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Effect of hormonal agents on monocyte chemotactic protein-1 expression by endometrial epithelial cells of women with endometriosis
FERTILITY AND STERILITY威 VOL. 74, NO. 5, NOVEMBER 2000 Copyright ©894 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Effect of hormonal agents on monocyte chemotactic protein-1 expression by endometrial epithelial cells of women with endometriosis Annie Boucher, M.S., Andre´ Lemay, M.D., Ph.D., and Ali Akoum, Ph.D. Unite´ d’Endocrinologie de la Reproduction, Centre de Recherche, Hoˆpital Saint-Franc¸ois d’Assise, Centre Hospitalier Universitaire de Que´bec, Universite´ Laval, Que´bec, Que´bec, Canada
Objective: To assess whether hormonal agents used in the medical treatment of endometriosis, such as danazol and GnRH agonist, exert direct regulatory action on monocyte chemotactic protein-1 (MCP-1) expression by endometrial epithelial cells. Design: Primary cultures of epithelial cells isolated from human endometrium were exposed to different concentrations of cytokines and steroid hormone analogs. Expression of MCP-1 was analyzed at the levels of protein and messenger RNA. Setting: Gynecology clinic and laboratory of endocrinology of reproduction. Patient(s): Women presenting for infertility or pelvic pain in whom endometriosis was diagnosed by using laparoscopy. Intervention(s): Endometrial tissue biopsy performed at laparoscopy. Main Outcome Measure(s): Secretion of MCP-1 protein was measured by using enzyme-linked immunosorbent assay, and mRNA steady-state levels were measured by performing Northern blot analysis. Result(s): Buserelin acetate, a GnRH agonist (0.1–10 ng/mL), had no significant effect on MCP-1 expression, whereas danazol (10⫺7–10⫺5 M), a testosterone analog, and dexamethasone, an anti-inflammatory glucocorticoid hormone (10⫺12–10⫺6 M), showed a direct and a dose-dependent inhibitory effect on MCP-1 expression. This effect occurred at the level of protein and mRNA. Conclusion(s): The findings of the study may affect understanding of the mechanisms by which hormonal treatments act on endometriosis and influence its clinical manifestations. (Fertil Steril威 2000;74:969 –75. ©894 by American Society for Reproductive Medicine.) Key Words: Danazol, endometriosis, endometrium, GnRH agonist, MCP-1 Received March 3, 2000; accepted June 1, 2000. Supported by grant MT14638 from the Medical Research Council of Canada (Dr. Akoum). Dr. Akoum is a ChercheurBoursier Senior of the Fonds de la Recherche en Sante´ du Que´bec. Reprint requests: Ali Akoum, Ph.D., Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hoˆpital SaintFranc¸ois d’Assise, 10 rue de l’Espinay, Local D0-711, Que´bec, Que´bec, Canada, G1L 3L5 (FAX: 418-5254481; E-mail: ali.akoum @crsfa.ulaval.ca). 0015-0282/⫺1006/$20.00 PII S0015-0282(00)01540-5
Endometriosis, the proliferation of endometrial-like tissue outside the uterus, is a common gynecologic disease that causes pelvic pain and infertility in about 10% of the female population (1). Endometriosis is associated with numerous immunologic changes in the eutopic endometrium of affected women (2); These include the presence of antibodies to specific endometrial antigens (3), increased secretion of interleukin-6 by stromal cells (4), elevated level of complement component C3 (5), marked leukocyte infiltration (6), increased expression of heat-shock protein 27 (7), and augmented release of intercellular adhesion molecule-1 by endometrial stromal cultures (8). According to our previous in vitro and in vivo studies, the
endometrial cells of women with endometriosis abnormally express increased levels of monocyte chemotactic protein-1 (MCP-1) (9, 10). This factor has the potent ability to chemoattract and activate monocytes and macrophages (11, 12) and may be involved in the increased recruitment of these cells into the endometrium of women with endometriosis. Endometriosis is an estrogen-dependent disorder that primarily affects women of reproductive age (13). Estradiol is believed to be the hormonal factor that plays a key role in development of the disease (13–15). Current medical therapy for endometriosis is based on suppression of ovarian function (16, 17). Danazol, a testosterone analog that inhibits secretion of 969
TABLE 1 Clinical characteristics of study patients and experiments performed on biopsy samples. Patient
Age
Cycle phase
Stage of endometriosis
Type of experiment
33 34 30 29 30 37 42 26 41 32 35 23 28
Proliferative Secretory Proliferative Proliferative Proliferative Secretory Proliferative Secretory Secretory Proliferative Secretory Proliferative Secretory
II IV II IV I III II I II III I I III
Interleukin-1 (time course and dose-response) Interleukin-1 (time course and dose-response) Interleukin-1 (time course and dose-response) Danazol Danazol Danazol Danazol Dexamethasone Dexamethasone Dexamethasone Buserelin acetate Buserelin acetate Buserelin acetate
1 2 3 4 5 6 7 8 9 10 11 12 13
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gonadotropins and suppresses gametogenesis and gonadal steroidogenesis, has been shown to have direct immunosuppressive properties (18, 19). On the other hand, gonadotropin-releasing hormone (GnRH) agonists, which are known to mainly act by desensitizing the pituitary gonadotropes with resultant reduction in circulating serum gonadotropin levels and inhibition of ovarian function, do not appear to directly affect immune function (16, 17).
Endometrial biopsy specimens were obtained by aspiration with an endometrial suction curette (Unimar Inc., Neuilly-En-Tchelle, France). They were immediately placed in sterile Hank’s balanced salt solution containing penicillin, 100 U/mL; streptomycin, 100 g/mL; and 0.25 amphotericin B, g/mL (GIBCO BRL, Burlington, Ontario, Canada) at 4°C and were transported to the laboratory.
Our previous data revealed enhancement of MCP-1 expression by endometrial cells of women with endometriosis in response to administration of ovarian steroids (Personal communication, Annie Boucher). These observations indicate a possible direct action of sex steroid on the inflammatory processes that take place in the endometrium. The objective of the present study was to investigate whether hormonal agents used in the medical treatment of endometriosis have a direct effect on MCP-1 expression by endometrial cells.
Endometrial tissue was minced into small pieces and incubated with collagenase (Sigma Chemical Co., St. Louis, MO) to dissociate epithelial glands from fibroblast-like cells. The material was further separated by differential sedimentation and adhesion as described elsewhere (9). Epithelial glands were dissociated by using 0.05% trypsin/0.53 mM ethylenediamine tetracetic acid (EDTA) (GIBCO BRL) for 10 minutes at 37°C, and 5% CO2; they were then cultured in 56-cm2 tissue culture dishes in Dulbecco’s modified Eagle medium F12 (GIBCO BRL) supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL); insulin, 10 g/mL (Sigma), transferrin, 5 g/mL (Sigma); and 1% antibiotics– antimycotics (GIBCO BRL). The purity of endometrial epithelial cell cultures has been verified morphologically by using phase-contrast microscopy and immunocytochemically on parallel coverslip cultures as described elsewhere (9). No CD-45 positive leukocytes were detected in culture, and less than 1% contamination by stromal vimentin–positive or endothelial factor VIII–positive cells was generally observed (data not shown).
MATERIALS AND METHODS Source and Handling of Tissue Endometrial biopsy specimens used in this study were obtained from 13 women with endometriosis (Table 1). Four women had stage I disease, 4 had stage II disease, 3 had stage III disease, and 2 had stage IV disease. All women gave signed informed consent, and the research protocol was approved by the Ethics Committee on Human Research of Hoˆpital Saint-Franc¸ois d’Assise. Participants were 23 to 42 years of age, had had consultation for infertility or pelvic pain (or both), were found to have endometriosis at the time of laparoscopy, had no endometrial hyperplasia or neoplasia, and had not received any anti-inflammatory or hormonal medication in the 3 months before laparoscopy. 970
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Tissue Dissociation and Cell Culture
Culture Stimulation and MCP-1 Synthesis Primary epithelial cell cultures grown to confluence were subcultured (using only one passage) with a split ratio of 1:2 in 56-cm2 petri dishes in Roosevelt Park Memorial Institute (RPMI) medium (GIBCO BRL) containing 10% dextranVol. 74, No. 5, November 2000
coated charcoal-treated FBS; insulin, 10 g/mL; transferrin, 5 g/mL; and 1% antibiotics–antimycotics. Optimization of Cell Stimulation with Interleukin-1 To evaluate the effects of hormonal therapeutic agents, we used interleukin (IL)-1 (Genzyme, Cambridge, MA) to induce MCP-1 expression by endometrial cells. To determine the optimal conditions of stimulation with IL-1, subcultured endometrial cells were grown to confluence and incubated overnight in FBS-free medium before being exposed to different concentrations of IL-1 (0.01–10 ng/mL) in a fresh FBS-free medium for varying times (0 –24 hours). Assays of the Agents Danazol (Sigma), 10⫺7–10⫺5 M, and GnRH agonist (buserelin acetate [Suprefact; Hoechst, Montreal, Quebec, Canada]), 0.1–10 ng/mL, were used. The ranges of concentrations were chosen according to previously established therapeutic plasma levels of these agents (19, 20). Treatment of endometrial cell cultures with danazol or with GnRHa was begun 2 days after cell passage. The culture medium was replaced with a fresh medium supplemented with hormones and was refreshed every 2 days. At confluence, cells were washed with serum-free RPMI, and incubation with hormones in RPMI medium enriched with 1% insulin-transferrin-selenium-linoleic acid (Becton-Dickinson Inc., Mississauga, Ontario, Canada) was performed for 3 more days. Cells were then rinsed and exposed (or not exposed) to IL-1, 0.1 ng/mL, for 6 hours. The culture supernatant was then collected and stored in small aliquots at ⫺20°C until MCP-1 assay using ELISA, as described elsewhere (21). Cells were dissociated with trypsin/ethylenediamine tetracetic acid and stored at ⫺80°C until Northern blot analysis. For dexamethasone (Sigma), cells grown to confluence were incubated overnight with serum-free RPMI, then with dexamethasone (10⫺12–10⫺6 M) (22) for 18 hours in the same medium. Cells were then exposed to IL-1, 0.1 ng/mL, for an additional 6 hours. Cells and culture supernatants were recovered as described above. The ranges of concentrations were chosen according to previously established therapeutic plasma levels of dexamethasone (23, 24).
Northern Blot Analysis Total RNA was extracted from cells with TRIzol reagent according to the manufacturer’s instructions (GIBCO BRL). The RNA was size-fractionated by using 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 minutes, prehybridized with a hybridization buffer composed of 5⫻ SSC; 5⫻ Denhardt’s solution; 50 mM NaH2PO4; sodium dodecylsulfate, 0.5%; salmon sperm DNA, 200 g/mL; and 50% formamide. It was hybridized with 32P-labeled MCP-1 complementary DNA (ATCC, FERTILITY & STERILITY威
Rockville, MD) in the hybridization buffer and washed with 1⫻ SSC (0.15 M sodium chloride and 0.015 M sodium citrate), 0.2⫻ SSC, and 0.1% sodium dodecylsulfate before being exposed to x-ray film (BioMax; Eastman Kodak, Rochester, NY) at ⫺80°C for about 18 hours. Staining with ethidium bromide (GIBCO BRL) and hybridization with 28S (Svedberg), cDNA probe (ATCC) were performed to ensure equal loading of RNA.
Statistical Analysis All experiments were repeated at least three times. Data were analyzed by using one-way analysis of variance; the Tukey test was used post hoc for multiple comparisons. P⬍.05 was considered statistically significant.
RESULTS Endometrial cells were first incubated with different concentrations of danazol in the absence of any stimulation. The effect of danazol was difficult to ascertain because spontaneous MCP-1 protein secretion in the culture medium and steady-state mRNA levels in nonstimulated cells were barely detectable (data not shown). On the basis of this finding, we assessed whether danazol or other therapeutic agents used to treat endometriosis affect MCP-1 expression by endometrial cells in response to IL-1, one of the major proinflammatory cytokines. Elevated concentrations of interleukin-1 were found in the peritoneal fluid of patients with endometriosis (25, 26). In addition, peripheral blood monocytes secrete higher levels of IL-1 in women with endometriosis (27), and an increased number of macrophages (known to be major source of IL-1) was detected in such patients (6). To determine the optimal concentration of IL-1 that can enable detection of possible effects of the hormonal agents, endometrial cells were first stimulated with different concentrations of IL-1 for varying periods. Dose–response data indicated dose-dependent stimulation of MCP-1 protein and mRNA expression during 6 hours of treatment, reaching maximal levels at 1 ng/mL (Fig. 1, A and B). Secretion of MCP-1 protein was detectable 2 hours after stimulation with 0.1 ng/mL of IL-1 and gradually increased during 24 hours of treatment. The highest level of mRNA was measured after 6 hours of stimulation; after this point, levels decreased over time (Fig. 1, C and D) (Table 1, patient 1). Similar cell response profiles were obtained in endometrial cell cultures of two additional endometrial biopsy specimens (Table 1, patients 2 and 3), even though MCP-1 secretion at baseline differed (data not shown). Therefore, an IL-1 concentration of 0.1 ng/mL and 6 hours of treatment with cytokine were subsequently used to evaluate the effect of hormonal therapeutic agents on IL-1–induced MCP-1 expression. Figure 2A shows that danazol (10⫺7–10⫺5 M) inhibited IL-1– induced MCP-1 protein secretion by endometrial cells in a dose-dependent manner. This effect was also observed on Northern blot analysis of MCP-1 mRNA steady-state levels 971
FIGURE 1
FIGURE 2
Dose-dependent course (A and B) and time course (C and D) of monocyte chemotactic protein (MCP)-1 expression by IL-1–treated endometrial epithelial cells. Confluent cultures were exposed to different concentrations of IL-1 (0.01–10 ng/mL) for increasing periods of time. At each time point, the culture supernatant was recovered for measurement of MCP-1 protein secretion by ELISA, and total cellular RNA was extracted to evaluate MCP-1 mRNA expression by using Northern blot analysis. (A) and (C) show MCP-1 protein secretion (expressed in pg/mL). (B) and (D) are autoradiographs showing hybridization of MCP-1 messenger RNA with 32Plabeled MCP-1 complementary DNA probe. Hybridization of 28S ribosomal RNA with a 32P-labeled 28S complementary DNA probe showed equal RNA loading (representative data from patient 1, Table 1).
Effect of danazol on interleukin (IL)-1–induced monocyte chemotactic protein (MCP)-1 expression by endometrial epithelial cells. Cells were treated with different concentrations of danazol (10⫺7 M–10⫺5 M) (see Materials and Methods) and with or without IL-1, 0.1 ng/mL, for 6 hours. (A), Secretion of MCP-1 protein in the culture medium was determined by using ELISA and is expressed as a percentage of control (conditioned medium from cultures were not subjected to hormonal treatment). Values are means (⫾SE) obtained from duplicate determinations in culture from four different endometrial biopsy specimens (patients 4, 5, 6, and 7, Table 1). **Significantly different from control (P⬍.01). (B), MCP-1 messenger RNA steady-state levels in endometrial cells were analyzed by using Northern blot analysis; an autoradiograph from a representative experiment is shown (patient 4, Table 1). Concomitant hybridization of 28S ribosomal RNA demonstrated equal RNA loading.
Boucher. MCP-1 regulation by hormonal agents. Fertil Steril 2000.
in endometrial cells, which declined as danazol concentrations increased in the culture medium (Fig. 2B). Interleukin1–induced MCP-1 protein secretion and mRNA steadystate levels were also inhibited in a dose-dependent manner by dexamethasone (10⫺12–10⫺6 M), an antiinflammatory agent known to inhibit MCP-1 expression in various cells (22, 28, 29) (Fig. 3). In contrast, buserelin acetate, a GnRH agonist, had no significant effect on MCP-1 expression by endometrial cells compared with benzalkonium chloride used at concentrations (0.01–1 ng/mL) equivalent to those present in the various busereline acetate dilutions (0.1–10 ng/mL) (Fig. 4).
DISCUSSION We show that danazol had a direct inhibitory effect on MCP-1 expression by IL-1–treated endometrial epithelial 972
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Boucher. MCP-1 regulation by hormonal agents. Fertil Steril 2000.
cells in women with endometriosis. Danazol significantly reduced MCP-1 protein secretion and mRNA steady-state levels after these cells were stimulated by IL-1. The danazol concentrations that produced an inhibitory effect were well within the range of plasma levels achieved after short-term and long-term oral administration of this agent in a clinical setting (18). Our findings may have interesting significance. Endometriosis is associated with a chronic immunoinflamVol. 74, No. 5, November 2000
FIGURE 3 Effect of dexamethasone on monocyte chemotactic protein (MCP)-1 expression by endometrial epithelial cells. Cells grown to confluence were incubated with different concentrations of dexamethasone (DXS) (10⫺12 M–10⫺6 M) for 18 hours and then with interleukin (IL)-1, 0.1 ng/mL, for 6 more hours. Conditioned media and cells were then recovered for ELISA and Northern blot analyses of MCP-1 protein and messenger RNA expression, respectively. (A), Secretion of MCP-1 protein was expressed as a percentage of control (cultures stimulated with IL-1, 0.1 ng/mL, without previous incubation with DXS). Values are means (⫾SE) obtained from duplicate determinations in cultures from three different endometrial biopsy specimens (patients 8, 9, and 10, Table 1). *P⬍.05 compared with control; **P⬍.01 compared with control. (B), Representative autoradiograph showing MCP-1 messenger RNA synthesis (patient 8, Table 1) and 28S ribosomal RNA showing equal RNA loading.
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matory process. Interleukin-1 is one of the major proinflammatory cytokines detected in elevated concentrations in the peritoneal cavity of patients with endometriosis (25, 26). The cytokine is mainly produced by activated macrophages, which are more actively recruited into the peritoneal cavity (30, 31) and infiltrate the ectopic and eutopic endometrial tissues of women with endometriosis (6, 32, 33). Peripheral blood monocytes from such women have been reported to release increased amounts of IL-1 as well (27). Taken together, these data strongly suggest that IL-1 may be involved FERTILITY & STERILITY威
FIGURE 4 Effect of a GnRH agonist (Suprefact) on monocyte chemotactic protein (MCP)-1 expression by endometrial cells. Cells were treated with agonist vehicle (benzalkonium chloride) (see Materials and Methods) and then with or without interleukin (IL)-1 for 6 hours. Secretion of MCP-1 protein in the culture medium was determined by using ELISA and is expressed as a percentage of control (conditioned medium from cultures were not subjected to hormonal treatment). (A), Values are means (⫾SE) obtained from duplicate determinations in cultures from three different endometrial biopsy specimens (patients 11, 12, and 13, Table 1). (B), MCP-1 messenger RNA steady-state levels in endometrial cells were analyzed by using Northern blot analysis; an autoradiograph of a representative experiment is shown (patient 16, Table 1). Concomitant hybridization of 28S ribosomal RNA demonstrated equal RNA loading.
Boucher. MCP-1 regulation by hormonal agents. Fertil Steril 2000.
locally in cell activation and may stimulate MCP-1 secretion by ectopic and eutopic endometrial tissues. Danazol, a testosterone analog, is one of the principal hormonal agents used in the medical management of endometriosis. It acts by inhibiting gonadotropin secretion and thus ovarian secretion of E2, creating a hypoestrogenic milieu inadequate for the development of endometriotic tissue. Moreover, this steroid analog is also known to have immuno-downregulating properties (18). Danazol has been shown to reduce serum levels of autoantibodies in women 973
with endometriosis and to inhibit secretion of proinflammatory cytokines by monocytes (34, 35). In previous studies, we have shown that E2 stimulates endometrial cell responsiveness to IL-1 and increases MCP-1 gene transcription (Personal communication, Annie Boucher). On the basis of these observations, it could be suggested that danazol inhibits MCP-1 expression by endometrial cells not only indirectly by reducing E2 levels, but directly through interaction with these cells. Of note, clinical data indicate that danazol therapy in patients with endometriosis reduced peripheral blood monocyte activation (36), the number of macrophages that infiltrate the eutopic endometrium, and peritoneal levels of proinflammatory cytokines produced by these cells (6, 26). These findings are in agreement with our in vitro data and indicate that inhibition of MCP-1 expression by danazol may account for its overall biological effects. Danazol has been shown to interact with androgens, progesterone, and the glucocorticoid receptors present on endometrial cells (19, 37) and to exert a direct inhibitory action on their growth (20). It is still unknown whether androgens have regulatory action on MCP-1 expression. Progesterone has been shown to inhibit MCP-1 expression by many types of cells (38, 39), but it seems to have no direct or measurable effect on MCP-1 expression by endometrial cells (Personal communication). These findings suggest that the direct down-regulation of MCP-1 expression in endometrial cells produced by danazol administration could be mediated at least in part by androgen and/or glucocorticoid receptor. In the present study, dexamethasone, an antiinflammatory glucocorticoid hormone, had potent inhibitory action on IL-1–induced MCP-1 expression by endometrial cells both at the level of protein and mRNA. According to recent studies, glucocorticoid receptor inhibits the activity of nuclear factor-B, one of the main transcriptional factors that mediates MCP-1 gene activation by IL-1, by physical interaction and by inducing inhibitory-B. Glucocorticoid receptor can also directly interact with cis-active elements in the promoter region of the MCP-1 gene (40, 41). Thus, it is plausible that both dexamethasone and danazol can inhibit MCP-1 gene expression at the transcriptional level. Further studies are needed to elucidate the mechanisms underlying the inhibitory action of danazol that we observed and to determine whether there is any effect on MCP-1 mRNA stability. We did not find a detectable direct effect of buserelin acetate on MCP-1 expression by endometrial epithelial cells. The presence of GnRH and GnRH receptor mRNA in human endometrium was recently demonstrated (42), but it is still unknown whether functional GnRH receptors are expressed by endometrial cells. On the other hand, the GnRH agonists widely used in the treatment of endometriosis are known to act mainly by inhibiting GnRH production and release of gonadotropin hormones, which suppresses steroidogenesis (16, 17). According to our results, which do not indicate a direct effect of GnRH agonist on MCP-1 974
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expression by endometrial epithelial cells, GnRH agonist would act by an indirect mechanism related to estrogen suppression. This mechanism of action is consistent with results of our recent studies demonstrating that E2 can potentiate MCP-1 expression by endometrial cells in response to IL-1 (Personal communication, Annie Boucher). In conclusion, we found a direct inhibitory action of danazol and dexamethasone on MCP-1 expression by eutopic endometrial cells of women with endometriosis and that busereline acetate, a GnRH agonist, had no detectable direct effect. These result may contribute to our understanding of the biochemical and biological mechanisms underlying the clinical effects of the hormonal therapeutic agents currently used to treat endometriosis. To our knowledge, glucocorticoids have rarely been used to treat endometriosis, but they appeared to decrease sera embryotoxicity in a clinical setting (43). The use of these drugs may be of clinical interest in view of the biological properties of MCP-1, levels of which are elevated in the serum, peritoneal fluid, and eutopic endometrium of women with endometriosis and in view of the immunoinflammatory process frequently associated with the disease (44 – 46).
Acknowledgments: The authors thank the group of investigation in gynecology, Centre Hospitalier Universitaire de Que´bec (CHUQ), Universite´ Laval, Que´bec, Canada (Drs. Franc¸ois Belhumeur, Jacques Bergeron, Jean Blanchet, Marc Bureau, Simon Carrier, Elphe`ge Cyr, Marle`ne Daris, JeanLouis Dube´, Jean-Yves Fontaine, Ce´line Huot, Pierre Huot, Johanne Hurtubise, Philippe Laberge, Rodolphe Maheux, Jacques Mailloux, Antonin Rochette, and Marc Villeneuve) for patient evaluation and providing endometrial biopsies, and Madeleine Desaulniers, Centre Hospitalier de l’Universite´ Laval, CHUQ, Universite´ Laval, Que´bec, Canada, Christine Jolicoeur, Monique Longpre´, and Johanne Pelletier, Unite´ d’Endocrinologie de la Reproduction, Centre de Recherche, Hoˆpital Saint-Franc¸ois d’Assise, CHUQ, Universite´ Laval, Que´bec, Canada, for technical assistance.
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scriptional induction of JE, a platelet-derived growth factor-inducible gene. J Biol Chem 1991;266:13261– 6. Loetscher P, Dewald B, Baggiolini M, Seitz M. Monocyte chemoattractant protein-1 and interleukin-8 production by rheumatoid synoviocytes. Effects of anti-rheumatic drugs. Cytokine 1994;6:162–70. Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1981;35:696 – 8. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333–7. Witz CA, Schenken RS. Pathogenesis of endometriosis. Semin Reprod Endocrinol 1997;15:199 –208. Bulmer JN, Jones RK, Searle RF. Intraepithelial leukocytes in endometriosis and adenomyosis: comparison of eutopic and ectopic endometrium with normal endometrium. Hum Reprod 1998;13:2910 –5. El-Roeiy A, Dmowski WP, Gleicher N, Radwanska E, Harlow L, Binor Z, et al. Danazol but not gonadotropin-releasing hormone agonists suppresses autoantibodies in endometriosis. Fertil Steril 1988;50:864 – 71. Mori H, M. Nakagawa T, Itoh N, Wada K, Tamaya K. Danazol suppresses the production of interleukin-1 and tumor necrosis factor by human monocytes. Am J Reprod Immunol 1990;24:45–50. Braun DP, Gebel H, Dmowski EP. Effect of danazol in vitro and in vivo on monocyte-mediated enhancement of endometrial cell proliferation in women with endometriosis. Fertil Steril 1994;62:89 –95. Barbieri RL, Ryan KJ. Danazol: endocrine pharmacology and therapeutic applications. Am J Obstet Gynecol 1981;141:453– 63. Kelly RW, Carr GG, Riley SC. The inhibition of synthesis of a beta-chemokine, monocyte chemotactic protein-1 (MCP-1), by progesterone. Biochem Biophys Res Commun 1997;239:557– 61. Arici A, Senturk LM, Seli E, Bahtiyar MO, Kim G. Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by estrogen and progesterone. Biol Reprod 1999;61:85–90. van der Burg B, van der Saag PT. Nuclear factor-kappa-B/steroid hormone receptor interactions as a functional basis of anti-inflammatory action of steroids in reproductive organs. Mol Hum Reprod 1996; 2:433– 8. Wissink S, van Heerde EC, van der Burg B, van der Saag PT. A dual mechanism mediates repression of NF-kappaB activity by glucocorticoids. Mol Endocrinol 1998;12:355– 63. Raga F, Casan EM, Kruessel JS, Wen Y, Huang HY, Nezhat C, et al. Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod 1998;59:661–9. Simon C, Gomez E, Mir A, de los Santos M, Pellicer A. Glucocorticoid treatment decreases sera embryotoxicity in endometriosis patient. Fertil Steril 1992;25:284 –9. Oral E, Olive DL, Arici A. The peritoneal environment in endometriosis. Hum Reprod Update 1996;2:385–98. McMaster MT, Lim KH, Taylor RN. Immunobiology of human pregnancy. Current Problems in Obstetrics, Gynecology and Fertility 1998; 21:1–24. Simon C, Caballero-Campo P, Garia-Velasco JA, Pellicer A. Potential implications of chemokines in reproductive function: an attractive idea. J Reprod Immunol 1998;38:169 –93.
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Effect of hormonal agents on monocyte chemotactic protein-1 expression by endometrial epithelial cells of women with endometriosis Annie Boucher, M.S., Andre´ Lemay, M.D., Ph.D., and Ali Akoum, Ph.D. Unite´ d’Endocrinologie de la Reproduction, Centre de Recherche, Hoˆpital Saint-Franc¸ois d’Assise, Centre Hospitalier Universitaire de Que´bec, Universite´ Laval, Que´bec, Que´bec, Canada
Objective: To assess whether hormonal agents used in the medical treatment of endometriosis, such as danazol and GnRH agonist, exert direct regulatory action on monocyte chemotactic protein-1 (MCP-1) expression by endometrial epithelial cells. Design: Primary cultures of epithelial cells isolated from human endometrium were exposed to different concentrations of cytokines and steroid hormone analogs. Expression of MCP-1 was analyzed at the levels of protein and messenger RNA. Setting: Gynecology clinic and laboratory of endocrinology of reproduction. Patient(s): Women presenting for infertility or pelvic pain in whom endometriosis was diagnosed by using laparoscopy. Intervention(s): Endometrial tissue biopsy performed at laparoscopy. Main Outcome Measure(s): Secretion of MCP-1 protein was measured by using enzyme-linked immunosorbent assay, and mRNA steady-state levels were measured by performing Northern blot analysis. Result(s): Buserelin acetate, a GnRH agonist (0.1–10 ng/mL), had no significant effect on MCP-1 expression, whereas danazol (10⫺7–10⫺5 M), a testosterone analog, and dexamethasone, an anti-inflammatory glucocorticoid hormone (10⫺12–10⫺6 M), showed a direct and a dose-dependent inhibitory effect on MCP-1 expression. This effect occurred at the level of protein and mRNA. Conclusion(s): The findings of the study may affect understanding of the mechanisms by which hormonal treatments act on endometriosis and influence its clinical manifestations. (Fertil Steril威 2000;74:969 –75. ©894 by American Society for Reproductive Medicine.) Key Words: Danazol, endometriosis, endometrium, GnRH agonist, MCP-1 Received March 3, 2000; accepted June 1, 2000. Supported by grant MT14638 from the Medical Research Council of Canada (Dr. Akoum). Dr. Akoum is a ChercheurBoursier Senior of the Fonds de la Recherche en Sante´ du Que´bec. Reprint requests: Ali Akoum, Ph.D., Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hoˆpital SaintFranc¸ois d’Assise, 10 rue de l’Espinay, Local D0-711, Que´bec, Que´bec, Canada, G1L 3L5 (FAX: 418-5254481; E-mail: ali.akoum @crsfa.ulaval.ca). 0015-0282/⫺1006/$20.00 PII S0015-0282(00)01540-5
Endometriosis, the proliferation of endometrial-like tissue outside the uterus, is a common gynecologic disease that causes pelvic pain and infertility in about 10% of the female population (1). Endometriosis is associated with numerous immunologic changes in the eutopic endometrium of affected women (2); These include the presence of antibodies to specific endometrial antigens (3), increased secretion of interleukin-6 by stromal cells (4), elevated level of complement component C3 (5), marked leukocyte infiltration (6), increased expression of heat-shock protein 27 (7), and augmented release of intercellular adhesion molecule-1 by endometrial stromal cultures (8). According to our previous in vitro and in vivo studies, the
endometrial cells of women with endometriosis abnormally express increased levels of monocyte chemotactic protein-1 (MCP-1) (9, 10). This factor has the potent ability to chemoattract and activate monocytes and macrophages (11, 12) and may be involved in the increased recruitment of these cells into the endometrium of women with endometriosis. Endometriosis is an estrogen-dependent disorder that primarily affects women of reproductive age (13). Estradiol is believed to be the hormonal factor that plays a key role in development of the disease (13–15). Current medical therapy for endometriosis is based on suppression of ovarian function (16, 17). Danazol, a testosterone analog that inhibits secretion of 969
TABLE 1 Clinical characteristics of study patients and experiments performed on biopsy samples. Patient
Age
Cycle phase
Stage of endometriosis
Type of experiment
33 34 30 29 30 37 42 26 41 32 35 23 28
Proliferative Secretory Proliferative Proliferative Proliferative Secretory Proliferative Secretory Secretory Proliferative Secretory Proliferative Secretory
II IV II IV I III II I II III I I III
Interleukin-1 (time course and dose-response) Interleukin-1 (time course and dose-response) Interleukin-1 (time course and dose-response) Danazol Danazol Danazol Danazol Dexamethasone Dexamethasone Dexamethasone Buserelin acetate Buserelin acetate Buserelin acetate
1 2 3 4 5 6 7 8 9 10 11 12 13
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gonadotropins and suppresses gametogenesis and gonadal steroidogenesis, has been shown to have direct immunosuppressive properties (18, 19). On the other hand, gonadotropin-releasing hormone (GnRH) agonists, which are known to mainly act by desensitizing the pituitary gonadotropes with resultant reduction in circulating serum gonadotropin levels and inhibition of ovarian function, do not appear to directly affect immune function (16, 17).
Endometrial biopsy specimens were obtained by aspiration with an endometrial suction curette (Unimar Inc., Neuilly-En-Tchelle, France). They were immediately placed in sterile Hank’s balanced salt solution containing penicillin, 100 U/mL; streptomycin, 100 g/mL; and 0.25 amphotericin B, g/mL (GIBCO BRL, Burlington, Ontario, Canada) at 4°C and were transported to the laboratory.
Our previous data revealed enhancement of MCP-1 expression by endometrial cells of women with endometriosis in response to administration of ovarian steroids (Personal communication, Annie Boucher). These observations indicate a possible direct action of sex steroid on the inflammatory processes that take place in the endometrium. The objective of the present study was to investigate whether hormonal agents used in the medical treatment of endometriosis have a direct effect on MCP-1 expression by endometrial cells.
Endometrial tissue was minced into small pieces and incubated with collagenase (Sigma Chemical Co., St. Louis, MO) to dissociate epithelial glands from fibroblast-like cells. The material was further separated by differential sedimentation and adhesion as described elsewhere (9). Epithelial glands were dissociated by using 0.05% trypsin/0.53 mM ethylenediamine tetracetic acid (EDTA) (GIBCO BRL) for 10 minutes at 37°C, and 5% CO2; they were then cultured in 56-cm2 tissue culture dishes in Dulbecco’s modified Eagle medium F12 (GIBCO BRL) supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL); insulin, 10 g/mL (Sigma), transferrin, 5 g/mL (Sigma); and 1% antibiotics– antimycotics (GIBCO BRL). The purity of endometrial epithelial cell cultures has been verified morphologically by using phase-contrast microscopy and immunocytochemically on parallel coverslip cultures as described elsewhere (9). No CD-45 positive leukocytes were detected in culture, and less than 1% contamination by stromal vimentin–positive or endothelial factor VIII–positive cells was generally observed (data not shown).
MATERIALS AND METHODS Source and Handling of Tissue Endometrial biopsy specimens used in this study were obtained from 13 women with endometriosis (Table 1). Four women had stage I disease, 4 had stage II disease, 3 had stage III disease, and 2 had stage IV disease. All women gave signed informed consent, and the research protocol was approved by the Ethics Committee on Human Research of Hoˆpital Saint-Franc¸ois d’Assise. Participants were 23 to 42 years of age, had had consultation for infertility or pelvic pain (or both), were found to have endometriosis at the time of laparoscopy, had no endometrial hyperplasia or neoplasia, and had not received any anti-inflammatory or hormonal medication in the 3 months before laparoscopy. 970
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Tissue Dissociation and Cell Culture
Culture Stimulation and MCP-1 Synthesis Primary epithelial cell cultures grown to confluence were subcultured (using only one passage) with a split ratio of 1:2 in 56-cm2 petri dishes in Roosevelt Park Memorial Institute (RPMI) medium (GIBCO BRL) containing 10% dextranVol. 74, No. 5, November 2000
coated charcoal-treated FBS; insulin, 10 g/mL; transferrin, 5 g/mL; and 1% antibiotics–antimycotics. Optimization of Cell Stimulation with Interleukin-1 To evaluate the effects of hormonal therapeutic agents, we used interleukin (IL)-1 (Genzyme, Cambridge, MA) to induce MCP-1 expression by endometrial cells. To determine the optimal conditions of stimulation with IL-1, subcultured endometrial cells were grown to confluence and incubated overnight in FBS-free medium before being exposed to different concentrations of IL-1 (0.01–10 ng/mL) in a fresh FBS-free medium for varying times (0 –24 hours). Assays of the Agents Danazol (Sigma), 10⫺7–10⫺5 M, and GnRH agonist (buserelin acetate [Suprefact; Hoechst, Montreal, Quebec, Canada]), 0.1–10 ng/mL, were used. The ranges of concentrations were chosen according to previously established therapeutic plasma levels of these agents (19, 20). Treatment of endometrial cell cultures with danazol or with GnRHa was begun 2 days after cell passage. The culture medium was replaced with a fresh medium supplemented with hormones and was refreshed every 2 days. At confluence, cells were washed with serum-free RPMI, and incubation with hormones in RPMI medium enriched with 1% insulin-transferrin-selenium-linoleic acid (Becton-Dickinson Inc., Mississauga, Ontario, Canada) was performed for 3 more days. Cells were then rinsed and exposed (or not exposed) to IL-1, 0.1 ng/mL, for 6 hours. The culture supernatant was then collected and stored in small aliquots at ⫺20°C until MCP-1 assay using ELISA, as described elsewhere (21). Cells were dissociated with trypsin/ethylenediamine tetracetic acid and stored at ⫺80°C until Northern blot analysis. For dexamethasone (Sigma), cells grown to confluence were incubated overnight with serum-free RPMI, then with dexamethasone (10⫺12–10⫺6 M) (22) for 18 hours in the same medium. Cells were then exposed to IL-1, 0.1 ng/mL, for an additional 6 hours. Cells and culture supernatants were recovered as described above. The ranges of concentrations were chosen according to previously established therapeutic plasma levels of dexamethasone (23, 24).
Northern Blot Analysis Total RNA was extracted from cells with TRIzol reagent according to the manufacturer’s instructions (GIBCO BRL). The RNA was size-fractionated by using 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 minutes, prehybridized with a hybridization buffer composed of 5⫻ SSC; 5⫻ Denhardt’s solution; 50 mM NaH2PO4; sodium dodecylsulfate, 0.5%; salmon sperm DNA, 200 g/mL; and 50% formamide. It was hybridized with 32P-labeled MCP-1 complementary DNA (ATCC, FERTILITY & STERILITY威
Rockville, MD) in the hybridization buffer and washed with 1⫻ SSC (0.15 M sodium chloride and 0.015 M sodium citrate), 0.2⫻ SSC, and 0.1% sodium dodecylsulfate before being exposed to x-ray film (BioMax; Eastman Kodak, Rochester, NY) at ⫺80°C for about 18 hours. Staining with ethidium bromide (GIBCO BRL) and hybridization with 28S (Svedberg), cDNA probe (ATCC) were performed to ensure equal loading of RNA.
Statistical Analysis All experiments were repeated at least three times. Data were analyzed by using one-way analysis of variance; the Tukey test was used post hoc for multiple comparisons. P⬍.05 was considered statistically significant.
RESULTS Endometrial cells were first incubated with different concentrations of danazol in the absence of any stimulation. The effect of danazol was difficult to ascertain because spontaneous MCP-1 protein secretion in the culture medium and steady-state mRNA levels in nonstimulated cells were barely detectable (data not shown). On the basis of this finding, we assessed whether danazol or other therapeutic agents used to treat endometriosis affect MCP-1 expression by endometrial cells in response to IL-1, one of the major proinflammatory cytokines. Elevated concentrations of interleukin-1 were found in the peritoneal fluid of patients with endometriosis (25, 26). In addition, peripheral blood monocytes secrete higher levels of IL-1 in women with endometriosis (27), and an increased number of macrophages (known to be major source of IL-1) was detected in such patients (6). To determine the optimal concentration of IL-1 that can enable detection of possible effects of the hormonal agents, endometrial cells were first stimulated with different concentrations of IL-1 for varying periods. Dose–response data indicated dose-dependent stimulation of MCP-1 protein and mRNA expression during 6 hours of treatment, reaching maximal levels at 1 ng/mL (Fig. 1, A and B). Secretion of MCP-1 protein was detectable 2 hours after stimulation with 0.1 ng/mL of IL-1 and gradually increased during 24 hours of treatment. The highest level of mRNA was measured after 6 hours of stimulation; after this point, levels decreased over time (Fig. 1, C and D) (Table 1, patient 1). Similar cell response profiles were obtained in endometrial cell cultures of two additional endometrial biopsy specimens (Table 1, patients 2 and 3), even though MCP-1 secretion at baseline differed (data not shown). Therefore, an IL-1 concentration of 0.1 ng/mL and 6 hours of treatment with cytokine were subsequently used to evaluate the effect of hormonal therapeutic agents on IL-1–induced MCP-1 expression. Figure 2A shows that danazol (10⫺7–10⫺5 M) inhibited IL-1– induced MCP-1 protein secretion by endometrial cells in a dose-dependent manner. This effect was also observed on Northern blot analysis of MCP-1 mRNA steady-state levels 971
FIGURE 1
FIGURE 2
Dose-dependent course (A and B) and time course (C and D) of monocyte chemotactic protein (MCP)-1 expression by IL-1–treated endometrial epithelial cells. Confluent cultures were exposed to different concentrations of IL-1 (0.01–10 ng/mL) for increasing periods of time. At each time point, the culture supernatant was recovered for measurement of MCP-1 protein secretion by ELISA, and total cellular RNA was extracted to evaluate MCP-1 mRNA expression by using Northern blot analysis. (A) and (C) show MCP-1 protein secretion (expressed in pg/mL). (B) and (D) are autoradiographs showing hybridization of MCP-1 messenger RNA with 32Plabeled MCP-1 complementary DNA probe. Hybridization of 28S ribosomal RNA with a 32P-labeled 28S complementary DNA probe showed equal RNA loading (representative data from patient 1, Table 1).
Effect of danazol on interleukin (IL)-1–induced monocyte chemotactic protein (MCP)-1 expression by endometrial epithelial cells. Cells were treated with different concentrations of danazol (10⫺7 M–10⫺5 M) (see Materials and Methods) and with or without IL-1, 0.1 ng/mL, for 6 hours. (A), Secretion of MCP-1 protein in the culture medium was determined by using ELISA and is expressed as a percentage of control (conditioned medium from cultures were not subjected to hormonal treatment). Values are means (⫾SE) obtained from duplicate determinations in culture from four different endometrial biopsy specimens (patients 4, 5, 6, and 7, Table 1). **Significantly different from control (P⬍.01). (B), MCP-1 messenger RNA steady-state levels in endometrial cells were analyzed by using Northern blot analysis; an autoradiograph from a representative experiment is shown (patient 4, Table 1). Concomitant hybridization of 28S ribosomal RNA demonstrated equal RNA loading.
Boucher. MCP-1 regulation by hormonal agents. Fertil Steril 2000.
in endometrial cells, which declined as danazol concentrations increased in the culture medium (Fig. 2B). Interleukin1–induced MCP-1 protein secretion and mRNA steadystate levels were also inhibited in a dose-dependent manner by dexamethasone (10⫺12–10⫺6 M), an antiinflammatory agent known to inhibit MCP-1 expression in various cells (22, 28, 29) (Fig. 3). In contrast, buserelin acetate, a GnRH agonist, had no significant effect on MCP-1 expression by endometrial cells compared with benzalkonium chloride used at concentrations (0.01–1 ng/mL) equivalent to those present in the various busereline acetate dilutions (0.1–10 ng/mL) (Fig. 4).
DISCUSSION We show that danazol had a direct inhibitory effect on MCP-1 expression by IL-1–treated endometrial epithelial 972
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Boucher. MCP-1 regulation by hormonal agents. Fertil Steril 2000.
cells in women with endometriosis. Danazol significantly reduced MCP-1 protein secretion and mRNA steady-state levels after these cells were stimulated by IL-1. The danazol concentrations that produced an inhibitory effect were well within the range of plasma levels achieved after short-term and long-term oral administration of this agent in a clinical setting (18). Our findings may have interesting significance. Endometriosis is associated with a chronic immunoinflamVol. 74, No. 5, November 2000
FIGURE 3 Effect of dexamethasone on monocyte chemotactic protein (MCP)-1 expression by endometrial epithelial cells. Cells grown to confluence were incubated with different concentrations of dexamethasone (DXS) (10⫺12 M–10⫺6 M) for 18 hours and then with interleukin (IL)-1, 0.1 ng/mL, for 6 more hours. Conditioned media and cells were then recovered for ELISA and Northern blot analyses of MCP-1 protein and messenger RNA expression, respectively. (A), Secretion of MCP-1 protein was expressed as a percentage of control (cultures stimulated with IL-1, 0.1 ng/mL, without previous incubation with DXS). Values are means (⫾SE) obtained from duplicate determinations in cultures from three different endometrial biopsy specimens (patients 8, 9, and 10, Table 1). *P⬍.05 compared with control; **P⬍.01 compared with control. (B), Representative autoradiograph showing MCP-1 messenger RNA synthesis (patient 8, Table 1) and 28S ribosomal RNA showing equal RNA loading.
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matory process. Interleukin-1 is one of the major proinflammatory cytokines detected in elevated concentrations in the peritoneal cavity of patients with endometriosis (25, 26). The cytokine is mainly produced by activated macrophages, which are more actively recruited into the peritoneal cavity (30, 31) and infiltrate the ectopic and eutopic endometrial tissues of women with endometriosis (6, 32, 33). Peripheral blood monocytes from such women have been reported to release increased amounts of IL-1 as well (27). Taken together, these data strongly suggest that IL-1 may be involved FERTILITY & STERILITY威
FIGURE 4 Effect of a GnRH agonist (Suprefact) on monocyte chemotactic protein (MCP)-1 expression by endometrial cells. Cells were treated with agonist vehicle (benzalkonium chloride) (see Materials and Methods) and then with or without interleukin (IL)-1 for 6 hours. Secretion of MCP-1 protein in the culture medium was determined by using ELISA and is expressed as a percentage of control (conditioned medium from cultures were not subjected to hormonal treatment). (A), Values are means (⫾SE) obtained from duplicate determinations in cultures from three different endometrial biopsy specimens (patients 11, 12, and 13, Table 1). (B), MCP-1 messenger RNA steady-state levels in endometrial cells were analyzed by using Northern blot analysis; an autoradiograph of a representative experiment is shown (patient 16, Table 1). Concomitant hybridization of 28S ribosomal RNA demonstrated equal RNA loading.
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locally in cell activation and may stimulate MCP-1 secretion by ectopic and eutopic endometrial tissues. Danazol, a testosterone analog, is one of the principal hormonal agents used in the medical management of endometriosis. It acts by inhibiting gonadotropin secretion and thus ovarian secretion of E2, creating a hypoestrogenic milieu inadequate for the development of endometriotic tissue. Moreover, this steroid analog is also known to have immuno-downregulating properties (18). Danazol has been shown to reduce serum levels of autoantibodies in women 973
with endometriosis and to inhibit secretion of proinflammatory cytokines by monocytes (34, 35). In previous studies, we have shown that E2 stimulates endometrial cell responsiveness to IL-1 and increases MCP-1 gene transcription (Personal communication, Annie Boucher). On the basis of these observations, it could be suggested that danazol inhibits MCP-1 expression by endometrial cells not only indirectly by reducing E2 levels, but directly through interaction with these cells. Of note, clinical data indicate that danazol therapy in patients with endometriosis reduced peripheral blood monocyte activation (36), the number of macrophages that infiltrate the eutopic endometrium, and peritoneal levels of proinflammatory cytokines produced by these cells (6, 26). These findings are in agreement with our in vitro data and indicate that inhibition of MCP-1 expression by danazol may account for its overall biological effects. Danazol has been shown to interact with androgens, progesterone, and the glucocorticoid receptors present on endometrial cells (19, 37) and to exert a direct inhibitory action on their growth (20). It is still unknown whether androgens have regulatory action on MCP-1 expression. Progesterone has been shown to inhibit MCP-1 expression by many types of cells (38, 39), but it seems to have no direct or measurable effect on MCP-1 expression by endometrial cells (Personal communication). These findings suggest that the direct down-regulation of MCP-1 expression in endometrial cells produced by danazol administration could be mediated at least in part by androgen and/or glucocorticoid receptor. In the present study, dexamethasone, an antiinflammatory glucocorticoid hormone, had potent inhibitory action on IL-1–induced MCP-1 expression by endometrial cells both at the level of protein and mRNA. According to recent studies, glucocorticoid receptor inhibits the activity of nuclear factor-B, one of the main transcriptional factors that mediates MCP-1 gene activation by IL-1, by physical interaction and by inducing inhibitory-B. Glucocorticoid receptor can also directly interact with cis-active elements in the promoter region of the MCP-1 gene (40, 41). Thus, it is plausible that both dexamethasone and danazol can inhibit MCP-1 gene expression at the transcriptional level. Further studies are needed to elucidate the mechanisms underlying the inhibitory action of danazol that we observed and to determine whether there is any effect on MCP-1 mRNA stability. We did not find a detectable direct effect of buserelin acetate on MCP-1 expression by endometrial epithelial cells. The presence of GnRH and GnRH receptor mRNA in human endometrium was recently demonstrated (42), but it is still unknown whether functional GnRH receptors are expressed by endometrial cells. On the other hand, the GnRH agonists widely used in the treatment of endometriosis are known to act mainly by inhibiting GnRH production and release of gonadotropin hormones, which suppresses steroidogenesis (16, 17). According to our results, which do not indicate a direct effect of GnRH agonist on MCP-1 974
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expression by endometrial epithelial cells, GnRH agonist would act by an indirect mechanism related to estrogen suppression. This mechanism of action is consistent with results of our recent studies demonstrating that E2 can potentiate MCP-1 expression by endometrial cells in response to IL-1 (Personal communication, Annie Boucher). In conclusion, we found a direct inhibitory action of danazol and dexamethasone on MCP-1 expression by eutopic endometrial cells of women with endometriosis and that busereline acetate, a GnRH agonist, had no detectable direct effect. These result may contribute to our understanding of the biochemical and biological mechanisms underlying the clinical effects of the hormonal therapeutic agents currently used to treat endometriosis. To our knowledge, glucocorticoids have rarely been used to treat endometriosis, but they appeared to decrease sera embryotoxicity in a clinical setting (43). The use of these drugs may be of clinical interest in view of the biological properties of MCP-1, levels of which are elevated in the serum, peritoneal fluid, and eutopic endometrium of women with endometriosis and in view of the immunoinflammatory process frequently associated with the disease (44 – 46).
Acknowledgments: The authors thank the group of investigation in gynecology, Centre Hospitalier Universitaire de Que´bec (CHUQ), Universite´ Laval, Que´bec, Canada (Drs. Franc¸ois Belhumeur, Jacques Bergeron, Jean Blanchet, Marc Bureau, Simon Carrier, Elphe`ge Cyr, Marle`ne Daris, JeanLouis Dube´, Jean-Yves Fontaine, Ce´line Huot, Pierre Huot, Johanne Hurtubise, Philippe Laberge, Rodolphe Maheux, Jacques Mailloux, Antonin Rochette, and Marc Villeneuve) for patient evaluation and providing endometrial biopsies, and Madeleine Desaulniers, Centre Hospitalier de l’Universite´ Laval, CHUQ, Universite´ Laval, Que´bec, Canada, Christine Jolicoeur, Monique Longpre´, and Johanne Pelletier, Unite´ d’Endocrinologie de la Reproduction, Centre de Recherche, Hoˆpital Saint-Franc¸ois d’Assise, CHUQ, Universite´ Laval, Que´bec, Canada, for technical assistance.
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