Cycle-dependent expression of macrophage migration inhibitory factor in the human endometrium

R. Kats1, M. Al-Akoum1, S. Guay1, C. Metz2 and A. Akoum1,3

1 Unité d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, Centre Hospitalier Universitaire de Québec, Université Laval, Québec, Canada, and 2 Institute for Medical Research at North Shore–LIJ, Manhasset, NY, USA

3 To whom correspondence should be addressed at: Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, 10 rue de l’Espinay, Local D0-711, Québec, Québec, Canada, G1L 3L5. E-mail: ali.akoum{at}crsfa.ulaval.ca


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that was shown to promote angiogenesis and tissue remodelling. Our previous studies identified MIF as one of the principal bioactive molecules involved in endothelial cell proliferation released by ectopic endometrial cells. METHODS AND RESULTS: In the present study, we examined the expression of MIF in the human endometrium and found an interesting distribution and temporal pattern of expression throughout the menstrual cycle. Immunoreactive MIF was predominant in the glands and surface epithelium. Dual immunofluorescence analysis further identified endothelial cells, macrophages and T-lymphocytes as cells markedly expressing MIF in the stroma. Quantitative assessment of MIF protein showed a regulated cycle phase-dependent expression pattern. MIF expression increased in the late proliferative/early Secretory phase of the menstrual cycle was moderate during the receptive phase or what is commonly called the implantation window before increasing again at the end of the cycle. This pattern paralleled MIF mRNA expression determined by northern blot. CONCLUSION: The cycle phase-specific expression of MIF suggests a tight regulation and perhaps different roles for this factor in the reparative, reproductive and inflammatory-like processes that occur in human endometrium during every menstrual cycle.

Key words: cytokine/endometrium/macrophage migration inhibitory factor/menstrual cycle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine with a wide variety of biological properties (Metz and Bucala, 1997Go; Calandra and Roger, 2003Go; Nishihira et al., 2003Go). Originally described as a product of activated T-lymphocytes that inhibited the random migration of cultured macrophages, MIF is now known as an important modulator of systemic as well as local inflammatory and immune responses (Metz and Bucala, 1997Go; Calandra and Roger, 2003Go; Nishihira et al., 2003Go). Many recent studies demonstrated MIF expression in a variety of cells other than activated T cells and macrophages, indicating its involvement within and beyond the immune system (Nishihira et al., 2003Go). In addition to inflammatory and immunological functions, MIF has been proposed to play an essential role in cell proliferation, cell differentiation, angiogenesis, and wound healing (Nishihira et al., 2003Go).

Human endometrium is known as an active site of cytokine production and action (von Wolff et al., 2000Go). During the menstrual cycle, the tissue undergoes a series of transformations including regeneration, proliferation, differentiation and disintegration at the end of the cycle should embryo implantation fail or does not occur. These events require active tissue remodelling and it is not therefore surprising to see cytokines mediating these events at different stages of the menstrual cycle.

Recent data from our laboratory identified MIF as one of the principal bioactive molecules involved in endothelial cell proliferation released by ectopic endometrial cells (Yang et al., 2000Go). In the present study, we examined the expression of MIF in the normal human endometrium and found a cycle phase-dependent expression. MIF expression increased in the late proliferative/early secretory phase of the menstrual cycle, was moderate during the receptive phase or what is commonly called the implantation window before increasing again at the end of the cycle.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
Sixty-eight women were included in this study. These women were aged between 23 and 43 years (mean ± SD: 35.2 ± 5.1 years). They were fertile, requested tubal ligation and had a normal and regular menstrual cycle. None had visible endometrial hyperplasia or neoplasia, inflammatory disease, or endometriosis at the time of clinical examination or laparoscopy. Women were not receiving any anti-inflammatory or hormonal medication at least 3 months before laparoscopy. The cycle day was determined according to the cycle history and histological criteria (Noyes et al., 1975Go). Twenty-six women were in the proliferative phase and 42 in the secretory phase. Women who participated in the study provided informed consent for a protocol approved by Saint-François d’Assise Hospital Ethics Committee on Human Research.

Collection of endometrial biopsies
Endometrial biopsies were obtained using sterile pipelle (Unimar Inc., Neuilly-en-Thelle, France). Samples were placed at 4°C in sterile Hanks’ balanced salt solution (HBSS) (Invitrogen Life Technologies, Burlington, ON, Canada) containing 100 IU/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin, immediately transported to the laboratory and washed twice in HBSS at 4°C. Only biopsies devoid of any visible blood contamination were included in the study. Biopsies were snap-frozen on dry ice and kept at –80°C in Eppendorf tubes for enzyme-linked immunosorbent assay (ELISA), western blot and northern blot analyses or in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN, USA) for immunohistochemical studies.

Immunohistochemistry
Immunohistochemical staining of MIF in endometrial tissue sections was performed according to our previously described procedure using a goat polyclonal anti-human-MIF antibody [0.66 mg/ml in phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA)/0.01% Tween 20 (PBS/BSA/Tween)] (R&D Systems, Minneapolis, MN, USA), a biotin-conjugated rabbit anti-goat antibody (1:500 in PBS/BSA/Tween) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), peroxidase-conjugated streptavidin (1:500 dilution in PBS/BSA/Tween) (Jackson ImmunoResearch Laboratories) and diaminobenzidine (DAB) as chromogen (3 mg DAB/0.03% H2O2 in PBS) (Kats et al., 2002Go). Sections were counterstained with haematoxylin, mounted in Mowiol (Calbiochem–Novabiochem Corp., La Jolla, CA, USA) and observed using a Leica microscope (Leica mikroskopie und systeme GmbH, Model DMRB; Postfach, Wetzlar, Germany) connected to an image analysis system (ISIS, Metasystems, Altlussheim, Germany). Sections incubated with goat immunoglobulins (IgG) at the same concentration as the primary antibody were used as negative controls in all experiments.

Dual immunofluorescent staining
Dual immunofluorescent staining was performed as reported previously (Kats et al., 2002Go). Briefly, tissue sections were first incubated with a goat polyclonal anti-human-MIF antibody (0.66 mg/ml in PBS/BSA/Tween) (R&D Systems) then with one of the following antibodies: mouse monoclonal anti-human CD68 to detect macrophages (1:50 dilution in PBS/BSA/Tween) (Dako Diagnostics Canada Inc., Mississauga, ON, Canada), mouse monoclonal anti-human CD3 to detect T lymphocytes (1:100 dilution in PBS/BSA/Tween) (gift from Dr W.Mourad, Laval University) and rabbit polyclonal anti-human von Willebrand Factor (vWF) to detect endothelial cells (Sigma–Aldrich Canada Ltd, Oakville, ON, Canada) (1:200 dilution in PBS/BSA/Tween). To detect MIF and CD68 or MIF and CD3, tissue sections were incubated simultaneously with fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat antibody (Jackson ImmunoResearch Laboratories) (1:50 dilution in PBS/BSA/Tween) and rhodamine-conjugated sheep anti-mouse antibody (Roche Diagnostics, Laval, QC, Canada) (1:10 dilution in PBS/BSA/Tween). To detect MIF and vWF, tissue sections were incubated simultaneously with FITC-conjugated donkey anti-goat antibody and rhodamine-conjugated mouse anti-rabbit antibody (Jackson ImmunoResearch Laboratories) (1:50 dilution in PBS/BSA/Tween). Slides were mounted in Mowiol containing 10% para-phenylenediamine (Sigma–Aldrich Canada Ltd), an anti-fading agent and observed under the Leica microscope equipped for fluorescence with a 100 W UV lamp (Leica mikroskopie und systeme GmbH) connected to an image analysis system (ISIS, Metasystems).

Western blotting
Protein extraction from endometrial tissue was performed according to our previously described procedure (Kats et al., 2002Go), and total protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories Ltd, Mississauga, Ontario, Canada). For western blot analysis, denatured proteins were separated by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis in 15% acrylamide slab gels and transferred onto 0.45 µm nitrocellulose membranes. Equal protein loading was confirmed by staining the membrane with Ponceau S (Sigma-Aldrich Canada. LTD) (2%). Nitrocellulose membranes (Pall Corporation, Pensacola, FL, USA) were then cut into strips, and incubated overnight at 4°C with a polyclonal goat anti-human MIF antibody (R&D Systems) at 2 µg/ml of blocking solution (0.1 mol/l Tris buffer, 0.9% NaCl/0.05% Tween 20 containing 5% non-fat dry milk (w/v) or with normal goat IgG (R&D Systems) at the same concentration. Strips were then washed in Tris-buffered saline 0.1% Tween 20, incubated for 1 h at room temperature with a peroxidase-conjugated rabbit anti-goat antibody (Jackson ImmunoResearch Laboratories), diluted 1:10 000 in the blocking solution, washed again in Tris-buffered saline 0.1% Tween 20, incubated for 1 min with an enhanced chemiluminescence system using BM chemiluminescence blotting substrate (POD) (Roche Diagnostics), and exposed to Kodak BioMax film for several time intervals allowing for an optimal detection (all bands visible but not overexposed).

MIF ELISA
MIF concentration in endometrial tissue protein extract was measured by ELISA according to a previously reported procedure (Calandra et al., 1995Go). Briefly, this technique uses a capture mouse monoclonal anti-human MIF antibody (R&D Systems), a rabbit polyclonal anti-human-MIF antibody for detection, alkaline phosphatase-conjugated goat anti-rabbit IgG (Chemicon International Inc., Temecula, CA, USA) and para-nitrophenyl phosphate as substrate (Sigma). The optical density (OD) was measured at 405 nm and MIF concentrations were extrapolated from a standard curve using recombinant human MIF (R&D Systems).

In situ hybridization
This was performed as described in our previous studies (Jolicoeur et al., 1998Go). Briefly, biotin-labelled complementary DNA (cDNA) probes were prepared by nick-translation from the entire plasmid vector (pBluescript SK) with the MIF cDNA (ATCC, Rockville, MD, USA), or from the plasmid vector alone (negative control), using a BioNick Labelling System (Invitrogen Life Technologies). Serial cryosections were fixed in 4% formaldehyde solution for 20 min at room temperature, treated with proteinase K (2 µg/ml of Tris/ethylenediaminetetra-acetic acid) for 15 min at 37°C, post-fixed in 4% formaldehyde, acetylated by immersion in 0.25% acetic anhydride in 0.1 mol/l triethanolamine pH 8 for 10 min at room temperature, rinsed in PBS and progressively dehydrated in alcohol baths (50 to 100%). Sections were prehybridized for 30 min at 37°C with the hybridization buffer devoid of probe containing 50% (v/v) formamide, 10% (v/v) dextran sulphate, 0.1% SDS, 2 x standard saline citrate (SSC), 1 x Denhardt’s solution [0.02% Ficoll, 0.02% human serum albumin (HSA), 0.02% polyvinylpyrrolidone and 40 mmol/l monosodium phosphate, pH 7]. Sections were then hybridized with 5 ng/µl of biotinylated probe, dissolved in the hybridization buffer, for 18 h at 37°C in a humidified chamber. Thereafter, slides were first immersed in 50% formamide/2 x SSC solution (two baths, 2 min each at 37°C), then in 2 x SSC and finally in PBS containing 0.25% HSA (two baths, 5 min each, at room temperature). Biotin was detected with a rabbit polyclonal anti-biotin antibody (1% dilution in PBS/0.25% HAS) (Enzo Diagnostics Inc., New York, USA), a biotinylated goat anti-rabbit polyclonal antibody (1% dilution in PBS/0.25% HSA) (Vector Laboratories, Burlingame, CA, USA) and fluorescein isothiocyanate-conjugated streptavidin (0.5% in PBS/0.25% HSA) (Invitrogen Life Technologies) respectively. Slides were then treated with propidium iodine (1.5 µg/ml of distilled water) (Sigma) which makes the nucleus visible in yellow-orange upon UV excitation, mounted in Mowiol containing 10% para-phenylenediamine and observed under the Leica microscope. As negative controls, sections from each tissue were incubated without cDNA probes or with non-specific DNA probes prepared by nick-translation from the plasmid vector alone, i.e. without the MIF cDNA.

Northern blot analysis
Total RNA was extracted from cells with TRIzol reagent according to the manufacturer’s instructions (Invitrogen life technologies). RNA was size-fractionated by electrophoresis on 1% agarose gels containing 10% formaldehyde and transferred electrophoretically to a Hybond-N+ membrane (Amersham, Oakville, ON, Canada). The membrane was then dehydrated at 37°C for 30 min, prehybridized overnight at 42°C with a hybridization buffer comprising 5xSSC, 5xDenhardt’s solution, 50 mmol/l NaH2PO4, 0.5% SDS, 200 µg/ml salmon sperm DNA and 50% formamide, hybridized overnight at 42°C with 32P-labelled MIF cDNA in the hybridization buffer and washed at room temperature with 1xSSC/0.1% SDS then 0.2xSSC/0.1% SDS, before being exposed to X-ray film (BioMax; Eastman Kodak, Rochester, NY, USA) at –80°C for ~18 h. Staining with ethidium bromide (Invitrogen Life Technologies) and hybridization with 28S cDNA probe (ATCC, Rockville, MD, USA) were performed to ensure equal loading of RNA. 32P-labelling of MIF and 28S cDNA used for hybridization was performed using isolated cDNA fragments (50 ng), dCTP32 and NEBlot kit (New England Biolabs Inc., Pickering, Ontario, Canada), and labelled probes were purified using QIAquick nucleotide removal Kit (Qiagen, Inc., Mississauga, Ontario, Canada). Data were analysed as ratios of the density of the hybridization signals of MIF to 28S, as determined by computer-assisted densitometry (BioImage; Visage 110s, Genomic Solutions Inc., AnnHarbour, MI, USA), and normalized by including an equal amount of a control RNA (a single RNA preparation from an endometrial tissue) in every northern blot experiment.

Statistical analyses
MIF protein concentrations and mRNA levels were normally distributed as assessed by Kolmogorov–Smirnov test. However, because of the statistically different SD of MIF mean values for each cycle phase, as assessed by Bartlett’s test for equal variances, the logarithmic (log) transformation of the data was used for one-way analysis of variance (ANOVA). Log values were normally distributed and did not show significant differences between variances (Bartlett’s test). The Newman–Keuls test was then used post hoc for multiple comparisons. Comparison of two groups was performed using the unpaired t-test. P < 0.05 was considered statistically significant. All analyses were performed using GraphPad Software, Prism 3.0 (GraphPad Software, San Diego, CA, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A polyclonal antibody was used to detect MIF protein in endometrial tissue sections. Examples of positive immunostaining with MIF antibody are shown in Figure 1. Immunoreactive MIF was detectable throughout endometrial tissue, but was more marked in the glands, surface epithelium and cell aggregates scattered throughout the stroma (Figure 1A). Normal goat IgG used at equivalent concentration as the primary goat polyclonal anti-MIF antibody (negative control) did not display any detectable immunoreactivity (Figure 1B).



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Figure 1. Immunohistochemical detection of macrophage migration inhibitory factor (MIF) in the endometrium. (A) Positive brown immunostaining in the glands and the stroma (day 26); Staining is virtually absent in a serial section from the same endometrial tissue incubated with normal goat immunoglobulins (IgG) instead of the primary antibody taken as negative control (B). Note the marked immunostaining in surface (arrow) (C) and glandular (D) epithelium, the moderate staining in mid-proliferative (day 9) (E) and mid-secretory (day 19) (G) phase tissues and the more intense staining in tissues from the early secretory (day 16) (F) and the late secretory (day 26) (H) phases. Scale bars = 50 µm.

 

Intense brown immunostaining could be seen in luminal (Figure 1C) and glandular (Figure 1D) epithelium from late secretory endometrial tissues. It is noteworthy that MIF immunostaining appeared to be intense in tissues from the late proliferative/early secretory and the late secretory phases of the menstrual cycle, whereas a less intense staining in tissues from the early proliferative and the mid-secretory phases of the cycle was noted (Figure 1E–H).

In order to identify cells expressing MIF in the stroma, dual immunofluorescence analysis was performed using antibodies specific to MIF and to vWF, CD68 and CD3. Representative photomicrographs exhibited in Figure 2 show a marked expression of MIF in vWF-positive endothelial cells, CD68-positive macrophages and CD3-positive T lymphocytes.



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Figure 2. Dual immunofluorescent staining of macrophage migration inhibitory factor (MIF) (A, B and C) and von Willebrand factor (vWF) (D), CD68 (E) or CD3 (F) in endometrial tissue. Endometrial tissue sections (day 27) were incubated with goat polyclonal anti-MIF antibody and with rabbit polyclonal anti-vWF, mouse monoclonal anti-CD68, or mouse monoclonal anti-CD3 antibody. Sections were then incubated simultaneously with rhodamine-conjugated mouse anti-rabbit antibody and fluorescein isothiocyanate-conjugated donkey anti-goat antibody to detect co-expression of MIF with vWF, or with rhodamine-conjugated sheep anti-mouse antibody and fluorescein isothiocyanate-conjugated donkey anti-goat antibody to detect co-expression of MIF with CD68 or CD3, Note the expression of MIF (green color) in vWF-, CD68- and CD3-positive endothelial cells, macrophages and T lymphocytes respectively (red colour). Superposition of fluorescein (green) and rhodamine (red) signals clearly shows co-expression (yellow signal) of MIF with vWF (G = A + D), CD68 (H = B + E) and CD3 (I = C + F). Scale bars = 20 µm.

 

Western blot analysis of proteins extracted from endometrial tissue using a goat polyclonal anti-human MIF antibody showed a single specific 12.5 kDa band corresponding to the known molecular weight of MIF (Figure 3). No band was observed following preabsorption of MIF antibody with an excess of recombinant human MIF (1 µg/ml) or replacement of the antibody with an equivalent concentration of goat IgG, thereby demonstrating specific detection of MIF.



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Figure 3. Representative western blot analysis of macrophage migration inhibitory factor (MIF) expression in endometrial tissue. Total proteins (40 mg) from endometrial tissue (day 28) were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis and western blotting with an affinity-purified polyclonal goat anti-MIF antibody preabsorbed with an excess of recombinant human MIF (1 µg/ml) (lane 1, an equivalent concentration of normal goat IgG instead of the primary antibody (lane 2) or the primary antibody (lane 3). The detected band has an estimated apparent molecular weight of ~12.5 kDa.

 

MIF expression in the endometrium was then studied by in situ hybridization in order to localize MIF mRNA. Figure 4 shows the appearance of endometrial glands and stroma with x10 (A1, B1, C1 and D1) and x40 (A2, B2, C2 and D2) objectives following hybridization and staining with propidium iodine (cycle day 21). The hybridization signal (green-yellow) could only be visualized at higher magnification using x100 objective as illustrated in the Figure 4, and was more pronounced in glandular (A3) and surface (B3) epithelial than in stromal (C3) cells. No hybridization was observed in negative controls, either omitting the biotinylated MIF cDNA probe or using biotinylated DNA probes obtained from the plasmid alone (D3). Similar patterns of staining were observed with biopsies from three different subjects (days 17, 18 and 21 of the menstrual cycle).



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Figure 4. Localization of macrophage migration inhibitory factor (MIF) mRNA in the endometrium by in situ hybridization. Sections from endometrial tissue (day 21) were hybridized with biotin-labelled complementary DNA probes. Detection of biotin was performed using a rabbit polyclonal anti-biotin antibody, a biotinylated goat anti-rabbit polyclonal antibody and fluorescein isothiocyanate-conjugated streptavidin respectively. Slides were treated with propidium iodine which makes the nucleus visible in yellow-orange upon UV excitation, and mounted in the presence of an anti-fading agent (para-phenylenediamine). Appearance of endometrial glands (A), surface epithelium (B) and stroma (C) with x10 (1) or x40 (2) objective following hybridization, and staining with propidium iodine. Note the green-yellow hybridization signal that could only be observed at higher magnification using x100 objective (3), predominantly in endometrial glands (A) and surface epithelium (B) as compared to the stroma (C). Biotinylated DNA probes obtained from the plasmid alone were used as negative control (D1, D2 and D3) where no hybridization could be seen (D3). Scale bars = 20 µm.

 

To quantify and examine MIF expression in the endometrium throughout the menstrual cycle, we measured MIF concentrations in total protein extracts by ELISA and determined the levels of mRNA using northern blot analysis.

As shown in Figure 5, MIF protein levels were low in the early–mid-proliferative phase of the menstrual cycle (P1, days 2–9, n = 9), increased during the late proliferative/early secretory phase (P2, days 10–18, n = 25) and declined to moderate levels in the mid-secretory phase (P3, days 19–23, n = 9) before increasing again in the late secretory phase (P4, days 24–28, n = 12). Statistical analysis of the data using ANOVA and the Newman–Keuls multiple comparison test showed that MIF protein levels found in P2 and P4 were significantly higher than those found in P1 (both P < 0.001) and P3 (P < 0.01 and P < 0.001 respectively). No statistically significant difference between P2 and P4 was noted (P = 0.18). Also there was no statistically significant difference within P1 between the menstrual phase (days 2–5, n = 3) and mid-proliferative days 6–9 (n = 6) (P = 0.27).



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Figure 5. Graphical illustration of macrophage migration inhibitory factor (MIF) protein concentrations according to the menstrual cycle phase. MIF was measured by enzyme-linked immunosorbent assay in total endometrial tissue protein extracts and expressed as ng/mg of total proteins. The box and whisker plot was used to illustrate the distribution of MIF concentrations. The box delimits values falling between the 25th and the 75th percentiles and the horizontal line within the box refers to the median scores. P1 represents the menstrual, early and mid-proliferative phases (days 2–9, n = 9); P2 represents the late proliferative and the early secretory phases (days 10–18, n = 25); P3 represents the mid-secretory phase (days 19–23, n = 9); P4 represents the late secretory phase (days 24–28, n = 12). Statistical analysis of the data using ANOVA and the Newman–Keuls multiple comparison test showed that MIF protein levels found in P2 and P4 were significantly higher than those found in P1 (both P < 0.001) and P3 (P < 0.01 and P < 0.001 respectively). No statistically significant difference between P2 and P4 was noted (P = 0.18). Also there was no statistically significant difference within P1 between the menstrual phase (days 2–5, n = 3) and mid-proliferative days 6–9 (n = 6) (P = 0.27).

 

Northern blot analysis of MIF mRNA in 30 endometrial biopsies across the menstrual cycle showed that MIF mRNA levels tended to be elevated in P1 (days 6–9, n = 4) and P2 (days 10–17, n = 16), and decreased in P3 (days 20–22, n = 4) before augmenting again in P4 (days 24–28, n = 6). However, no statistically significant differences between the different phases were found (Figure 6A) as analysed with ANOVA and the Newman–Keuls multiple comparison test. A representative example of MIF mRNA levels as analysed by northern blotting is shown in Figure 6B.



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Figure 6. Macrophage migration inhibitory factor (MIF) mRNA expression in endometrial tissue throughout the menstrual cycle. (A) Graph of MIF mRNA relative levels in the endometrium throughout the menstrual cycle. The box and whisker plot was used to illustrate the distribution of MIF mRNA levels. The box delimits values falling between the 25th and the 75th percentiles and the horizontal line within the box refers to the median scores. P1, days 6–9, n = 4; P2, days 10–17, n = 16; P3, days 20–22, n = 4; P4, days 24–28, n = 6. (B) Representative autoradiogram showing MIF mRNA synthesis, and 28S ribosomal RNA demonstrating equal RNA loading. (1) day 6/28, (2) day 13/28, (3) day 24/28 and (4) day 27/28.

 


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
During the menstrual cycle, human endometrium undergoes profound and dynamic changes orchestrated in cyclic events of cell proliferation, differentiation and shedding. Many of these changes are reminiscent of those associated with the inflammatory and the reparative processes, which make plausible the involvement of proinflammatory cytokines. It is now evident that the effects of ovarian steroids are mediated and further modulated by peptide hormones, growth factors and cytokines locally produced within the endometrium, and by a variety of endometrial cell types (Lim et al., 1998Go; von Wolff et al., 2000Go; Fazleabas and Strakova, 2002Go).

Our present study located MIF protein throughout the human endometrial tissue both in epithelial and stromal compartments, but immunostaining was more obvious in surface and glandular epithelial cells. This was confirmed by in situ hybridization showing high levels of MIF mRNA in endometrial glands and surface epithelium. Dual immunofluorescence analysis identified endothelial cells, macrophages and T-lymphocytes as cells markedly expressing MIF in the stroma. These findings clearly show that MIF can be produced by a variety of cell types in the endometrial tissue and suggest a deep involvement in endometrial physiology.

Our data further revealed a regulated cycle-dependent expression of MIF in the human endometrium both at the protein and the mRNA levels. MIF protein concentration showed an interesting temporal distribution and varied at specific moments of the cycle. Being low at the beginning of the proliferative phase of the menstrual cycle, MIF protein concentration increased during the mid–late proliferative phase, reached a maximum around ovulation, then declined progressively to moderate levels in the mid-secretory phase, before undertaking a final augmentation in the late secretory phase. This pattern paralleled MIF mRNA expression determined by northern blot, which, however, showed no significant differences between the cycle phases. Such a discrepancy cannot at the present time be explained with certainty. However, one could speculate that the ELISA procedure is more sensitive than the northern blot analysis and that more patients were analysed by ELISA (n = 55) than by northern blotting (n = 30) which increased the statistical power of the study. Furthermore, endometrial biopsies were not similarly distributed across the menstrual cycle. For instance, for mRNA analysis, P1 included no tissue in the early proliferative days 2–5 where MIF protein concentrations were the lowest, although not significantly different from those found in the mid-proliferative days 6–9.

The temporal expression of MIF is interesting considering the biological properties of this factor. In fact, recent evidence shows pleiotropic effects for MIF and different cell targets. The factor exerts potent proinflammatory properties and modulates the immune responses by acting on different immune cells, but was shown to directly and indirectly affect cell proliferation, angiogenesis and tissue remodelling (Nishihira, 1998Go; Chesney et al., 1999Go; Calandra and Roger, 2003Go; Nishihira et al., 2003Go). Interestingly, according to recent data MIF seems to be implicated in critical reproductive events such as ovulation, implantation and pregnancy and to act as an endocrine and local regulator of reproductive functions (Suzuki et al., 1996Go; Arcuri et al., 2001Go; Matsuura et al., 2002Go; Saitoh, 2003Go). Our findings showing a regulated cycle phase-dependent and varying MIF expression in the human endometrium is consistent with that of Suzuki et al. (1996)Go who showed that endometrial MIF expression varied in pro- and post-estrus of mice. However, our results differ from those of Arcuri et al. (2001)Go who compared MIF expression in proliferative, early and late secretory phases and found no significant differences albeit an increased secretion in late secretory phase. In the present study, there were more patients included and four different phases based on the kinetics of MIF expression throughout the cycle. Furthermore, in our study no statistically significant difference between proliferative, early and late secretory phases was noted. However, we found a significant difference between the mid-secretory decline in MIF protein expression and the increase observed in mid–late proliferative and late secretory phases.

MIF expression and distribution in endometrial tissue across the menstrual cycle is remarkable for several reasons.

First, the increase of MIF protein expression during the proliferative phase is of interest as the human endometrium undergoes active tissue remodelling, angiogenesis and cell growth during this estrogen-dependent phase of the cycle (Tabibzadeh, 1991Go; von Wolff et al., 2000Go; Gargett and Rogers, 2001Go; Gambino et al., 2002Go), and suggests that MIF may be involved in endometrial growth and regeneration. In fact, numerous recent studies indicate a role for MIF in the reparative processes, cell proliferation and show the factor as a potent mediator of angiogenesis in vivo and endothelial cell proliferation in vitro (Nishihira, 1998Go; Chesney et al., 1999Go; Yang et al., 2000Go; Nishihira et al., 2003Go). It is still, however, not known whether estradiol influences MIF expression in the endometrium and up-regulates MIF expression by endometrial cells. On the other hand, it is quite possible that the LH surge occurring at ovulation may contribute to MIF augmentation considering that HCG displays a dose-dependent effect on MIF expression (Akoum et al., 2005Go) and that LH and HCG act via the same receptors (Rao, 2002Go).

Second, the significant diminution in MIF levels in the mid-secretory phase further suggests that MIF secretion is subjected to subtle chronological regulation in the human endometrial tissue. This is remarkable as the phase of reduced secretion correlates to a putative ‘implantation window’ (Bergh and Navot, 1992Go; Psychoyos, 1993Go; Simon et al., 1998Go; Tabibzadeh, 1998Go). The relative decrease in MIF expression at this specific time of the cycle where embryonic attachment and implantation may occur indicates a possible dose-dependent action for MIF in the endometrium and suggests that moderate levels of MIF would be required for the initial stages of interaction between maternal and embryonic cells and the establishment of an endometrial period of receptivity. However, the mechanisms underlying such a well-timed regulation remain to be elucidated.

Third, the late secretory phase increase in MIF expression may have a considerable significance as, in the absence of implantation, the endometrial tissue undergoes a process of necrosis and disintegrates at the end of the menstrual cycle (Tabibzadeh, 1991Go). The elevated expression of MIF observed in the late secretory phase may play a key role in the inflammatory-like process arising during the pre-menstrual and menstrual periods. In fact, MIF is known for retaining macrophages (Calandra et al., 1994Go; Metz and Bucala, 1997Go) whose increased recruitment into the endometrium during this specific period of the menstrual cycle is believed to contribute to the menstrual process (Critchley et al., 2001Go). Macrophages release numerous proteases and potent proinflammatory mediators such as interleukin (IL)-1 and tumour necrosis factor-{alpha} which were shown to be implicated in menstrual shedding (Simon et al., 1998Go; von Wolff et al., 2000Go; Critchley et al., 2001Go). Macrophages are also known to secrete MIF (Nishihira et al., 2003Go). However, other inflammatory cells such as neutrophils which have been shown to produce MIF (Baumann et al., 2003Go; Riedemann et al., 2004Go), may also contribute to the increased MIF secretion as their numbers increase substantially in the late secretory phase (Critchley et al., 2001Go).

It is noteworthy that our previous studies showed a similar temporal expression of another inflammatory molecule, IL-1 receptor type II (IL-1RII), across the menstrual cycle (Boucher et al., 2001Go). IL-1RII specifically down-regulates the biological effects of IL-1 (Boraschi et al., 1996Go; Mantovani et al., 2003Go), and up-regulation of IL-1RII expression by cells/tissues could be understood as a local reaction to buffer IL-1 effects. Available evidence indicates that IL-1 circulating levels increase around ovulation and that the cytokine levels increase in late secretory phase endometrial tissue (Cannon and Dinarello, 1985Go; Simon et al., 1993Go, 1994Go). One could speculate therefore that increased MIF expression/secretion may at least in part be related to IL-1 as our most recent data showed a significant stimulation of MIF by IL-1 in endometrial stromal cells (unpublished data).

In summary, MIF expression in the endometrium is an interesting and dynamic process. The molecule appeared to be expressed and released predominantly by glandular and luminal epithelial cells. This is of additional interest as first interactions between the embryo and the endometrium occur at the level of the luminal epithelial cells during the adhesion process. Furthermore, MIF expression appeared to follow a regulated cycle-dependent pattern and to undergo subtle chronological regulation throughout the menstrual cycle. In view of the molecule’s proinflammatory, immunomodulatory, growth regulatory and tissue remodelling effects, our findings suggest a tight regulation and perhaps different roles for this factor in the reparative, reproductive and inflammatory-like processes that occur in human endometrium during every menstrual cycle.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to 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 Rodolphe Maheux, Jacques Mailloux and Marc Villeneuve for patient evaluation and providing endometrial biopsies, Madeleine Desaulniers, Monique Longpré, Johanne Pelletier, Sylvie Pleau and Marie-Josée Therriault for technical assistance. This research was supported by grant MOP-77737 to A.Akoum from The Canadian Institutes of Health Research. A.A. is a ‘Chercheur Boursier National’ of the ‘Fonds de la Recherche en Santé du Québec (FRSQ)’.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Akoum A, Metz CN and Morin M (2005) Marked increase in macrophage migration inhibitory factor synthesis and secretion in human endometrial cells in response to human chorionic gonadotropin hormone. J Clin Endocrinol Metab 90,2904–2910.[Abstract/Free Full Text]

Arcuri F, Ricci C, Ietta F, Cintorino M, Tripodi SA, Cetin I, Garzia E, Schatz F, Klemi P, Santopietro R and Paulesu L (2001) Macrophage migration inhibitory factor in the human endometrium: expression and localization during the menstrual cycle and early pregnancy. Biol Reprod 64,1200–1205.[Abstract/Free Full Text]

Baumann R, Casaulta C, Simon D, Conus S, Yousefi S and Simon HU (2003) Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway. FASEB J 17,2221–2230.[Abstract/Free Full Text]

Bergh PA and Navot D (1992) The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril 58,537–542.[ISI][Medline]

Boraschi D, Bossu P, Macchia G, Ruggiero P and Tagliabue A (1996) Structure–function relationship in the IL-1 family. Front Biosci 1, d270–308.[Medline]

Boucher A, Kharfi A, Al-Akoum M, Bossu P and Akoum A (2001) Cycle-dependent expression of interleukin-1 receptor type II in the human endometrium. Biol Reprod 65,890–898.[Abstract/Free Full Text]

Calandra T and Roger T (2003) Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol 3,791–800.[CrossRef][ISI][Medline]

Calandra T, Bernhagen J, Mitchell RA and Bucala R (1994) The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 179,1895–1902.[Abstract/Free Full Text]

Calandra T, Bernhagen J, Metz CN, Spiegel LA, Bacher M, Donnelly T, Cerami A and Bucala R (1995) MIF as a glucocorticoid-induced modulator of cytokine production. Nature 377,68–71.[CrossRef][ISI][Medline]

Cannon JG and Dinarello CA (1985) Increased plasma interleukin-1 activity in women after ovulation. Science 227,1247–1249.[ISI][Medline]

Chesney J, Metz C, Bacher M, Peng T, Meinhardt A and Bucala R (1999) An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma. Mol Med 5,181–191.[ISI][Medline]

Critchley HO, Kelly RW, Brenner RM and Baird DT (2001) The endocrinology of menstruation—a role for the immune system. Clin Endocrinol (Oxf) 55,701–710.[CrossRef][ISI][Medline]

Fazleabas AT and Strakova Z (2002) Endometrial function: cell specific changes in the uterine environment. Mol Cell Endocrinol 186,143–147.[CrossRef][ISI][Medline]

Gambino LS, Wreford NG, Bertram JF, Dockery P, Lederman F and Rogers PA (2002) Angiogenesis occurs by vessel elongation in proliferative phase human endometrium. Hum Reprod 17,1199–1206.[Abstract/Free Full Text]

Gargett CE and Rogers PA (2001) Human endometrial angiogenesis. Reproduction 121,181–186.[Abstract/Free Full Text]

Jolicoeur C, Boutouil M, Drouin R, Paradis I, Lemay A and Akoum A (1998) Increased expression of monocyte chemotactic protein-1 in the endometrium of women with endometriosis. Am J Pathol 152,125–133.[Abstract]

Kats R, Metz CN and Akoum A (2002) Macrophage migration inhibitory factor is markedly expressed in active and early-stage endometriotic lesions. J Clin Endocrinol Metab 87,883–889.[Abstract/Free Full Text]

Lim KJ, Odukoya OA, Ajjan RA, Li TC, Weetman AP and Cooke ID (1998) Profile of cytokine mRNA expression in peri-implantation human endometrium. Mol Hum Reprod 4,77–81.[Abstract]

Mantovani A, Bonecchi R, Martinez FO, Galliera E, Perrier P, Allavena P and Locati M (2003) Tuning of innate immunity and polarized responses by decoy receptors. Int Arch Allergy Immunol 132,109–115.[CrossRef][ISI][Medline]

Matsuura T, Sugimura M, Iwaki T, Ohashi R, Kanayama N and Nishihira J (2002) Anti-macrophage inhibitory factor antibody inhibits PMSG-hCG-induced follicular growth and ovulation in mice. J Assist Reprod Genet 19,591–595.[CrossRef][ISI][Medline]

Metz CN and Bucala R (1997) Role of macrophage migration inhibitory factor in the regulation of the immune response. Adv Immunol 66,197–223.[ISI][Medline]

Nishihira J (1998) Novel pathophysiological aspects of macrophage migration inhibitory factor (review). Int J Mol Med 2,17–28.[ISI][Medline]

Nishihira J, Ishibashi T, Fukushima T, Sun B, Sato Y and Todo S (2003) Macrophage migration inhibitory factor (MIF): its potential role in tumor growth and tumor-associated angiogenesis. Ann NY Acad Sci 995,171–182.[Abstract/Free Full Text]

Noyes RW, Hertig AT and Rock J (1975) Dating the endometrial biopsy. Am J Obstet Gynecol 122,262–263.[Medline]

Psychoyos A (1993) Uterine receptivity for egg-implantation and scanning electron microscopy. Acta Eur Fertil 24,41–42.[Medline]

Rao CV (2002) [LH receptors: follicle and endometrium]. J Gynecol Obstet Biol Reprod (Paris) 31,1S7–1S11.[Medline]

Riedemann NC, Guo RF, Gao H, Sun L, Hoesel M, Hollmann TJ, Wetsel RA, Zetoune FS and Ward PA (2004) Regulatory role of C5a on macrophage migration inhibitory factor release from neutrophils. J Immunol 173,1355–1359.[Abstract/Free Full Text]

Saitoh H (2003) [The role of macrophage migration inhibitory factor (MIF) in follicle growth and ovulation]. Hokkaido Igaku Zasshi 78,329–338.[Medline]

Simon C, Piquette GN, Frances A and Polan ML (1993) Localization of interleukin-1 type I receptor and interleukin-1 beta in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab 77,549–555.[Abstract]

Simon C, Piquette GN, Frances A, el-Danasouri I, Irwin JC and Polan ML (1994) The effect of interleukin-1 beta (IL-1 beta) on the regulation of IL-1 receptor type I messenger ribonucleic acid and protein levels in cultured human endometrial stromal and glandular cells. J Clin Endocrinol Metab 78,675–682.[Abstract]

Simon C, Caballero-Campo P, Garcia-Velasco JA and Pellicer A (1998) Potential implications of chemokines in reproductive function: an attractive idea. J Reprod Immunol 38,169–193.[CrossRef][ISI][Medline]

Suzuki H, Kanagawa H and Nishihira J (1996) Evidence for the presence of macrophage migration inhibitory factor in murine reproductive organs and early embryos. Immunol Lett 51,141–147.[CrossRef][ISI][Medline]

Tabibzadeh S (1991) Human endometrium: an active site of cytokine production and action. Endocr Rev 12,272–290.[ISI][Medline]

Tabibzadeh S (1998) Molecular control of the implantation window. Hum Reprod Update 4,465–471.[Abstract/Free Full Text]

von Wolff M, Thaler CJ, Strowitzki T, Broome J, Stolz W and Tabibzadeh S (2000) Regulated expression of cytokines in human endometrium throughout the menstrual cycle: dysregulation in habitual abortion. Mol Hum Reprod 6,627–634.[Abstract/Free Full Text]

Yang Y, Degranpre P, Kharfi A and Akoum A (2000) Identification of macrophage migration inhibitory factor as a potent endothelial cell growth-promoting agent released by ectopic human endometrial cells. J Clin Endocrinol Metab 85,4721–4727.[Abstract/Free Full Text]

Submitted on February 4, 2005; resubmitted on June 27, 2005; accepted on July 5, 2005.





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