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BOR - Papers in Press, published online ahead of print May 18, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038331
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BIOLOGY OF REPRODUCTION 73, 565–570 (2005)
DOI: 10.1095/biolreprod.104.038331
© 2005 by the Society for the Study of Reproduction, Inc.

Stimulation of Macrophage Migration Inhibitory Factor Expression in Endometrial Stromal Cells by Interleukin 1, beta Involving the Nuclear Transcription Factor NF{kappa}B1

W.-G. Cao 3, M. Morin 3, C. Metz 4, R. Maheux 3, and A. Akoum 2, 3

Unité d'endocrinologie de la reproduction,3 Centre de Recherche, Hôpital Saint-François d'Assise, Centre Hospitalier Universitaire de Québec, Faculté de Médecine, Université Laval, Québec, Canada G1L 3L5 Institute for Medical Research at North Shore-LIJ,4 Manhasset, New York 11030


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endometriosis, the ectopic development of endometrial tissue, is, particularly in peritoneal endometriosis, believed to result from tubal reflux of menstrual tissue. The release of cytokines and growth factors by refluxed endometrial cells in response to peritoneal inflammatory stimuli may enhance the capability of endometrial cells to implant and grow into the peritoneal host tissue. Herein we report that interleukin 1 (IL1), a major proinflammatory cytokine that is overproduced by endometriosis women-derived peritoneal macrophages and found in elevated concentrations in the peritoneal fluid of patients with endometriosis, stimulates the synthesis and the secretion of macrophage migration inhibitory factor (MIF) by human endometrial stromal cells. IL1B (0.1–100 ng/ml) exerted dose- and time-dependent effects of MIF protein secretion and mRNA synthesis, as shown by ELISA and reverse transcription-polymerase chain reaction, respectively. IL1B appeared to induce MIF gene transcription via the {kappa}B nuclear transcription factor (NF{kappa}B), as shown by electrophoretic mobility shift assay and Western blot analysis of I{kappa}B phosphorylation. Curcumin (10–8 M), which is known for inhibiting NF{kappa}B activation, inhibited IL1B-induced MIF secretion as well as NF{kappa}B nuclear translocation and DNA binding. Taken together, these findings clearly show that IL1B up-regulates the expression of MIF in endometrial stromal cells in vitro and acts via NF{kappa}B. This may play an important role in the physiology of the human endometrium and the pathophysiology of endometriosis considering the immunomodulatory properties of MIF as well as its role in cell growth, angiogenesis and tissue remodeling.

cytokines, endometrial cells, endometriosis, female reproductive tract, IL1B, menstrual cycle, MIF, NF{kappa}B, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endometriosis, one of the most common gynecologic diseases, affects women at reproductive age and is frequently associated with pain and infertility [1]. The mechanisms underlying endometriosis pathogenesis are not well elucidated, but it is believed that the disease, at least peritoneal endometriosis, arises from the ectopic growth of endometrial tissue, which during menstruation, reaches the peritoneal cavity by tubal reflux [2]. Numerous factors, including genetic, hormonal, and immunologic factors, may contribute to the aberrant ectopic growth of endometrial tissue [3]. We believe that the release of cytokines and growth factors by refluxed endometrial cells in response to peritoneal inflammatory stimuli may enhance the capability of endometrial cells to implant and grow into the peritoneal host tissue [4]. Our previous studies showed a marked expression of macrophage migration inhibitory factor (MIF) in ectopic endometrial implants and an increased concentration of MIF in the peritoneal cavity of patients with endometriosis [5, 6]. First described as a cytokine endowed with the property of retaining and activating macrophages [7, 8], MIF rather appears as a pivotal regulator of the host innate immunity and an integral component of host immune system that promotes the proinflammatory functions of immune cells [9]. MIF has recently been shown to be implicated in tumorigenesis as well as in the pathogenesis of many inflammatory and autoimmune diseases [9–11]. Interleukin 1 (IL1), a major proinflammatory cytokine released primarily by macrophages, is believed to play a considerable role in endometriosis pathophysiology [12–18]. In endometriosis, peritoneal macrophages release increased amounts of IL1 [15, 16, 19]. Elevated concentrations of IL1 were detected in the peritoneal cavity of patients with endometriosis [15, 16], where endometrial tissue migrates and develops into endometriotic lesions. The aim of the present study therefore was to assess whether IL1 may influence MIF secretion by endometrial cells. Interaction between IL1 and MIF, owing to the properties of these two major and multifunctional cytokines, may favor ectopic endometrial tissue growth and play a considerable role in the pathophysiology of endometriosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Hanks balanced salt solution (HBSS) without calcium and magnesium, Dulbecco modified Eagle medium-F12 (DMEM-F12), fetal bovine serum (FBS), antibiotics-antimycotics, and TRIzol reagent were from Invitrogen Life Technologies (Burlington, ON, Canada). Recombinant human (rh) IL1B, mouse monoclonal, and goat polyclonal anti-hMIF antibodies were from R&D Systems (Minneapolis, MN). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat antibody and horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulins (Igs) were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Curcumin, insulin, para-phenylenediamine, para-nitrophenyl phosphate (pNPP), transferrin, monoclonal antibody specific to tubulin, and goat IgGs were from Sigma (St. Louis, MO). Nuclear extract kit and mouse anti-human phosphorylated (p)-I{kappa}B were from Active Motif (Carlsbad CA). The chemiluminescence kit was from Roche Diagnostics (Laval, QC, Canada). The protein assay kit was from Bio-Rad Laboratories Ltd. (Mississauga, ON, Canada). T4 polynucleotide kinase was from Promega (Madison, WI). ProbeQuant G-50 microcolumn and 32P-ATP were from Amersham Biosciences (Baie d'Urfe, QC, Canada). SYBR Green PCR Master Mix was from Applied Biosystems (Foster City, CA).

Subjects and Tissue Handling

Endometrial biopsies were obtained during laparoscopy for tubal ligation from normal fertile women who had not received hormonal or anti-inflammatory therapy for at least 3 mo before surgery (mean age ± SD, 32.9 ± 4.1 yr; n = 12). A written informed consent was obtained from these women under a study protocol approved by the Ethical Committee on Human Research at Laval University, Quebec, Canada. Biopsies were obtained by aspiration with the use of a Pipelle (Unimar Inc., Prodimed, Neuilly-En-Tchelle, France), immediately placed at 4°C in sterile HBSS containing 1% antibiotics-antimycotics, and transported to the laboratory.

Cell Culture and Stimulation

Endometrial stromal cells were obtained and characterized according to our previously described procedure [20]. Extensive characterization of cell cultures prepared using this protocol previously confirmed >95% purity with cells retaining cytoskeletal markers of their endometrial stromal origin. For ELISA and reverse transcription-polymerase chain reaction (RT-PCR) studies, endometrial cells were plated in 12-well plates in DMEM-F12 medium (80 000 cells per 1.5 ml/well), supplied with 10% FBS, 5 µg/ml insulin, 5 µg/ml transferrin, and 1% antibiotics-antimycotics (complete DMEM-F12 medium). Medium was changed every 48 h. When cells proliferated to confluence, the culture medium was replaced overnight with serum-free medium, then with serum-free and phenol red-free medium containing various concentrations of IL1B (0–100 ng/ml) for different time periods (0–24 h). For electrophoretic mobility shift assay (EMSA) and Western blot analysis of p-I{kappa}B, cells were plated in 100-mm Petri dishes at 106 cells/plate and cultured in complete DMEM-F12 medium (10 ml/plate). In some cultures, curcumin (10–8 M), an extract from a plant known for inhibiting NF{kappa}B [21–25], was added 1 h before IL1B. Each experimental setup was repeated at least on three occasions using cells obtained from different patients.

Immunocytofluorescence

Briefly, endometrial stromal cells were seeded in chamber slides (Nalge Nunc International, Naperville, IL) at 104 cells/well and cultured in complete DMEM-F12 medium. At confluence, cells were incubated overnight with serum-free medium and exposed for 24 h to 0.1 ng/ml IL1B in serum-free and phenol red-free medium. Cells were then fixed in formaldehyde (3.5% v/v in PBS) for 15 min at room temperature, rinsed in PBS-0.01% Tween-20, incubated with a goat anti-human MIF antibody (0.66 µg/ml in PBS-0.2% BSA-0.01% Tween-20) for 90 min at room temperature, rinsed in PBS-0.01% Tween-20, incubated with FITC-conjugated anti-goat antibody (1/50 dilution in PBS-BSA-Tween), rinsed in PBS-0.01% Tween-20, and mounted in Mowiol containing 10% para-phenylenediamine. Cells incubated without the primary antibody or with goat IgGs at the same concentration as the primary antibody were included as negative controls. Slides were observed under a microscope equipped for fluorescence (Leica mikroskopie und systeme GmbH, Model DMRB; Postfach, Wetzlar, Germany) and photographed.

Enzyme-Linked Immunosorbent Assay

Migration inhibitory factor ELISA was performed as previously described [26] using a mouse monoclonal anti-human MIF antibody as a capture antibody, a rabbit polyclonal anti-human-MIF antibody for detection, an alkaline phosphatase-conjugated goat anti-rabbit antibody, and pNPP as substrate. The optical density (OD) was measured at 405 nm and MIF concentrations were extrapolated from a standard curve using recombinant human MIF. The sensitivity limit of the assay was 300 pg/ml, with intraassay and interassay coefficients of variation <4%.

Reverse Transcription-Polymerase Chain Reaction

Briefly, cells were cultured until confluence in 12-well plates. Following appropriate treatment with IL1B, cells were washed with ice-cold PBS, total RNA was extracted using the TRIzol reagent according to the manufacturer's instructions, and reverse transcribed in the presence of random primers. The resulting cDNA was amplified with oligonucleotide primers specific to human MIF (amplimer size, 255 base pairs [bp]) and to human glyceraldehyde phosphate dehydrogenase (GAPDH) used as internal control (amplimer size, 240 bp). The PCR reaction products were then separated on 1.2% agarose gel by electrophoresis for qualitative analysis of mRNA expression as described previously [6].

Quantitative real-time PCR reactions were carried out in an ABI 7000 Thermal Cycler (Applied Biosystems). Each standard PCR reaction contained 2.5 µl of RT product, 0.5 µl of each primer (final concentration, 0.1 µM), 12.5 µl SYBR Green PCR Master Mix consisting of Taq DNA polymerase reaction buffer, dNTP mix, SYBR green I, MgCl2, and Taq DNA polymerase. Following a 95°C denaturation for 10 min, the reactions were cycled 40 times with a 95°C denaturation for 15 sec and a 60°C annealing for 60 sec. Primers for MIF (forward primer, 5'-CGCAGAACCGCTCCTACAG-3'; reverse primer, 5'-GGAGTTGTTCCAGCCCACAT-3'; amplimer size, 125 bp) and GAPDH (forward primer, 5'-CAGGGCTGCTTTTAACTCTGG-3'; reverse primer, 5'-TGGGTGGAATCATATTGGAACA-3'; amplimer size, 102 bp) were designed using Primer Express version 2.0 (Applied Biosystems). The primers were designed to span intron-exon boundaries to avoid amplification of genomic DNA, and selected to have compatible Tm values (59–61°C). Quantification of MIF mRNA was performed by using a relative quantification method. For each experimental sample, MIF mRNA levels were normalized to GAPDH mRNA levels. After each run, melting curve analysis (55–95°C) was performed to verify the specificity of the PCR reaction. All samples were tested in triplicate, and each run included no-template and no-RT controls.

Western Blotting

Endometrial cells were plated in 100-mm Petri dishes and cultured as described above. Stimulation was performed with 1 ng/ml IL1B in phenol red-free DMEM-F12 medium without serum. The medium was then recovered and cells were washed with ice-cold PBS before being lysed with 80 µl of lysis buffer (50 mM Tris-HCl, 125 mM NaCl, 0.1% Nonidet P-40, 5 mM ethylenediamine tetraacetic acid, 50 mM NaF, 0.1% phenylmethylsulfonyl fluoride, and protease inhibitors). Equal amounts of cell lysates (20 µg) were separated by electrophoresis on a 10% gradient polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was washed with 0.1% Tween-20-PBS and incubated with 5% dry nonfat skimmed milk powder in 0.1% Tween-20-PBS pH 7.4 for 1 h, then with mouse anti-human p-I{kappa}B (2 µg/ml in 0.1% Tween-20-PBS for 1.5 h), rinsed in 0.1% Tween-20-PBS, and incubated with HRP-labeled anti-mouse IgGs for 1 h. After intensive wash with 0.1% Tween-20-PBS, a chemiluminescence kit was used for detection following the manufacturer's instructions. Membranes were stripped and reblotted with a monoclonal antibody specific to tubulin (1:50 000 dilution in Tween-20-PBS), which was used as an internal control for protein loading and transfer.

Extraction of Nuclear Proteins and EMSA

Extraction of nuclear proteins After appropriate treatment of cells with IL1B in the absence or the presence of curcumin as described above, a nuclear extract kit was used for nuclear extraction. Briefly, endometrial stromal cells were harvested in 1 ml of ice-cold PBS and centrifuged for 1 min at 2000 x g at 4°C. The cell pellet was lysed in 50 µl of hypotonic buffer (buffer A) containing 10 mM Hepes pH 7.9, 10 mM KCl, 300 mM sucrose, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.4% protease inhibitor cocktail containing 0.5% nonidet P-40 for 5 min on ice, and the samples were vortexed vigorously for 15 sec before centrifuging at 14 000 x g for 5 sec at 4°C. The pellet was washed a second time in 100 µl of buffer A and centrifuged at 14 000 x g for 5 sec at 4°C. The pellet was then resuspended in 100 µl of hypertonic buffer (buffer B) composed of 20 mM Hepes pH 7.9, 1 mM KCl, 100 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1% protease inhibitor cocktail, and 20% (v/v) glycerol; mixed vigorously by vortexing for 15 sec; sonicated for 10 sec; and centrifuged at 14 000 x g for 5 sec at 4°C. Protein concentration of the nuclear extracts was determined using the Bio-Rad DC Protein assay kit. Nuclear extracts were stored at –80°C until use.

Electrophoretic mobility shift assay DNA binding of NF{kappa}B was examined using the consensus oligonucleotide of NF{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'). The oligonucleotide was end-labeled with (32P){gamma}ATP using T4 polynucleotide kinase. The labeled probe was purified using a ProbeQuant G-50 micro-column (3000 x g for 1 min) and recovered in Tris-EDTA buffer pH 8.0. Binding reactions included 5 µg of nuclear proteins in incubation buffer containing 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 2.5 mM dithiothreitol, 2.5 mM EDTA, 5 mM MgCl2, 0.25 mg/ml poly(dI-dC)poly(dI-dC), and 20% glycerol. After 10 min the labeled oligonucleotide (4 x 105 cpm) was added and the mixture was incubated for 20 min at room temperature in a final volume of 10 µl. In some assays, unlabeled NF{kappa}B was added 20 min before the incubation with the labeled oligonucleotide as control for specific binding. Immediately after binding, the nucleoprotein-oligonucleotide complexes were separated from unbound oligonucleotide by electrophoresis on a nondenaturing 4% acrylamide gel at 100 V for 4 h using 0.5 M Tris-borate-EDTA (TBE) buffer. The gel was then dried and exposed overnight at –80°C to x-ray films (BioMax, Eastman Kodak, Rochester, NY).

Statistical Analysis

Data were expressed as mean ± SEM. An unpaired t-test was used to compare two means, whereas one-way analysis of variance followed by the Dunnett test was performed for multiple comparisons using GraphPad Software, Prism 3.0 (GraphPad Software, San Diego, CA). Differences were considered as statistically significant for P values < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endometrial stromal cells were first examined for MIF protein expression in response to IL1B by immunocytofluorescence using anti-MIF antibody. As shown in Figure 1, only a faint staining was observed in cells incubated with the culture medium containing no stimulus (Fig. 1A). However, cell incubation with IL1B (0.1 ng/ml) for 24 h showed a marked increase in the intensity of staining (Fig. 1B). No staining was observed in cells incubated with goat IgGs instead of anti-MIF antibody or with anti-MIF antibody preabsorbed with an excess rhMIF (2 µg/ml) (data not shown).



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  		FIG. 1. IL1B up-regulates MIF expression in human endometrial stromal cells. Cells cultured in chamber slides were incubated for 24 h with culture medium alone (A) or containing 0.1 ng/ml IL1B (B). MIF was detected by immunocytofluorescence staining using an MIF-specific polyclonal goat antibody. Bar = 30 µm

We then assessed MIF secretion by endometrial stromal cells in response to IL1B by ELISA. Data illustrated in Figure 2 showed a dose- and time-dependent effect of IL1B on MIF secretion. Such a secretion was statistically significant compared with that of control (culture medium without stimulus) for 0.1 ng/ml IL1B and 6 h of stimulation (P < 0.05), and was more markedly significant for higher IL1B concentrations (1–100 ng/ml) and longer time periods (12 and 24 h) (P < 0.001).



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  		FIG. 2. IL1B induces MIF secretion by human endometrial stromal cells. Cells were stimulated with different concentrations of IL1B (0–100 ng/ml) for varying time periods (6–24 h). MIF secretion in the culture supernatants (pg/ml, mean ± SEM) was measured by ELISA. *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively

The effects of IL1B on MIF gene expression in endometrial stromal cells was assessed by RT-PCR. As shown in Figure 3, MIF mRNA was detectable in endometrial cells cultured without stimuli. However, cell exposure to IL1B (0–100 ng/ml) for 6 h considerably increased MIF mRNA levels, which varied as a function of IL1B doses. Quantitative analysis of MIF mRNA expression using real-time PCR confirmed these data and further showed a significant increase in MIF mRNA levels in endometrial stromal cells in response to 1 (P < 0.05), 10 (P < 0.01), and 100 (P < 0.01) ng/ml IL1B.



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  		FIG. 3. IL1B stimulates MIF mRNA expression in cultured human endometrial stromal cells. Cells were treated with IL1B (0, 0.1, 1, 10, and 100 ng/ml) for 6 h. Total RNA was extracted and reverse transcribed, then MIF and GAPDH (internal control) cDNAs were amplified by PCR as described in Materials and Methods. A) Analysis of amplified cDNAs by electrophoresis and ethidium bromide staining. B) Quantitative real-time PCR analysis. IL1B effects were statistically significant by the Dunnett test; *P < 0.05, **P < 0.01. An arbitrary unit is defined as the ratio of MIF to GAPDH mRNA in cells incubated with the control medium (i.e., the medium alone without stimuli)

The transcription factor NF{kappa}B plays a pivotal role in activation of multiple inflammatory molecules and appeared to play a important role in mediating cell activation by IL1 [27]. However, it is still unknown whether NF{kappa}B is involved in MIF gene transcription. Data depicted in Figure 4 showed that preincubation of endometrial stromal cells with curcumin (10–8 M), a known NF{kappa}B inhibitor [21–25], significantly reduced IL1B-induced MIF secretion, although not to a level comparable to that of the medium alone without stimuli (control) (P < 0.05). Analysis of NF{kappa}B activation and translocation to the nucleus by EMSA in the same cultures further showed a marked increase in NF{kappa}B-DNA binding activity in cells treated with IL1B, whereas nuclear extracts from untreated cells showed negligible binding activity. The specificity of this reaction was confirmed by adding an excess of cold oligonucleotide, which eliminated the reactive band. Addition of antibodies directed against the 50-kDa subunit (p50) of NF{kappa}B induced a supershift of the binding complex. The IL1B-induced NF{kappa}B-DNA binding was suppressed by pretreating cells with curcumin (10–8 M) (Fig. 5).



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  		FIG. 4. Curcumin suppresses IL1B-induced MIF secretion. Confluent endometrial stromal cells were preincubated for 1 h with or without curcumin (10–8 M) before adding IL1B (1 ng/ml) for a further 24 h. MIF concentration in the supernatants (pg/ml, mean ± SEM) was measured by ELISA. *Significant inhibition of IL1B-induced MIF secretion in the presence of curcumin (P < 0.05)



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  		FIG. 5. IL1B induces NF{kappa}B activation and nuclear translocation in human endometrial stromal cells. Confluent cell cultures were exposed to medium alone, medium with IL1B (1 ng/ml), or medium with IL1B (1 ng/ ml) and curcumin (10–8 M) for 24 h. Nuclear protein extracts (5 µg) were incubated with radiolabeled NF{kappa}B oligonucleotide. The resulting complexes were separated on 4% nondenaturing polyacrylamide gel. To test for specificity of NF{kappa}B binding, supershift analysis with antibody against the p50 subunit of NF{kappa}B and competition experiments with unlabeled oligonucleotide were carried out

Activation of NF{kappa}B is usually associated with phosphorylation of I{kappa}B, followed by its degradation by the proteasome and NF{kappa}B nuclear translocation. Western blot analysis of protein extracts from endometrial stromal cell cultures using an anti-p-I{kappa}B antibody clearly showed that IL1B induced p-I{kappa}B phosphorylation. No p-I{kappa}B band was observed in cells pretreated with (10–8 M) curcumin before stimulation with IL1B (Fig. 6).



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  		FIG. 6. IL1B induces I{kappa}B phosphorylation in human endometrial stromal cells. Confluent cell cultures were exposed for 15 min to medium alone or to IL1B at 1 ng/ml with and without curcumin (10–8 M). Proteins were separated by electrophoresis on a 10% gradient polyacrylamide gel and immunoblotted using anti-pI{kappa}B antibody; (p-I{kappa}B, 43 kDa). Tubulin levels are shown to demonstrate equal protein loading


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These results show that IL1B up-regulates MIF gene expression and protein synthesis in endometrial stromal cells and further suggest that NF{kappa}B is involved in IL1B-induced MIF gene transcription. The nuclear transcription factor NF{kappa}B is normally bound to I{kappa}B in cytosol; this binding prevents its translocation into the nucleus [28]. It is known that phosphorylation of I{kappa}B and its degradation are essential for liberation of NF{kappa}B from binding with I{kappa}B [28]. Proinflammatory stimuli induce the phosphorylation of I{kappa}B, which leads to its degradation by proteasomes and the release of NF{kappa}B. The latter translocates into the nucleus, where it is known to induce the transcription of proinflammatory cytokines and the proteins/enzymes involved in generating reactive oxygen species, and to thereby modulate the molecular and cellular mechanisms involved in inflammation [29]. We demonstrated here that NF{kappa}B activation is involved in the induction of MIF secretion in human endometrial stromal cells. IL1B clearly induced NF{kappa}B translocation into the nucleus and DNA binding as shown by EMSA, and induced the phosphorylation of the NF{kappa}B inhibitor I{kappa}B. Furthermore, adding curcumin, which is known for inhibiting NF{kappa}B activation [21–25], blocked NF{kappa}B translocation into the nucleus and DNA binding, and inhibited MIF secretion as measured in the supernatants of the same cell cultures. However, as shown by EMSA, a reduced band was obtained when endometrial cell cultures were treated with curcumin and IL1B compared with IL1B alone. In addition, curcumin did not completely block IL1B-induced MIF secretion, suggesting the involvement of additional or other transcriptional pathways.

IL1 was first identified as a proinflammatory cytokine secreted by activated monocytes/macrophages. IL1 is now known as a pluripotent mediator that promotes the production of cytokines and growth factors in various cells [30].

Human endometrium is an active site of cytokine production and action [31]. IL1 was shown to be involved in several reproductive processes [32, 33]. IL1 was reported to act as an embryonic signal and to promote embryo implantation [33]. Increased concentrations of IL1 occurring locally within the endometrium at the end of the menstrual cycle are believed to play an important role in the inflammatory-like process associated with menstrual shedding [31]. Our findings showing that IL1B stimulates MIF mRNA synthesis and protein secretion by human endometrial cells makes it plausible that MIF is one of the factors that mediates IL1 effects in the human endometrium. This is all the more plausible as recent studies showed MIF expression in the human endometrium [34] and suggest an important role for this factor in a variety of reproductive functions [34, 35]. MIF was first reported in 1932 and was described as a cytokine produced by T lymphocytes, which inhibited the random migration of microphages [7, 8]. New evidence, however, showed a wide spectrum of immunomodulatory properties and direct involvement of MIF in cell growth, angiogenesis, and tissue remodeling [9, 10, 36]. Of particular interest is the ability of MIF to stimulate endothelial cell growth [37, 38] and to induce matrix metalloproteinase (MMP) secretion by a variety of cell types [39], which makes plausible its involvement in the dynamic and physiologic processes of tissue regeneration and disintegration taking place within the endometrium during the normal menstrual cycle.

Numerous studies support an important role for IL1 in endometriosis pathophysiology. We demonstrated that IL1B promoted the production of cytokines in eutopic and ectopic endometrial cells of women with endometriosis [13, 40, 41]. Lebovic et al. [17] showed that IL1B induces an angiogenic phenotype in endometriotic cells by stimulating the production of angiogenic factors such as vascular endothelial growth factor. IL1 may therefore play an important role in endometriosis-associated immunoinflammatory changes and tissue remodeling observed in the peritoneal cavity of patients with endometriosis. In endometriosis, the number and activation level of peritoneal macrophages were reported to be increased [42–44]. These inflammatory cells were shown to overproduce IL1 compared with that of cells from normal women [19]. IL1 concentrations were also reported to be increased in the peritoneal fluid of patients with endometriosis as well as in eutopic endometrial tissue [15, 16]. Consequently, our data showing a marked stimulatory effect of IL1B on MIF secretion by endometrial stromal cells suggest that IL1 may interact with endometrial cells in the eutopic endometrial tissue (i.e., before they migrate into the peritoneal cavity following retrograde menstruation, and after they migrate as well), thereby enhancing MIF expression in eutopic and ectopic locations. In fact, our previous data showed increased concentrations of MIF in the peritoneal fluid of patients with endometriosis [5] and ectopic endometrial implants [6]. Studies have been undertaken to assess MIF expression in the endometrial tissue of women with endometriosis. Nevertheless, MIF, which for instance is known for being involved in the inhibition of macrophage migration, for inhibiting NK cell activity, and for stimulating tissue remodeling and angiogenesis [9, 11, 45, 46] may contribute to the accumulation of peritoneal macrophages and NK immunosuppression observed in patients with endometriosis as well as to the active tissue remodeling required for the ectopic implantation of endometrial cells [4, 47–49].

In conclusion, our study showed an IL1B-induced MIF synthesis and secretion by human endometrial stromal cells occurring possibly via NF{kappa}B activation. This may have significant relevance to the physiology of the human endometrium and to the dynamic changes occurring within this tissue at every normal menstrual cycle, and play an important role in the chronic immunoinflammatory reaction and tissue remodeling observed in endometriosis, not only in the peritoneal cavity where endometrial tissue migrates and implants, but also in the eutopic endometrial tissue, where it originates.


   ACKNOWLEDGMENTS
 
The authors thank Drs. François Belhumeur, Jean Blanchet, Marc Bureau, Simon Carrier, Elphège Cyr, Marlène Daris, Jean-Louis Dubé, Jean-Yves Fontaine, Céline Huot, Pierre Huot, Johanne Hurtubise, Jacques Mailloux, and Marc Villeneuve for patient evaluation and providing endometrial biopsies; Christian Bellehumeur, Madeleine Desaulniers, Yves Lajeunesse, Monique Longpré, Johanne Pelletier, Sylvie Pleau, and Marie-Josée Therriault for technical assistance.


   FOOTNOTES
 
1 Supported by grant MOP-37921 to A.A. from The Canadian Institutes for Health Research. A.A. is Chercheur National from the Fonds de la Recherche en Santé du Québec (FRSQ). W.-G.C. is a recipient of a Wyeth-Ayerst CIHR/Rx&D Fellowship. Back

2 Correspondence: Ali Akoum, 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. FAX: 418 525 4195; ali.akoum{at}crsfa.ulaval.ca Back

Received: 22 November 2004.

First decision: 21 December 2004.

Accepted: 9 May 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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