Molecular Human Reproduction, Vol. 7, No. 9, 859-866,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro
1 Centre de Recherche, Hôpital Saint-François d'Assise, Centre Hospitalier Universitaire de Québec, Faculté de Médecine, Université Laval, Québec, Canada and 2 Department of Molecular Biosciences, University of Adelaide, Australia
Abstract
Endometriosis, an oestrogen-dependent disorder affecting women of reproductive age, is associated with active angiogenesis and an increased recruitment of leukocyte into the peritoneal cavity where the implants often develop. The role of oestrogens in the development of endometriosis has been clearly established, but the biochemical mechanisms of their action are still not clearly elucidated. The present study shows that interleukin-1 (IL-1) induces interleukin-8 (IL-8) secretion by endometriotic cells and that oestradiol enhances endometriotic cell responsiveness to IL-1. In contrast, no significant cell responsiveness to progesterone either alone in the culture medium or in combination with oestradiol was noted. Positive immunostaining for IL-8 was observed throughout endometriotic tissue, and no perceptible difference in the intensity of staining regarding the menstrual cycle phase was observed. Together with the in-vitro data, this suggests that IL-8 expression in endometriotic tissue is not subject to cyclic variation. Furthermore, this study provides evidence that oestradiol indirectly up-regulates the expression by ectopic endometrial cells of IL-8, a cytokine endowed with neutrophil chemotactic and angiogenic properties. This may contribute to peritoneal leukocyte recruitment and to the growth of endometriotic implants, and may be a new mechanism for oestradiol action in endometriosis.
endometriosis/oestradiol/interleukin-8/progesterone
Introduction
Endometriosis, defined by the presence of functional endometrial glands and stroma outside the uterine cavity, is a very frequent gynaecological disorder occurring during the reproductive age, and a common cause of infertility and chronic pelvic pain (Strathy et al., 1982
).
Both clinical and laboratory evidence indicates that endometriosis is oestrogen-dependent. The disease arises during the reproductive phase of life, but can be sporadically found in postmenopausal women with relatively high concentrations of oestrogens (Di Zerga et al., 1980; Meldrum, 1985; Gleicher et al., 1987). Ovarian steroids appear to be essential for the maintenance of endometriosis in vivo (Di Zerga et al., 1980). Endometriotic lesions express sex steroid receptors and can undergo cyclical changes which, however, do not proceed as clearly or as uniformly as in the uterine mucosa (Shaw, 1993). The main purpose of current endometriosis medical therapy is to reduce the level of oestrogen production and to induce a menopausal state (Barbieri and Ryan, 1981; Shaw, 1992; Lemay, 1993).
Endometriosis is associated with an immuno-inflammatory process which has been commonly observed in the peritoneal cavity where endometriotic lesions are frequently found. Elevated concentrations of pro-inflammatory mediators (Vernon et al., 1986; Fakih et al., 1987; Isaacson et al., 1989; Taketani et al., 1992) and increased concentrations of inflammatory cells (Halme et al., 1983; Hill et al., 1988) have been reported in the peritoneal fluid of women with endometriosis. Ectopic endometrial implants may provide a major source for the peritoneal inflammation seen in patients with endometriosis. This may be elicited by menstrual bleeding of haemorrhagic lesions (Halme, 1991), but also by numerous secretions such as prostaglandins (Vernon et al., 1986), pro-inflammatory cytokines (Rier et al., 1995; Akoum et al., 1996; Tseng et al., 1996; Hornung et al., 1997) and activation of Complement (Isaacson et al., 1989), which may alter the peritoneal environment and contribute to endometriosis- associated pain and infertility (Haney et al., 1981; Halme et al., 1983; Fakih et al., 1987; Halme, 1991; Taketani et al., 1992; Falcone and Hemmings, 1996; Marcoux et al., 1997).
Interleukin-8 (IL-8) is one of the inflammatory cytokines found in elevated concentrations in the peritoneal fluid of patients with endometriosis, particularly in the mild but most active stages of the disease (Ryan et al., 1995; Gasvani et al., 1998). The molecule mediates neutrophil migration and activation (Baggiolini et al., 1989), induces the expression of leukocyte adhesion molecules (Koch et al., 1992), promotes endometrial stromal cell proliferation (Arici et al., 1998b) and possesses angiogenic properties (Koch et al., 1992). IL-8 could therefore play an important role in leukocyte recruitment into the peritoneal cavity of women with endometriosis, and may contribute through its mitogenic and angiogenic activities to the ectopic growth of endometrial cells.
IL-8 can be produced by a variety of cell types, including monocytes (Yoshimura et al., 1987), endothelial cells (Strieter et al., 1989), fibroblasts (Larsen et al., 1989) and mesothelial cells (Goodman et al., 1992). Endometrial cells produce IL-8 (Arici et al., 1996) and the refluxed endometrial tissue or associated fluid may well represent a source of IL-8 in the peritoneal fluid. However, the presence of inflammation and neovascularization in and around ectopic endometrial implants and the appearance of inflammatory neutrophils in these lesions (Khorram et al., 1993) make plausible a local production of IL-8 by endometriotic implants.
In the present study, we have examined IL-8 expression in endometriotic tissue and investigated the effects of IL-1 and ovarian hormones on the regulation of that expression by endometriotic cells. Our study has revealed a marked expression of IL-8 throughout endometrial tissues regardless of the menstrual cycle phase. Furthermore, oestradiol increased the responsiveness of endometriotic cells to IL-1 by enhancing IL-8 production, whereas progesterone had no significant regulatory effect.
Materials and methods
Source and handling of tissue
Endometriotic tissue specimens used in this study (Table I) were obtained with written informed consent for a research protocol approved by the Saint-François d'Assise Hospital Ethics Committee on Human Research. These women, 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 anti-inflammatory 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 (American Fertility Society, 1985). The cycle phase was determined according to the cycle history of patients and to progesterone concentrations measured in the serum. Endometriotic biopsies were immediately placed at 4°C in sterile Hanks' balanced salt solution (HBSS) containing 100 IU/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, USA) and stored at –80°C until analysed by immunohistochemistry, or were directly used for cell culture. Some endometriotic specimens were embedded in paraffin for pathological investigations. All endometriotic biopsies were characterized histologically by the presence of endometrial-like glands.
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Immunohistochemistry
For immunohistochemical analysis, both paraffin-embedded and frozen endometriotic biopsies were used. Paraffin sections (4 µm) were first immersed in two successive toluene baths (3 min each) to dissolve paraffin. They were then gradually rehydrated by a series of alcohols (100, 95, 70%) and water baths (3 min each). Cryosections (5 µm) were immediately fixed in 4% formaldehyde solution (Fisher Scientific, Fair Lawn, NJ, USA) for 20 min at room temperature. Sections were then permeabilized with Triton X-100 [1% in phosphate-buffered saline (PBS)] for 20 min at room temperature, and treated with 0.3% H2O2 in absolute methanol for 20 min at room temperature to eliminate endogenous peroxidase. Immunostaining was performed using a polyclonal rabbit anti-IL-8 antibody [10 µg/ml in PBS containing 1% bovine serum albumin (BSA)] (Biosource International, Camarillo, CA, USA), a biotin-conjugated affinity-purified goat anti-rabbit antibody (H+L) (2 µg/ml) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), peroxidase-conjugated streptavidin (1.3 µg/ml) (Jackson ImmunoResearch Laboratories), diaminobenzidine (Sigma Chemical Co, St Louis, MO, USA) as chromogen and haematoxylin for counterstaining. Sections incubated without the primary antibody or with normal rabbit immunoglobulins (IgG) used at the same concentration as the primary antibody were included as negative controls in all experiments. Slides were viewed using a Leica microscope (Leica mikroskopie und systeme GmbH, Model DMRB) and photomicrographs were taken with Kodak 100 ASA film.
Tissue dissociation and cell culture
Endometriotic tissue was minced into small pieces and dissociated with collagenase as previously described (Akoum et al., 1995). Cells were pelleted by centrifugation (200 g/10 min), and plated in Dulbecco's modified Eagle's medium (DMEM)–Ham's F-12 medium containing 10 µg/ml insulin, 5 µg/ml transferrin and 10% fetal bovine serum (FBS) at 37°C, 5% CO2. In this study, no attempt was made to separate epithelial and stromal fibroblast-like cells. These cells were identified morphologically in culture by light microscopy and immunocytochemically with specific monoclonal antibodies to cytokeratins and vimentin as previously described (Akoum et al., 1995). No leukocytes were detected in the endometriotic cells detached from culture dishes and assessed by flow cytometry (data not shown).
Culture stimulation and IL-8 synthesis
Endometriotic cells grown to confluence were used after one passage. Tadpole-shaped glandular epithelial cells were present in our different endometriotic cell cultures, but cultures appeared morphologically to contain more stromal than epithelial cells. Cells were seeded at 20 000 cells/cm2 in 25 cm2 culture flasks in Roswell Park Memorial Institute medium (Gibco BRL, Burlington, ON, Canada) containing 10% dextran-coated charcoal-treated FBS (FBS-DC), 10 µg/ml insulin, 5 µg/ml transferrin and 1% antibiotics–antimycotics. For stimulation with IL-1ß, cells were grown to confluence and incubated overnight with FBS-free medium before being exposed to different concentrations of IL-1ß (0–10 ng/ml) (Genzyme, Cambridge, MA, USA) in a fresh FBS-free medium for varying periods of time (0–24 h). For treatment with ovarian steroids [progesterone: 4-pregnen-3, 20-dione and 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 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% ITS+ (insulin–transferrin–selenium–linoleic acid) (Becton Dickinson Inc., Mississauga, Ontario, Canada) and the incubation with hormones was further continued in this medium for 42 h. Finally, cells were or were not exposed to IL-1ß which was added to the culture medium to reach a final concentration of 0.1 ng/ml. The culture supernatant was collected 6 h later and kept in small aliquots at –80°C until use in the IL-8 assay by enzyme-linked immunosorbent assay (ELISA), whereas the 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 a final concentration 100 µg/ml at the same time as IL-1ß.
IL-8 ELISA
IL-8 concentrations were measured using an ELISA procedure developed in the laboratory. This assay uses a mouse monoclonal anti-human IL-8 antibody and a rabbit polyclonal anti-human IL-8 antibody (Biosource International). Briefly, 96-well plates were coated overnight at 4°C with the mouse monoclonal antibody. Plates were washed four times with 0.01 mol/l PBS containing 0.1% Tween 20 (washing buffer), and recombinant human IL-8 (R and D Systems, Minneapolis, MN, USA), used at concentrations ranging from 100 pg/ml to 6.4 ng/ml, or samples diluted in serum-free RPMI were then added to the plates. After 60 min incubation at 37°C, the plates were washed and incubated for 60 min at 37°C with the IL-8 polyclonal antibody (1/8000 dilution in PBS containing 0.5% BSA). The plates were then washed and incubated for 60 min at 37°C with a goat anti-rabbit IgG peroxidase conjugate (Zymed Laboratories Inc., San Francisco, CA, USA) (1/2000 dilution in PBS/0.5% BSA). After a final wash, 100 µl of TMB (3,3',5,5',-tetramethylbenzidine)–peroxidase substrate (Bio-Rad Laboratories Ltd, Mississauga, Ontario, Canada) was added to each well, the enzymatic reaction was terminated by the addition of 50 µl of 2 N H2SO4 and the optical density was determined at 450 nm. IL-8 concentrations were calculated by interpolation from the standard curve. The sensitivity limit of the assay was 50 pg/ml, with mean intra- and inter-assay coefficients of variation ~8%.
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 5xSSC (0.15 mol/l sodium chloride and 0.015 mol/l sodium citrate), 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 IL-8 cDNA 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 28S cDNA probe (American Type Culture Collection, Rockville, MD, USA) were performed to ensure equal loading of RNA.
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. Differences were considered as statistically significant for P values < 0.05.
Results
IL-8 expression in endometriotic lesions
IL-8 immunoreactivity was intense and localized both in the stroma and epithelial glands of endometriotic lesions (Figure 1). The intensity and distribution of IL-8 immunostaining were comparable in tissue sections from peritoneal endometriotic foci (Figure 1A) and the inner lining of ovarian endometriomas (Figure 1B). Furthermore, no apparent difference related to the menstrual cycle phase was noted. Control experiments performed on serial sections of endometriotic tissue confirmed the specificity of the immunoreaction as no immunostaining was observed when the primary antibody was replaced by normal rabbit IgG (C and D) or preabsorbed with an excess of IL-8 prior to incubation with tissue sections, or when it was completely omitted (data not shown).
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Effect of IL-1ß and ovarian hormones on IL-8 expression
Stromal fibroblast-like cells were predominant in endometriotic cell cultures, as endometriotic tissue generally contains fewer glands than eutopic endometrial tissue. However, endometriotic stromal fibroblast-like and epithelial cells were cultured without any prior cell separation or enrichment as previously described (Akoum et al., 1995
). This has the advantage of preserving cell interactions, but also better reflects in vitro the global response of endometriotic tissue. Both stromal and epithelial cells from endometriotic tissues express oestrogen and progesterone receptors (Bergqvist and Ferno, 1993; Nisolle et al., 1994).
Endometriotic cells released low but detectable amounts of IL-8 in the culture medium in the absence of any stimulus (Figure 2). Cell exposure to IL-1ß resulted in a dose- and time-dependent increase in IL-8 mRNA steady-state levels and protein secretion in the culture medium. Stimulation of IL-8 protein secretion was observed at 0.01 ng/ml IL-1ß, and both IL-8 protein and mRNA levels increased gradually at higher concentrations (up to 10 ng/ml) (Figure 2). The protein secretion increased 6 h following stimulation with 0.1 ng/ml of IL-1ß and continued to increase during the 24 h of treatment, whereas the highest level of mRNA was found after 6 h of stimulation and diminished subsequently over time (Figure 3). Cell treatment with cycloheximide, an inhibitor of protein synthesis, in combination with IL-1ß (0.1 ng/ml) considerably reduced IL-8 protein secretion, but caused a marked increase in IL-8 mRNA steady-state levels as compared to IL-1ß alone (Figure 2). Therefore, IL-1ß exerts a direct effect on endometriotic cell steady-state IL-8 mRNA expression that does not necessarily require de-novo protein synthesis.
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To investigate whether ovarian hormones may be involved in the regulation of IL-8 expression in endometriotic lesions, we used an established long-term cell culture treatment protocol in which endometriotic cells were exposed first to oestradiol at the onset of culture, then to combined oestradiol and progesterone following cell confluence. Ovarian steroid doses (10–8 mol/l for oestradiol and 10–6 mol/l for progesterone) 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 (De Leon et al., 1986
; McLaren et al., 1996). Endometriotic cells were first treated with ovarian hormones without any subsequent stimulation with IL-1ß. The results of a representative experiment are illustrated in Figure 4. No detectable effect on IL-8 expression, either at the level of protein secretion (Figure 4A) or at the level of mRNA synthesis (Figure 4B) was observed under those conditions. In parallel experiments, cells were pretreated with ovarian hormones as indicated above, before being exposed after confluence to 0.1 ng/ml IL-1ß for 6 h. As illustrated in Figure 4A and B, treatment with oestradiol enhanced IL-8 protein secretion and mRNA steady-state levels by endometriotic cells in response to IL-1ß. However, treatment with progesterone, either alone or in combination with oestradiol, did not significantly affect IL-8 expression by endometriotic cells, although a very slight attenuation of oestradiol action in the combined treatment could be seen. Results from three independent experiments were analysed. For IL-8 secretion, the increase over the control (cells stimulated only with IL-1ß) was statistically significant for oestradiol (68 ± 11%) (P < 0.01) and for oestradiol + progesterone (62 ± 9%) (P < 0.01), whereas for progesterone no significant effect (7 ± 3%) was observed. For IL-8 mRNA, steady-state levels, expressed as the ratio of IL-8 hybridization signal density to that of 28S, increased by 108 ± 19% (P < 0.01) after the addition of oestradiol, by 97 ± 22% (P < 0.01) after the addition of progesterone to oestradiol-treated cells and by 2 ± 5% (P > 0.05) after the addition of progesterone alone.
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Discussion
In this study we have shown that IL-8 was markedly expressed both in the glands and in the stroma in endometriotic tissue. In culture, endometriotic cells produced high concentrations of IL-8 following stimulation with IL-1ß. Cell responsiveness to IL-1ß was detectable at 10 pg/ml which is in the lowest range of IL-1 concentrations in the peritoneal fluid of women presenting with endometriosis (Taketani et al., 1992). IL-1 concentrations significantly increase in the peritoneal fluid of women with endometriosis (Fakih et al., 1987; Taketani et al., 1992). The cytokine may be produced by several cell types (Dinarello, 1997), but mainly by macrophages which have been shown to infiltrate endometriotic implants and to be actively recruited into the peritoneal cavity of endometriosis patients (Haney et al., 1981; Halme et al., 1983, 1988; Zeller et al., 1987; Witz and Schenken, 1997). Therefore, endometriotic implants themselves may represent a potential source of IL-8, and may produce this cytokine in response to local pro-inflammatory stimuli such as IL-1.
In the present study we have also shown that oestradiol increased endometriotic cell responsiveness to IL-1ß. Cell pretreatment with oestradiol by itself had no significant effect on IL-8 expression, but increased IL-1ß-induced IL-8 protein secretion and mRNA synthesis. These data indicate that oestrogens can up-regulate IL-8 expression by endometriotic cells, but act through an indirect mechanism. Moreover, they show a synergistic stimulatory action between oestradiol and the pro-inflammatory cytokine IL-1, exerted at the level of endometriotic cells, and this may actually occur in endometriotic implants.
Oestrogens are believed to be essential for the maintenance and growth of ectopic endometrium, and may play a major role in the disease-associated biological changes and clinical manifestations. Among these manifestations are chronic pelvic inflammatory reactions (Halme, 1991; Oral et al., 1996) and obvious neovascularization in most active endometriotic lesions (Nisolle et al., 1993; Shaw, 1993; Wiegerinck et al., 1993). However, little work has been done to investigate the biochemical mechanisms of oestrogen actions in endometriosis. Oestradiol has been reported to promote vascular endothelial growth factor (VEGF) production by peritoneal fluid macrophages (McLaren et al., 1996). This steroid, in concert with progesterone, has also been shown to stimulate VEGF expression in endometrial stromal cells of women with endometriosis (Shifren et al., 1996). However, in those studies, oestradiol appeared to exert a direct regulatory action. Recent studies have indicated that oestradiol may be abnormally produced in endometrial tissue of women with endometriosis, and in ectopic endometrial implants (Noble et al., 1996). This suggests that oestradiol may contribute to the up-regulation of IL-8 expression in endometriotic tissue by endocrine as well as by paracrine mechanisms.
To survive and proliferate in ectopic locations, endometriotic implants require the establishment of a new blood supply, therefore an active neovascularization. Endometriotic lesions have been shown to produce VEGF (Donnez et al., 1998), and oestradiol has been shown to induce VEGF production by macrophages (Witz and Schenken, 1997). IL-8, a potent chemoattractant and activating factor for neutrophils, also has the capability of promoting endometrial as well as endothelial cell growth (Koch et al., 1992; Arici et al., 1998). Our data, showing a local production of IL-8 in endometriosis lesions that is induced by IL-1 and oestradiol, makes it plausible that IL-8 acts as an autocrine factor which may facilitate, both by direct and indirect mechanisms, the survival and the growth of ectopic endometrial tissue, and contribute to the accumulation of inflammatory cells within the endometriotic implants.
IL-8 expression by endometriotic cell cultures was not significantly modified following exposure to progesterone either alone, or in combination with, oestradiol as this normally occurs during the secretory phase of the menstrual cycle. These in-vitro data are consistent with immunohistochemical analysis of IL-8 expression in endometriotic tissue, which has not revealed any noticeable difference between tissues from the proliferative and the secretory phase of the menstrual cycle. Investigations have been carried out to localize the site of IL-8 protein synthesis in the endometrium, but the data are conflicting. Arici et al. (1998) has been described cycle-dependent glandular and surface epithelial localization of IL-8 protein, whereas another study (Milne et al., 1999) reported perivascular IL-8 mRNA expression in late secretory endometrium. Studies regarding the effect of progesterone on IL-8 production by endometrial cells have also led to conflicting data. An inhibitory effect of progesterone on IL-8 production by endometrial explants has been described (Kelly et al., 1994), whereas another group (Arici et al., 1996), using stromal cells, have shown that progesterone enhances the action of IL-1 to increase the levels of IL-8 mRNA. However, it is noteworthy that these studies were performed on endometrial cells of normal women, and the effects of ovarian hormones on eutopic endometrial cells of women with endometriosis remain unknown.
To our knowledge, there is no report describing the mechanisms of oestrogen action on IL-8 production, and it remains unclear how oestradiol enhances IL-1-mediated IL-8 expression by endometriotic cells. Oestradiol seems even to have a cell-type dependent effect, as it has been shown to inhibit IL-8 secretion by human osteoclast-like cells (Sunyer et al., 1999). Further investigations will be necessary in order to elucidate whether oestradiol acts by increasing the IL-1-induced IL-8 gene transcription and/or by post-transcriptional mechanisms.
In summary, our study has revealed that IL-8, a potent chemotactic and activating factor for neutrophils, is markedly expressed in endometriotic lesions and that oestradiol indirectly up-regulates the expression of this chemokine by ectopic endometrial cells. Oestradiol appeared to augment endometriotic cell responsiveness to IL-1ß by increasing the IL-1ß-induced IL-8 production, thereby revealing a hormonal regulation of IL-8 expression in ectopic endometrial cells and illustrating a close relationship between immune and endocrine dysfunction in endometriosis. This may be of relevance to the understanding of endometriosis pathophysiology, considering the pro-inflammatory and angiogenic properties of IL-8, its elevated concentrations in the peritoneal fluid of women with endometriosis, and the major role that oestradiol, which may be abnormally produced in eutopic and ectopic endometrial tissues, plays in the pathophysiology of the disease. The mechanisms of oestradiol's stimulatory action on IL-8 expression remain to be further elucidated. In addition, it will be of interest to investigate the effect of current hormonal therapeutic agents or that of anti-oestrogens in this system, as this may be of substantial therapeutic interest.
Acknowledgements
The authors wish to thank Annie Boucher, Christine Jolicoeur, Monique Longpré and Johanne Pelletier for technical assistance. A.A. 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 A.A. 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-François d'Assise, 10 rue de l'Espinay, Local D0-711, Québec, Québec, Canada, G1L 3L5. E-mail: ali.akoum{at}crsfa.ulaval.ca 
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Submitted on January 25, 2001; accepted on July 7, 2001.
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