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Original Studies |
Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Pavillon Saint-François d’Assise, Centre Hospitalier Universitaire de Québec, 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.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endometriosis is known to be estrogen dependent. The disease usually arises at menarche and most often regresses spontaneously after menopause. However, it has been occasionally diagnosed in postmenopausal women with relatively high levels of estrogens (3, 4, 5). In a primate model, surgically induced endometriosis persisted only in the presence of ovarian steroids (3). Histological studies of endometriosis lesions during the menstrual cycle indicated that ectopic glands did not fully demonstrate the classic endocrine-mediated changes that manifest eutopic endometrium (6). Nevertheless, endometriotic lesions express sex steroid receptors and can undergo cyclical changes similar, but not identical, to those of the intrauterine endometrium (7). Moreover, current medical treatment of endometriosis is based on the suppression of ovarian function and the induction of hypoestrogenism (8, 9, 10), which, although often resulting in temporary involution of the disease, have been shown to reduce symptomatology and to reduce endometriotic lesion volume (10, 11).
According to numerous studies endometriosis is associated with immunological changes that were detected in the eutopic endometrium and the peripheral blood (reviewed in Refs. 12, 13, 14) but, in particular, were observed locally in the peritoneal cavity where the disease often develops. The peritoneal fluid of endometriosis patients was shown to contain elevated levels of autoantibodies (4, 15) and proinflammatory mediators (16, 17, 18, 19, 20) and to present increased infiltration by inflammatory cells (21), especially activated macrophages (20, 22). Macrophages are the predominant nucleated cells in the peritoneal fluid and represent the first line host response to inflammatory stimuli. However, in endometriosis, instead of favoring the elimination of misplaced endometrial cells, peritoneal macrophages may paradoxically contribute to their maintenance and growth by producing growth and angiogenic factors (23, 24, 25) and have a deleterious effect on the reproductive process through a direct cell-mediated damage or the release of embryotoxic substances (16, 17, 26, 27).
Ectopic endometrial implants may be a major source of the peritoneal inflammation seen in patients with endometriosis. This may be triggered by in situ menstruation and breakthrough bleeding (28), but also by numerous secretions, such as PGs (18), complement components (19), and proinflammatory cytokines (29, 30, 31, 32), which may alter the peritoneal environment and contribute to endometriosis-associated pain and infertility (16, 17, 20, 22, 27, 28, 33).
Using cell culture, we have recently been able to demonstrate that eutopic endometrial cells of women with endometriosis have the intrinsic faculty to secrete increased amounts of monocyte chemotactic protein-1 (MCP-1) compared to those from normal women (34) and found that ectopic endometrial cells also abundantly secrete this factor (35). Subsequently, we found that MCP-1 concentrations were elevated in the peritoneal fluid of patients with endometriosis, particularly in the mild but most active stages of the disease (36). MCP-1, a chemokine with potent chemotactic and activation properties toward monocytes (37, 38), could therefore play a key role in macrophage activation and their recruitment into the peritoneal cavity of patients with endometriosis and may also contribute to the infiltration of endometriotic implants by macrophages reported by many investigators (39).
Despite the undeniable role of estrogens in the maintenance and progression of endometriosis, the biochemical mechanisms of their action in the disease and their possible link to the immunoinflammatory process occurring in women with endometriosis have been poorly investigated. In the present study we report that estradiol (E2) enhances the responsiveness of ectopic endometrial cells to the proinflammatory cytokine interleukin-1ß (IL-1ß) by amplifying IL-1ß-induced MCP-1 expression at the level of both messenger ribonucleic acid (mRNA) synthesis and protein secretion, and we demonstrate that this effect is exerted at the transcriptional level.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue specimens used in this study (Table 1
) were obtained from women who signed a
written informed consent for a research protocol approved by the
Saint-François d’Assise Hospital ethics committee on human
research. These women (mean age ± SD, 36 ± 5
yr; n = 13), consulting for infertility and/or pelvic pain, were
found to have endometriosis during laparoscopy or laparotomy, had no
endometrial hyperplasia or neoplasia, and had not received any
antiinflammatory or hormonal medication during a period of at least 3
months before surgery. Endometriosis was staged during the operation
according to the revised American Fertility Society classification
system (40). The cycle phase [proliferative (PF) or secretory (SR)]
was determined according to the cycle history of patients and to the
serum progesterone (P) levels measured. Endometriotic tissues were
obtained from ovarian endometriomas (n = 6, 3 PF and 3 SR) and
peritoneal foci (n = 7, 3 PF and 4 SR). Endometriotic biopsies
were immediately placed at 4 C in sterile Hanks’ Balanced Salt
Solution containing 100 U/mL penicillin, 100 µg/mL streptomycin, and
0.25 µg/mL amphotericin; snap-frozen in liquid nitrogen with
Tissue-Tek OCT compound (Miles, Inc., Elkhart, IN); and stored at -80
C until analyzed by immunohistochemistry or immediately used for cell
culture experiments. Immunohistochemical analyses were performed on 12
of the 13 endometriotic tissue biopsies. However, for cell culture
experiments, sufficient tissue was available from only 5 ovarian
endometrioma cyst linings and 3 peritoneal endometriotic biopsies.
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Serial 4- to 5-µm cryosections were first fixed in 4% formaldehyde solution (Fisher Scientific, Pittsburgh, PA) got 20 min at room temperature, then permeabilized with Triton X-100 (1% in phosphate-buffered saline for 20 min at room temperature) and treated with 0.3% H2O2 in absolute methanol for 20 min at room temperature to eliminate endogenous peroxidase. Immunostaining was performed using a monoclonal mouse anti-MCP-1 antibody (10 µg/mL in phosphate-buffered saline containing 1% BSA; R&D Systems, Minneapolis, MN) and a Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA), diaminobenzidine (Sigma, St. Louis, MO) as the chromogen, and hematoxylin for counterstaining. The specificity of the immunoreactivity shown by the anti-MCP-1 antibody (primary antibody) was examined by preabsorption with an excess of MCP-1 (50 µg/mL) before incubation with endometriotic tissue sections. Sections incubated without the primary antibody or with mouse Ig of the same Ig class and concentration as the primary antibody were included as negative controls in all experiments. Endometriotic glands were identified using monoclonal antibodies specific to cytokeratins (AE1:AE3 Mix, ICN Biomedical, St. Laurent, Canada). Slides were viewed using a Leica Corp. microscope (model DMRB, Leica Corp.), and photomicrographs were taken with Kodak 100 ASA film (Eastman Kodak Co., Rochester, NY).
Tissue dissociation and cell culture
Endometriotic tissue was minced into small pieces and dissociated with collagenase as previously described (35). Cells were pelleted by centrifugation (200 x g, 10 min); resuspended in DMEM-Ham’s F-12 containing 10 µg/mL insulin, 5 µg/mL transferrin, and 10% FBS; and plated in 100-mm diameter culture dishes, then grown at 37 C in 5% carbon dioxide. In this study no attempt was made to separate epithelial cells from stromal fibroblast-like cells. These were identified morphologically in culture by light microscopy and immunocytochemically with specific monoclonal antibodies to cytokeratins and vimentin as previously described (35). No leukocytes were detected in the endometriotic cells detached from culture dishes and assessed by flow cytometry (data not shown).
Culture stimulation and MCP-1 synthesis
Endometriotic cells grown to confluence were seeded at 20,000 cells/cm2 in 25-cm2 culture flasks in RPMI medium (Life Technologies, Inc., Gaithersburg, MD) containing 10% dextran-coated charcoal-treated FBS, 10 µg/mL insulin, 5 µg/mL transferrin, and 1% antibiotics-antimycotics. For stimulation with IL-1ß, cells grown to confluence were incubated overnight with FBS-free medium before being exposed to different concentrations of IL-1ß (0–10 ng/mL; Genzyme Corp., Cambridge, MA) in a fresh FBS-free medium for varying periods of time (0–24 h). For treatment with ovarian steroids [P and 1,3,5,(10)-estratrien-3,17ß-diol 3-benzoate; Sigma], the culture medium was removed 2 days after cell passage and replaced with a fresh phenol red-free medium containing different concentrations of hormones. Cells were maintained in culture for 7–8 days (until confluence), and the medium was changed every 2 days. At confluence, cells were washed with serum-free RPMI enriched with 1% insulin-transferrin-selenium-linoleic acid (Becton Dickinson and Co., Mississauga, Canada), and incubation with hormones was continued in this medium for 42 h. Finally, cells were or were not exposed to IL-1ß, which was added to the culture medium to reach a final concentration of 0.1 ng/mL. Six hours later, the culture supernatant was collected and kept in small aliquots at -80 C until use in the MCP-1 assay by enzyme-linked immunosorbent assay (ELISA) as previously reported (36), whereas cells were dissociated with trypsin/ethylenediamine tetraacetate (EDTA) and kept at -80 C until use for Northern blot analysis. To determine the combined effect of E2 and P, cells were first treated with E2 alone until confluence, then with P and E2 together before stimulation, or not, with IL-1ß. In some experiments, cycloheximide (Sigma) was added to the cell culture at the same time as IL-1ß, at a final concentration 100 µg/mL.
Northern blot analysis
Total RNA was extracted from cells with Trizol reagent according to the manufacturer’s instructions (Life Technologies, Inc.). RNA was size fractionated by electrophoresis on 1% agarose gels containing 10% formaldehyde and transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech, Oakville, Canada). The membrane was then dehydrated at 37 C for 30 min; prehybridized with a hybridization buffer comprised of 5 x SSC (standard saline citrate)-5 x Denhardt’s solution, 50 mmol/L NaH2PO4, 0.5% SDS, 200 µg/mL salmon sperm DNA and 50% formamide; hybridized with 32P-labeled MCP-1 complementary DNA (cDNA; American Type Culture Collection, Manassas, VA) in the hybridization buffer; and washed with 1 x SSC, 0.2 x SSC, and 0.1% SDS, respectively, before being exposed to x-ray film (BioMax, Eastman Kodak Co.) at -80 C for about 18 h. Staining with ethidium bromide (Life Technologies, Inc.) and hybridization with 28S cDNA probe (American Type Culture Collection, Manassas, VA) were performed to ensure equal loading of RNA. Data were analyzed as ratios of the density of the hybridization signals of MCP-1 to 28S, as determined by computer-assisted densitometry (BioImage, Visage 110s, Genomic Solutions, Inc., Ann Arbor, MI).
mRNA stability and half-life experiments
Cells were treated with hormones, as described above, and incubated with IL-1ß (0.1 or 1 ng/mL) for 6 h. Transcription was then stopped with actinomycin D (10 µg/mL), and cells were harvested after different times of incubation with actinomycin D for RNA extraction and Northern blot analysis.
Nuclear run-on assay
Cell culture and treatment with hormones and IL-1ß were
performed as mentioned above. At the end of the treatment, cells were
scraped in a lysis buffer containing 0.25 mol/L sucrose, 10 mmol/L
HEPES (pH 8.0), 10 mmol/L MgCl2, 2 mmol/L
dithiothreitol (DTT), and 0.1% (vol/vol) Triton X-100, and homogenized
in a Dounce homogenizer (Kontes Co., Vineland, NJ) on ice. Nuclei were
isolated by centrifugation at 600 x g for 5 min at 4
C, washed twice by homogenization in fresh buffer, collected by
centrifugation, and stored in 80 µL glycerol storage buffer [50
mmol/L HEPES (pH 8.0), 40% (vol/vol) glycerol, 5 mmol/L
MgCl2, 0.1 mmol/L EDTA, and 2 mmol/L DTT]. For
in vitro transcription, nuclei were resuspended in 200 µL
reaction buffer containing 20 mmol/L HEPES (pH 8.0); 5 mmol/L
MgCl2; 90 mmol/L NH4Cl; 0.5
mmol/L MnCl2; 16% (vol/vol) glycerol; 0.04
mmol/L EDTA; 2 mmol/L DTT; 0.4 mmol/L each of ATP, CTP, GTP (Life Technologies, Inc.); and 0.25 mCi
[
-32P]UTP (3000 Ci/mmol). The reaction was
arrested by digestion with 100 µg/mL ribonuclease-free
deoxyribonuclease I (Life Technologies, Inc.) and 100
µg/mL proteinase K (Life Technologies, Inc.) in the
presence of 10 mmol/L CaCl2 and 25 µg yeast
transfer RNA (Roche, Mannheim, Germany) for 20 min at 37
C, followed by the addition of EDTA (15 mmol/L) and SDS (0.5%, wt/vol)
and a further incubation for 20 min at 37 C. RNA was extracted twice
with phenol/chloroform (1:1, vol/vol), precipitated overnight at -20 C
with 100% ethanol (2:1, vol/vol) in the presence of 7.5 mol/L ammonium
acetate (1:2, vol/vol), and collected by centrifugation at 12,000 rpm
at 4 C for 15 min. The process of enzyme digestion, phenol/chloroform
extraction, and ethanol precipitation was repeated; the RNA was
precipitated again with ammonium acetate and ethanol and finally
dissolved in 850 µL hybridization buffer containing 50 mmol/L PIPES
(pH 7.0), 0.5 mol/L NaCl, 2 mmol/L EDTA, 0.4% (wt/vol) SDS, and 33%
(vol/vol) formamide. The radioactive RNA was used to probe
alkali-denatured plasmid DNA (5 µg) or insert DNA (1 µg)
immobilized on nylon membranes using a slot blot apparatus
(Hoefer, San Francisco, CA). Hybridization was carried out
for 3 days at 42 C using 5–10 million cpm/mL hybridization buffer.
Membranes were washed four times with 2 x SSC-0.1% SDS at 65 C
for 30 min, incubated with 10 µg/mL ribonuclease A, and 100 µg/mL
proteinase K for 30 min at 37 C, respectively, and washed twice again
with 2 x SSC-0.1% SDS at 65 C for 30 min before being exposed to
x-ray films (BioMax) at -80 C.
Statistical analysis
All experiments were repeated at least three times. Data were analyzed using one-way ANOVA, and the Tukey test was used post-hoc for multiple comparisons. Differences were considered as statistically significant for P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the intrauterine endometrium, MCP-1 is expressed mostly in the
glandular epithelium in the secretory phase of the menstrual cycle
(41). Surprisingly, this was not the case in ectopic endometrium.
Intense MCP-1 immunoreactivity was localized in both endometriotic
glands and stroma (Fig. 1). A similar
pattern of immunostaining was observed whether the tissue sections were
from the inner lining of ovarian endometriomas (Fig. 1A) or from
peritoneal endometriotic foci (Fig. 1B), albeit it was relatively less
intense in the two white lesions included in the study. Furthermore, no
apparent difference related to the menstrual cycle phase was noted. The
epithelial glands of endometriotic lesions were identified because they
reacted positively with mouse monoclonal antibodies specific to
cytokeratins, whereas stromal cells did not show any positive
immunostaining for these epithelial cell-specific markers (data not
shown). Control experiments performed on serial sections of
endometriotic tissue verified the specificity of the immunolocalization
of MCP-1, as there was no noticeable immunostaining with normal mouse
Igs (C and D), with the primary antibody after preabsorption with an
excess of MCP-1, or when the primary antibody was omitted (data not
shown).
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Using an established protocol, endometriotic stromal and epithelial cells were cultured without any attempt of separation to preserve stromal/epithelial cell interactions and to evaluate the whole outcome of hormone treatment. According to previous investigations, differentiation and hormonal responsiveness of epithelial cells in vitro are dependent on an appropriate stromal environment (42, 43).
IL-1 is one of the major proinflammatory cytokines found in elevated
concentrations in the peritoneal fluid of patients with endometriosis
(16, 17). The protein is mainly secreted by activated macrophages,
which are more numerous in the peritoneal fluid of patients and show a
marked infiltration of endometriosis implants compared to eutopic
endometrium (39). In vitro experiments of the present study
showed that without stimulation, cultured endometriotic cells did not
express detectable amounts of MCP-1 mRNA by Northern blot. However, the
addition of IL-1ß to the culture medium induced both MCP-1 mRNA and
protein expression by endometriotic cells in a dose- and time-dependent
manner. The dose-response data depicted in Fig. 2 showed detectable stimulation of MCP-1
mRNA and protein expression at as low as 0.01 ng/mL IL-1ß for a 6-h
period of treatment and a maximal stimulation at 1 ng/mL. The levels of
protein secretion increased 2 h after stimulation with 0.1 ng/mL
IL-1ß and rose exponentially during the 24 h of treatment,
whereas the highest level of mRNA was found after 6 h of
stimulation and dropped afterward over time (Fig. 3). The addition of cycloheximide, an
inhibitor of protein synthesis, together with IL-1ß (0.1 ng/mL)
completely abolished MCP-1 protein secretion, whereas a superinduction
of the steady state levels of MCP-1 mRNA compared to those in cells
exposed to IL-1ß alone was observed (Fig. 2). 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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show that
endometriotic cell treatment with E2 and/or P had
no significant effect on MCP-1 protein secretion (Fig. 4A) and mRNA
steady state levels (Fig. 4B). Only MCP-1 protein secretion increased
slightly in some cultures exposed to E2 alone or
in combination with P (Fig. 4A).
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, C and D, treatment
with E2 enhanced MCP-1 protein secretion and mRNA
steady state levels by endometriotic cells in response to IL-1ß.
However, treatment of endometriotic cells with P, either alone or in
combination with E2, had no significant effect on
the IL-1ß-induced MCP-1 expression by these cells. Data from four
independent experiments were analyzed. For MCP-1 secretion, the
increase over the control value (cells stimulated only with IL-1ß),
expressed as the mean ± SEM, was statistically
significant for E2 (65 ± 10%;
P < 0.01) and E2 plus P (77
± 8%; P < 0.01), whereas for P (-4 ± 3%) no
significant effect was observed. MCP-1 concentrations in the
conditioned medium ranged from 11.5–80.4 ng/mL in the control,and from
20.1–124.7, 21.4–136.5, and 10.3–75.4 ng/mL in the presence of
E2, E2 plus P, and P,
respectively. For MCP-1 mRNA, steady state levels, expressed as the
ratio of MCP-1 hybridization signal density to that
of 28S, had increased by 201 ± 23% (P < 0.01)
after the addition of E2, by 223 ± 52%
(P < 0.01) after the addition of P to
E2-treated cells, and by 16 ± 11%
(P > 0.05) after the addition of P alone. Similar
results were obtained after simultaneous treatment with
E2 and P starting from the culture initiation or
with P concentrations varying between
10-8–10-6 mol/L (data
not shown). A similar pattern of response was obtained whether cells
were derived from ovarian cyst linings or from peritoneal endometriotic
biopsies.
To determine whether the E2-mediated
up-regulation of MCP-1 mRNA steady state levels in endometriotic cells
in response to IL-1ß, occurred at the transcriptional and/or the
posttranscriptional level, we evaluated MCP-1 mRNA stability and
nuclear transcription in cells pretreated, or not, with
E2 (10-8 mol/L) before
stimulation with 0.1 ng/mL IL-1ß for 6 h, as previously
described. As illustrated by Fig. 5, which depicts a representative autoradiogram, treatment with
E2 had no detectable effect on MCP-1 mRNA
kinetics of degradation after the arrest of de novo RNA
transcription by actinomycin D (10 µg/mL). However, as shown by
nuclear run-on analysis, the level of MCP-1 transcription in cells
exposed to E2 before stimulation with IL-1ß was
markedly higher (2.9 ± 0.7; mean ± SD
of three independent experiments) than that in cells maintained without
any previous hormonal treatment (Fig. 6),
suggesting a transcriptional regulation of MCP-1 expression by
E2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Little work has been performed to investigate the interactions between steroid hormones and the components of the immune system and their implications in the pathophysiology of endometriosis. E2 and P have been shown to stimulate vascular endothelial growth factor (VEGF) production by nonactivated and activated peritoneal fluid macrophages, which are more frequently found in patients with endometriosis (23). These steroids have also been shown to up-regulate VEGF expression by uterine endometrial stromal cells of women with endometriosis (47). However, in these studies the ovarian hormones appeared to exert a direct regulatory action. According to recent data, both eutopic endometrium of women with endometriosis and ectopic endometrial tissue may have the inherent capability to produce E2 locally, as they were found to express P450 aromatase, which catalyses C19 steroid conversion to estrogens (48). This suggests that E2 may contribute to the up-regulation of MCP-1 expression in the endometriotic tissue not only by an endocrine pathway, but also by a paracrine mechanism.
The findings of the present study are of additional interest as they extend our recently reported data revealing increased secretion of MCP-1 by eutopic endometrial cells of women with endometriosis (34) and elevated in situ expression of this factor in the eutopic endometrial tissue in the presence of the disease (41). Our results illustrate the complexity of MCP-1 regulation in endometriosis and provide a new insight into the debate regarding biochemical and functional differences between endometrial cells implanted in ectopic locations and their eutopic counterparts. In the endometrium, MCP-1 expression was up-regulated in endometrial glands, particularly during the secretory phase of the menstrual cycle (41). Immunohistochemical staining of MCP-1 in endometriosis tissues indicates that the chemokine is highly expressed in both the stromal and glandular compartments regardless of the menstrual cycle phase. Interestingly, cell culture experiments revealed that ectopic endometrial cells do respond to E2 by increasing IL-1-induced MCP-1 expression as we observed previously for eutopic endometrial cells, but, in contrast, they show no noticeable responsiveness to P either alone or in combination with E2, as this normally occurs during the secretory phase of the menstrual cycle. On the one hand, these results suggest a dysregulation of MCP-1 gene in endometrial stromal cells implanted in ectopic locations. On the other hand, they may explain the absence of cyclic variation in MCP-1 expression in endometriosis lesions compared to intrauterine endometrium (41) and suggest a reduced sensitivity to P in ectopic endometrial cells, which probably could be ascribed to the decreased P receptors observed in endometriosis tissues (44). Moreover, it is noteworthy that the lack of P-mediated regulation of MCP-1 expression by endometriotic cells and the particular cell responsiveness to E2 displayed in our in vitro culture model were consistently observed regardless of whether cells originated from ovarian endometriomas or from pelvic peritoneal red vesicles. Further studies will be necessary to investigate the hormonal regulation of MCP-1 in cells from other types of endometriosis lesions.
The findings of the present study may have an interesting physiological significance, as estrogens are believed to exacerbate the progression and effects of endometriosis, and MCP-1 is known as a major mediator of monocyte activation and recruitment into inflammatory sites (37, 38). Previous works from other laboratories and from our own have shown that MCP-1 concentrations and chemotactic activity for monocytes are elevated in the peritoneal fluid of women with endometriosis and varied according to the severity of the disease (36, 49), supporting an important role for this factor in the local peritoneal inflammation related to endometriosis. MCP-1 could be produced by numerous cell types, including mesothelial cells (49), activated macrophages themselves (37, 38), and endometrial cells, which may spill into the peritoneal cavity by tubal reflux and show an inherent property to secrete increased levels of MCP-1 in endometriosis, as we have previously shown (41). However, the high level of expression of MCP-1 that we observed in endometriosis implants in both epithelial glands and stroma and the ability of E2 to amplify MCP-1 synthesis and secretion by endometriotic cells strongly suggest that these cells could induce macrophage activation and recruitment and contribute to the local inflammatory process taking place in the disease. Interestingly, increased infiltration of macrophages was observed in endometriotic implants compared to eutopic endometrium (39), which makes plausible the involvement of MCP-1 in enhanced macrophage recruitment into the ectopic endometrial tissue. It should be pointed out that in the present study, ectopic endometrial cells were responsive to as little as 10 pg/mL IL-1ß. IL-1 is one of the major proinflammatory cytokines found in elevated concentrations in the peritoneal fluid of patients with endometriosis (16, 17). The data reported by Taketani et al. (17) indicated that IL-1ß production is also increased in endometriotic tissue and may therefore account for the high expression of MCP-1 found in this tissue and the release of the chemokine in the peritoneal environment .
Other substances endowed with chemotactic activity for macrophages, such as complement component 3 (C3) (19) and the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted) (29), have been found to be expressed by ectopic endometrial tissue. The hormonal modulation of C3 has not been documented, but added sex steroids had no apparent effect on RANTES expression (29). This is in keeping with our data, as in our in vitro model, neither E2 nor P by themselves demonstrated any significant effect on MCP-1 expression, but E2 rather clearly presented an indirect action. Nevertheless, these observations taken together suggest that ectopic endometrial tissue can contribute to a feedforward cascade of events that activates the immune system and perpetuates the infiltration of inflammatory cells associated with endometriosis and may be involved in endometriosis-associated infertility. In fact, recent clinical data from our group revealed that laparoscopic resection or ablation of minimal and mild endometriosis enhanced fecundity in infertile women (33).
The mechanisms by which E2 enhances MCP-1
expression by ectopic endometrial cells in response to IL-1ß remain
unclear. It is well documented that the induction of MCP-1 gene
transcription by IL-1ß involves transcriptional factors, such as
activating protein-1 and particularly nuclear factor-
B, which,
according to a recent study, is essential for IL-1-induced MCP-1 gene
transcriptional activity (50). The MCP-1 gene promoter region sequence,
although yet not complete, does not contain the palindromic
estrogen-responsive elements necessary to
E2/E2 receptor complex
binding and activation of gene transcription. Even if such
cis-acting elements were present, E2
treatment without subsequent exposure to IL-1ß did not have any
significant effect on MCP-1 mRNA steady state levels, making unlikely
any direct mechanism involving E2 receptor
binding to the MCP-1 gene regulatory region. However, nuclear run-on
analyses showed an enhancement at the transcriptional level of the
IL-1ß-induced MCP-1 gene expression by ovarian hormones. This
suggests a mechanism by which E2 may activate a
target gene whose products, in turn, may interact with IL-1ß-
induced transcription signals.
In summary, the results of the present study show that E2 up-regulates, although indirectly, the expression of a potent chemotactic and activating factor for monocytes by ectopic endometrial cells of women with endometriosis and that this chemokine is highly expressed by endometriotic lesions without noticeable cyclic variations. E2 enhances the responsiveness of endometriotic cells to the proinflammatory cytokine IL-1ß by increasing IL-1ß-induced MCP-1 production and exerts its action at the transcriptional level. These findings may have an interesting significance in view of the biological properties of MCP-1, whose levels are elevated in the serum, peritoneal fluid, and eutopic endometrium of women with endometriosis, and the paramount role attributed to E2 in the pathophysiology of endometriosis, which, in addition, is thought to be abnormally produced locally in eutopic and ectopic endometria. They also show for the first time a hormonal regulation of MCP-1 expression in ectopic endometrial cells and reveal a new method of interaction between the endocrine and the immune systems. Further investigations are needed to elucidate the mechanisms underlying the E2 stimulatory action on MCP-1 expression and the effect of commonly used hormonal therapeutic agents or antiestrogens on that expression, which may have a great potential therapeutic interest.
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Acknowledgments |
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Footnotes |
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2 Chercheur-Boursier Senior of the Fonds de la Recherche en
Santé du Québec.
Received August 2, 1999.
Revised October 18, 1999.
Accepted October 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and
Sp1 regulate transcription of the human monocyte chemoattractant
protein-1 gene. J Immunol. 153:2052–2063.[Abstract]
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M. Nishida, K. Nasu, J. Fukuda, Y. Kawano, H. Narahara, and I. Miyakawa Down-Regulation of Interleukin-1 Receptor Type 1 Expression Causes the Dysregulated Expression of CXC Chemokines in Endometriotic Stromal Cells: A Possible Mechanism for the Altered Immunological Functions in Endometriosis J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5094 - 5100. [Abstract] [Full Text] [PDF]
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O. Yoshino, Y. Osuga, Y. Hirota, K. Koga, T. Hirata, T. Yano, T. Ayabe, O. Tsutsumi, and Y. Taketani Endometrial Stromal Cells Undergoing Decidualization Down-Regulate Their Properties to Produce Proinflammatory Cytokines in Response to Interleukin-1{beta} via Reduced p38 Mitogen-Activated Protein Kinase Phosphorylation J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2236 - 2241. [Abstract] [Full Text] [PDF]
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A. Akoum, A. Lemay, and R. Maheux Estradiol and Interleukin-1{beta} Exert a Synergistic Stimulatory Effect on the Expression of the Chemokine Regulated upon Activation, Normal T Cell Expressed, and Secreted in Endometriotic Cells J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5785 - 5792. [Abstract] [Full Text] [PDF]
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S.-J. Tsai, M.-H. Wu, C.-C. Lin, H. S. Sun, and H.-M. Chen Regulation of Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Endometriotic Stromal Cells J. Clin. Endocrinol. Metab., December 1, 2001; 86(12): 5765 - 5773. [Abstract] [Full Text] [PDF]
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 D. Hornung, K. Klingel, K. Dohrn, R. Kandolf, D. Wallwiener, and R. N. Taylor Regulated on Activation, Normal T-Cell-Expressed and -Secreted mRNA Expression in Normal Endometrium and Endometriotic Implants : Assessment of Autocrine/Paracrine Regulation by in Situ Hybridization Am. J. Pathol., June 1, 2001; 158(6): 1949 - 1954. [Abstract] [Full Text] [PDF]
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, Effect of cycloheximide
(100 µg/mL) on MCP-1 secretion (A) and mRNA steady state levels (B)
in response to IL-1ß (0.1 ng/mL).
