio of RANTES hybridization signal density to that of 28S had increased by 79.9% ± 8.9% (P < 0.05) after the addition of E2, 72.8% ± 13% (P < 0.05) after the addition of P to E2-treated cells, and 10.7% ± 21.8% (P > 0.05) after the addition of P alone.



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  		Figure 5. Effect of cell pretreatment with E2 (10-8 M) and P (10-6 M) on IL-1ß-mediated RANTES expression. Cells were treated with E2 and/or P from the culture onset until stimulation with IL-1ß (6 pM for 6 h), as described in Materials and Methods. A, RANTES protein secretion in the culture medium as determined by ELISA. B, RANTES mRNA steady state levels in endometriotic cells as analyzed by Northern blot. M, Medium only (control). *, Significant increase of RANTES secretion, compared with control (cells exposed to IL-1ß without prior treatment with ovarian steroids, P < 0.05).

 
To determine whether the effect of E2 was exerted at the transcriptional and/or the posttranscriptional levels, we evaluated RANTES mRNA half-life and gene transcription. Cells were pretreated or not with E2 (10-8 M) before stimulation with 6 pM IL-1ß for 6 h, as previously described. Figure 6Go shows that pretreatment with E2 retarded RANTES mRNA degradation following arrest of de novo RNA transcription by actinomycin D (8 µM). Without pretreatment with E2, the half-life of RANTES mRNA was approximately 3.6 h, whereas in E2-pretreated cells, it was approximately 6.7 h (data from three separate experiments). Nuclear run-on analysis showed that E2 pretreatment also increased RANTES nuclear transcription in response to IL-1ß (Fig. 7Go). The mean increase ± SEM in RANTES gene transcription as determined by densitometric analysis of data from three experiments was 61.7% ± 11.1%.



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  		Figure 6. Effect of E2 on RANTES mRNA stability. Cells were pretreated with E2 (10-8 M) and stimulated with 6 pM IL-1ß for 6 h. Actinomycin D (8 µM) was added to stop de novo RNA synthesis and cells were harvested after 0, 2, 4, and 8 h of incubation with actinomycin D for RNA extraction and Northern blot analysis. A, Kinetics of RANTES mRNA degradation. The ratio of RANTES mRNA steady state level to that of 28S RNA, as quantified by densitometric analysis of RANTES and 28S hybridization signals, was determined at each time point, and data were expressed as the percentage ± SEM of RANTES mRNA level in endometriotic cells at time 0. B, An autoradiogram showing a representative experiment.

 


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  		Figure 7. Nuclear run-on analysis of RANTES gene expression by endometriotic cells following pretreatment with E2 and stimulation with 6 pM IL-1ß. DNA samples immobilized onto nylon membranes are as follows: 1, 28S cDNA; 2, RANTES cDNA; 3, pUC18 plasmid DNA. Radioactive transcripts were from cells having not been submitted to any hormonal pretreatment before exposure to IL-1ß (6 pM) (A) and cells pretreated with E2 (10-8 M) before stimulation with IL-1ß (B).

 
Discussion

Endometriosis is dependent on estrogen for its development and evolution. The disease arises at menarche, regresses after menopause, and can be present in postmenopausal women with elevated estrogen levels (32, 33). Animal models proved estrogen plays a critical role in the establishment and growth of endometriotic implants (32). Recent studies uncovered an autocrine mechanism that favored abnormal production of estrogen in endometriotic tissue (35). However, in spite of these facts, the mechanisms by which estrogen contributes to the pathogenesis of endometriosis are still poorly understood.

The present study revealed that E2 and IL-1ß act in synergy to stimulate RANTES production by endometriotic cells. In fact, E2 alone slightly affected RANTES protein secretion and had no detectable effect on RANTES mRNA expression. These data are in accordance with those of Hornung et al. (26, 30), who reported that sex steroids had no apparent effect on RANTES expression by endometriotic stromal cells. However, the present study showed that E2 enhances endometriotic cell responsiveness to IL-1ß and significantly increases RANTES protein secretion and mRNA steady state levels in response to the proinflammatory cytokine. Further analysis of E2 effect showed that the hormone acts both at the transcriptional and the posttranscriptional levels by increasing RANTES gene transcription and mRNA stability. However, although one would expect these two mechanisms to be additive, the fact that the mean increase in RANTES mRNA transcription rate following E2 treatment (61.7% over control) and the augmentation in RANTES mRNA stability (86.1% over control) are not fully reflected by the increase in RANTES mRNA steady state levels induced by E2 (77.9% over control) is difficult to explain with certainty. This might presumably be due to interpatient variations and/or differences between lesions regarding RANTES expression by endometriotic cells and cell sensitivity to E2 and IL-1.

These findings may have an interesting significance. First, they indicate that E2 may, by enhancing endometriotic cell secretion of RANTES in response to IL-1ß, indirectly contribute to the enhanced leukocyte recruitment into the peritoneal cavity of endometriosis patients and endometriotic tissue itself. In fact, one of the most known property of RANTES is that of T-lymphocyte and monocyte/macrophage chemoattraction (28). These cells, which were shown to infiltrate endometriotic lesions and be present in increased numbers in the peritoneal environment (5, 6, 7, 8, 9, 10, 11), are believed through their secretion of proteases, growth factors, reactive oxygen species, prostaglandins, and inflammatory and embryotoxic cytokines to contribute to endometriotic tissue growth and endometriosis-associated pain and infertility (14, 15, 16, 17, 22, 42). This strongly suggests that in synergy with proinflammatory cytokines, such as IL-1ß, E2 may exacerbate the immunoinflammatory process taking place in the disease locally in the peritoneal cavity and within endometriotic implants. This is all the more plausible because E2 appeared to be abnormally synthesized in endometriotic tissue (35) and may therefore act by endocrine and autocrine mechanisms. It is noteworthy that E2 was also found to stimulate vascular endothelial growth factor production by peritoneal fluid macrophages (17) and uterine endometrial stromal cells of women with endometriosis (43) and favor endometriosis growth by potentiating angiogenesis.

Second, our data give further support to the emerging concept tying the endocrine and immune dysfunctions in endometriosis pathophysiology. In fact, endometriotic epithelial cells were induced to secrete the chemokine eotaxin after treatment with cytokines and ovarian steroids (44). According to our previous data, E2 has an enhancing effect on IL-1-mediated IL-8 and monocyte chemotactic protein-1 (MCP-1) production by ectopic endometrial cells (24, 25). Interestingly, although E2 appeared to enhance MCP-1 gene transcription (25), its effect on RANTES expression was also manifest at the posttranscriptional level, which is suggestive of different and cytokine-dependent regulatory mechanisms.

It is of interest to note that estrogen effects appear to be cell type dependent and vary according to the pathological state. Thus, although E2 enhances cytokine production in endometriosis, it was reported to decrease cytokine production in some pathological states and increase it in others. For instance, estrogen was found to up-regulate IL-1ß-induced IL-6 production in fibroblastlike synoviocytes from rheumatoid arthritis patients (45) and prolong T-cell survival in systemic lupus erythematosus (46). On the other hand, estrogen appeared to decrease MCP-1 production by monocytes and coronary artery smooth muscle cells and is believed to play a protective effect against atherosclerosis (47, 48). However, the mechanisms underlying such opposite actions remain completely unknown.

RANTES expression by endometriotic cell cultures was not significantly modified following exposure to P alone or in the presence of E2 as in the secretory phase of the menstrual cycle. These in vitro data are consistent with immunohistochemical analysis of RANTES expression in endometriotic tissue, which has not revealed any noticeable difference between tissues from the proliferative and secretory phase of the menstrual cycle.

RANTES expression in the normal endometrium has previously been reported (26, 30). According to these studies, the chemokine is expressed throughout the ovulatory cycle in vivo, and sex steroids had no apparent effect on its expression by endometrial cells in vitro. Recent data from the same group (49) have, however, shown that long-term treatment with medroxyprogesterone acetate resulted in a significant decrease in RANTES secretion or mRNA synthesis by endometrial cells. Interestingly, Attia et al. (50) showed that the stimulatory P receptor isoform B is quite well expressed in the eutopic but is decreased or absent only in the ectopic endometrial tissue. This may relate to defective P sensitivity associated with endometriosis, as indicated by our study, and also suggests that eutopic endometrial cells have the capability of responding to P. In contrast, the responsiveness of endometrial cells to E2 alone or in combination with IL-1 remains to be elucidated.

The mechanisms by which E2 enhances RANTES expression by ectopic endometrial cells in response to IL-1ß remain unclear. It is well documented that IL-1ß induction of RANTES gene expression in endometriotic stromal cells is associated with the activation of the transcriptional factor nuclear factor {kappa}B and depends on a nuclear factor-{kappa}B site in the proximal promoter (31). We found no estrogen-responsive element in the known sequence of the RANTES gene proximal promoter (51) using the Transcription Element Search System (www.cbil.upenn.edu/tess/). However, E2 treatment, without subsequent stimulation with IL-1ß, had no detectable effect on RANTES mRNA steady state levels, which makes less probable a direct mechanism involving E2 receptor binding to the RANTES gene regulatory region. Our nuclear run-on analysis showed an enhancement at the transcriptional level of the IL-1ß-induced RANTES gene expression by E2. This suggests a mechanism by which E2 may activate a target gene whose products in turn may interact with IL-1ß-induced transcription signals. On the other hand, E2 was also shown to increase RANTES mRNA stability. This may be interesting because cytokines are known to have short-life mRNAs, and by prolonging RANTES mRNA half-life, E2 shows up a new way by which it may influence endometriosis pathophysiology and contribute to maintain endometriosis-associated local inflammation. The mechanisms of such E2-mediated actions are currently being investigated in our laboratory.

In summary, the results of the present study show that E2 up-regulates, although indirectly, the expression of a potent chemotactic factor for monocytes and T lymphocytes by endometriotic cells and that this chemokine is expressed in endometriotic lesions without noticeable cyclic variation. E2 enhances endometriotic cell responsiveness to the proinflammatory cytokine IL-1ß and increases RANTES gene transcription and mRNA stability. These findings may have an interesting significance in view of the biological properties of RANTES whose levels are elevated in the peritoneal fluid of women with endometriosis and the paramount role attributed to E2 in the pathophysiology of endometriosis, which in addition has been found to be abnormally produced locally in endometriotic tissue. Furthermore, they provide a new insight in the mechanisms of estrogen action in endometriosis and put forward a close relationship between immune and endocrine dysfunctions observed in this disease.

Acknowledgments

We thank Dr. Shaun McColl (Department of Microbiology and Immunology, University of Adelaide, Australia) for providing RANTES human recombinant protein and cDNA; Dr. Michel Marois, head of the Department of Pathology (Hôpital Saint-François d’Assise, Québec, Canada), for pathological investigations on endometriotic tissue biopsies; Drs. Jacques Bergeron and Marc Villeneuve for patient evaluation and providing endometriotic biopsies; and François Bigonnesse, Annie Boucher, Christine Jolicoeur, Monique Longpré, Johanne Pelletier, and Sylvie Pleau for technical assistance.

Footnotes

This work was supported by Grant MOP-14638 from Medical Research Council of Canada (to A.A.). A.A. is a "Chercheur Boursier National" of the Fonds de la Recherche en Santé du Québec (FRSQ).

Abbreviations: DTT, Dithiothreitol; E2, estradiol; FBS, fetal bovine serum; HBSS, Hanks’ balanced salt solution; MCP-1, monocyte chemotactic protein-1; P, progesterone; RANTES, regulated upon activation, normal T cell expressed, and secreted; SSC, saline sodium citrate.

Received January 28, 2002.

Accepted September 12, 2002.

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