Increased soluble interleukin-1 receptor type II proteolysis in the endometrium of women with endometriosis

C. Bellehumeur1,2, T. Collette1,2, R. Maheux1,2, J. Mailloux2, M. Villeneuve2 and A. Akoum1,2,3

1 Centre de Recherche, Hôpital Saint-François d'Assise, Centre Hospitalier Universitaire de Québec, Faculté de Médecine, Université Laval, 2 Département d'Obstétrique et Gynécologie, Faculté de Médecine, Université Laval, Québec, Canada

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Numerous functional changes were observed in the intrauterine endometrial tissue of women with endometriosis. Our previous studies revealed a marked decrease in the expression of interleukin-1 receptor type 2 (IL-1RII), a decoy receptor known for its ability to buffer IL-1 effects. The aim of the present study was to assess whether post-translational mechanisms such as proteolysis may contribute to the down-regulation of IL-1RII levels. Our data showed that soluble IL-1RII (sIL-1RII) concentrations released by freshly cultured endometrial tissue were significantly lower in women with endometriosis than in normal women (P < 0.01) and further revealed a statistically significant correlation between increased proteolysis and decreased sIL-1RII levels (P<0.05; r=–0.47). 125I-labelled soluble recombinant human IL-1RII ([125I]srhIL-1RII) was significantly more degraded in culture supernatant of tissues from women with endometriosis compared to normal women (P < 0.05), and natural tissue inhibitor of matrix metalloproteinase (TIMP)-1 inhibited [125I]srhIL-1RII degradation. Incubation of srhIL-1RII with active rhMMP-9 resulted in a dose-dependent degradation of srhIL-1RII as analysed by western blotting. Dual immunofluorescence showed an increased immunostaining for matrix metalloproteinase-9 in situ in the endometrial tissue of women with endometriosis compared to normal women and a decreased immunostaining for IL-1RII. The present study showed a reduced release of sIL-1RII by the endometrial tissue of women with endometriosis and revealed a proteolytic post-translational mechanism which may be involved in the down-regulation of IL-1RII levels. This may enhance IL-1-mediated activation of endometrial cells and contribute to the local immuno-inflammatory process observed in endometriosis patients.

Key words: endometriosis/endometrium/IL-1RII/proteolysis/MMP-9


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Endometriosis is one of the most common gynaecological diseases in women of reproductive age and frequently associated with infertility and pelvic pain (Strathy et al., 1982Go; Goldman and Cramer, 1990Go). The disease is characterized by an abnormal development of endometrial tissue outside the uterus, mainly in the peritoneal cavity, and associated with an aberrant immuno-inflammatory process that takes place not only in ectopic sites where endometrial tissue abnormally implants, but even in the eutopic intrauterine endometrium (Sharpe-Timms, 2001Go).

Interleukin-1 (IL-1), a major proinflammatory cytokine, is believed to have an important role in endometriosis pathophysiology. Increased concentrations of IL-1 were found in the peritoneal fluid of endometriosis patients (Fakih et al., 1987Go). Peripheral blood monocytes as well as peritoneal macrophages of women with endometriosis secrete elevated concentrations of IL-1 (Zeller et al., 1987Go; Mori et al., 1992Go). An up-regulation of IL-1 expression was recently observed in the eutopic endometrial tissue of endometriosis patients (Bergqvist et al., 2001Go). IL-1 induces an angiogenic phenotype in endometriotic cells and stimulates the secretion of vascular endothelial growth factor and IL-6 (Lebovic et al., 2000Go). According to our own data, both ectopic and eutopic endometrial tissue of women with endometriosis are more responsive to IL-1 and secrete increased amounts of monocyte chemotactic protein-1 (MCP-1), IL-8, and the chemokine regulated upon activation, normal T cell expressed and secreted (RANTES) (Akoum et al., 1995aGo,bGo, 2001bGo, 2002Go). Interestingly, our subsequent studies revealed a marked decrease in the expression of IL-1 receptor type II (IL-1RII) in the eutopic endometrial cells of endometriosis women (Akoum et al., 2001aGo), whereas no significant change in IL-1RI expression was noted (unpublished data). Evidence available to date indicates that IL-1 exerts its biological effects via IL-1RI, the functional signalling receptor, whereas IL-1RII acts as decoy receptor for IL-1, thereby buffering the cytokine's effects on target cells (Colotta et al., 1993Go; Sims et al., 1993Go; Greenfeder et al., 1995Go). Actually, the membrane-bound receptor can be cleaved and released in a soluble form from the cell surface following proteolysis (Orlando et al., 1997Go; Cui et al., 2003Go). However, both the soluble and the membrane forms of the receptor keep their ability to bind IL-1 and to neutralize its effects (Dunne and O'Neill, 2003Go). Consequently, decreased IL-1RII expression in the endometrial tissue of endometriosis patients reveals a deficiency in the capability of endometrial and endometriotic cells to down-regulate IL-1 effects, which may play an important role in the aberrant inflammatory process described in ectopic and eutopic endometrial sites.

In a first attempt to elucidate the mechanisms underlying such a deficiency in IL-1RII protein expression observed in the endometrial tissue of endometriosis patients, we studied IL-1RII mRNA expression and found a significant decrease in women with endometriosis as compared to normal women (Kharfi et al., 2002Go). This points toward a defect in IL-1RII gene transcription and/or to decreased mRNA stability, and may at least in part explain IL-1RII reduced protein expression. More recently, we found an increased proteolysis and a significant increase in protease release in the culture supernatant of endometrial tissue from women with endometriosis and identified matrix metalloproteinase-9 (MMP-9) as one of the major overproduced proteases (Collette et al., 2004Go). It is now well documented that proteolysis is one of the major mechanisms involved in the regulation of cytokine activity and inflammation, and the role of MMP in this process has been reported (McQuibban et al., 2000Go, 2002Go; Van Den Steen et al., 2000Go, 2003Go; Nelissen et al., 2003Go). It is also known that proteases such as MMP are involved in IL-1RII cleavage and shedding (Orlando et al., 1997Go; Cui et al., 2003Go). The objective of the present study was therefore to investigate whether post-translational mechanisms such as proteolysis may be implicated in the degradation of sIL-1RII. This may further reduce sIL-1RII availability, amplify IL-1-mediated cell activation and contribute to functional changes in the eutopic endometrium of endometriosis patients.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemicals
Recombinant human (rh) IL-1{beta}, soluble recombinant human IL-1 type II receptor (srhIL-1RII), tissue inhibitor of matrix metalloproteinase (rhTIMP-1) and goat polyclonal anti-shIL-1RII antibody were purchased from R&D Systems (USA). Peroxidase-conjugated rabbit anti-goat antibody, biotin-conjugated rabbit anti-goat antibody and peroxidase-conjugated streptavidin were supplied by Jackson ImmunoResearch Laboratories, Inc. (USA). Active rhMMP-9 and sheep polyclonal anti-hMMP-9 antibody were obtained from Oncogene research product (USA). Hanks' balanced salt solution (HBSS) without calcium and magnesium, Dulbecco's modified Eagle's medium F-12 (DMEM–F-12) and antibiotics–antimycotics were purchased from Invitrogen Life Technologies (Canada). Monoclonal mouse anti-hMMP-9 antibody was obtained by Novocastra Laboratories, Inc. (Canada). [125I]Na was supplied by NEN, Perkin Elmer (Canada). Fluorescein isothiocyanate-conjugated streptavidin and rhodamine-conjugated sheep anti-mouse antibody were obtained from Sigma (USA).

Subjects and tissue collection
The women recruited in this study provided informed consent for a research protocol approved by the Ethics Board for human research of the Saint-François d'Assise Hospital. Endometriosis was identified during laparoscopy or laparotomy in women consulting for infertility and/or pelvic pain. Cultured endometrial biopsies were from women with endometriosis (n=17; mean age 32.7±4.6 years) who had no other pelvic condition. Six were at the proliferative phase and 11 at the secretory phase. The stage of endometriosis was determined according to the revised classification of the American Society for Reproductive Medicine (1997)Go. Five had endometriosis stage I, 10 endometriosis stage II and two endometriosis stage III. Control tissues were from normal women (n=14; mean age=35.0±5.4 years) who were fertile, requesting tubal ligation, and exhibiting no visible evidence of endometriosis upon laparoscopy. Six patients were at the proliferative phase and eight at the secretory phase. Endometrial biopsies used for immunohistochemical analyses were from 10 women with endometriosis (mean age 32.6±2.6 years) and nine normal women (mean age 34.6±5.5 years). Five women with endometriosis were at the proliferative phase and five at the secretory phase. Six had endometriosis stage I and four endometriosis stage II. Five biopsies from normal women used for immunohistochemistry were at the proliferative phase and four at the secretory phase. The cycle phase (proliferative or secretory) was determined based on the patient's cycle history, serum progesterone, and histological criteria of Noyes et al. (1975)Go. Endometrial samples were collected with a curette before laparoscopy. The tissue was placed in cold, sterile HBSS containing 1% antibiotics, then immediately transported to the laboratory. A part of the biopsy was taken for explant culture, and the remaining was snap-frozen with Tissue-Tek OCT compound (Miles, Inc., USA) and stored at –80 °C until analysed by immunohistochemistry.

Culture of endometrial tissue
Biopsies used in this study were devoid of any visible blood contamination. Tissues were immediately washed with cold HBSS and cut into pieces of ~1 mm3. Six pieces of tissue were put in each well (24-well plates) and incubated for 24 h at 37 °C, 5% CO2 with phenol red-free DMEM–F-12 medium containing 100 IU/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin. The culture medium was then collected on ice, centrifuged in order to eliminate cell debris, aliquoted, and stored at –80 °C for future use. Endometrial tissue explants were recuperated, and total proteins were extracted as described previously (Bigonnesse et al., 2001Go), and protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories Ltd, Canada).

IL-1RII enzyme-linked immunosorbent assay (ELISA)
Soluble IL-1RII concentrations were measured according to our previously reported procedure (Kharfi and Akoum, 2001Go).

Protease assay
Protease activity was determined according to an original procedure described by Millet (1977)Go, which was modified in our laboratory so as to allow the use of small sample volume and a 96-well microplate reader as previously described (Collette et al., 2004Go). The proteolytic activity was extrapolated from a standard curve using Trypsin (Gibco BRL) as reference and expressed in USP (United States Pharmacopeia) units/µg of tissue proteins.

MMP-9 ELISA
MMP-9 concentrations in the explant culture medium were measured using an ELISA procedure developed in the laboratory as previously described (Collette et al., 2004Go).

Evaluation of radiolabelled srhIL-1RII degradation
Radiolabelling of carrier-free srhIL-1RII with [125I]Na was conducted using the chloramine-T method (Hunter and Greenwood, 1962Go) and stored at 4 °C in the presence of 0.02% NaN3 and 0.1% bovine serum albumin (BSA). Radiolabelled srhIL-1RII (12 500 cpm) in a total volume of 5 µl phosphate-buffered saline (PBS)–0.1% BSA were incubated in the presence of 25 µl of culture supernatants at 37 °C for 1–6 h. Radiolabelled srhIL-1RII incubated or not with the culture medium for the same time-periods were included as controls. The reaction was stopped by heating samples in 5x loading buffer [1.25 mol/l Tris–HCl pH 6.8, 50% (v/v) glycerol, 25% {beta}-mercaptoethanol, 10% (w/v) sodium dodecyl sulphate (SDS), and 0.01% (w/v) Bromophenol Blue]. Samples were then separated by SDS–polyacrylamide gel electrophoresis (PAGE) on a 12% (w/v) acrylamide linear-gradient slab gel, fixed for 20 min in 10% acetic acid, 10% methanol, 1% glycerol, dried using a gel dryer (Bio-Rad, USA) for 2 h at 80 °C under vacuum and exposed to a Bio-max MR Kodak film (Kodak, USA) for 1–16 h before development. For inhibition of MMP-9, incubation of [125I]srhIL-IRII with the culture supernatant was also carried out in the presence rhTIMP-1 (5 ng/ml), anti-hMMP-9 sheep polyclonal antibody (500 ng/ml), or with equal concentration of IgG. The intensity of [125I]srhIL-1RII was determined by computer-assisted densitometry (Quantity One quantitation software; Bio-Rad). Data were normalized to equal endometrial tissue proteins and expressed as percentage of degradation which was determined according to the following equation: (1–CS/CM)x100, where CS=[125I]srhIL-1RII signal following incubation with a culture supernatant and CM = [125I]srhIL-1RII signal following incubation with the basic culture medium taken as control.

Western blot analysis
For western blot analysis of srhIL-1RII degradation by rhMMP-9, 10 ng of srhIL-1RII were incubated for 1 h at 37 °C with different amounts of active rhMMP-9 (0, 100, 200, 400 and 600 ng) in 30 µl of 50 nmol/l Tris buffer pH 7.0 containing 200 mmol/l NaCl, 1 µmol/l ZnCl2, 5 mmol/l CaCl2, 0.5% NaN3 and 0.1% BSA. Incubation was stopped by adding 6 µl of 5x loading buffer and heating samples in a boiling water bath. In subsequent experiments, rhMMP-9 (200 ng) was preincubated for 1 h at 37 °C with different amounts of rhTIMP-1 (35, 70 and 140 ng), polyclonal sheep anti-hMMP-9 antibody (1000, 2000 or 4000 ng) or with equivalent concentrations of normal sheep IgG before being incubated with 5 ng of srhIL-1RII for 1 h at 37 °C. Samples were then separated by SDS–PAGE in 12% (w/v) acrylamide linear gradient gel slabs and transferred onto 0.45 µm nitrocellulose membranes (Schleicher & Schuell, Keene, USA) using an electrophoretic transfer cell (Bio-Rad).

For western blot analysis of sIL-1RII in the culture supernatant of endometrial tissue, samples were denatured and separated by SDS–PAGE in 12% (w/v) acrylamide linear gradient gel slabs before being transferred onto 0.45 µm nitrocellulose membranes.

For IL-1RII detection, the nitrocellulose membranes were immersed in PBS containing 5% (w/v) skimmed milk and 0.1% Tween 20 (blocking solution) for 1 h at room temperature and cut into strips. Membrane strips were successively incubated with a polyclonal goat anti-shIL-1RII antibody (0.1 µg/ml in PBS, 0.1%–Tween 20) for 1 h at room temperature, peroxidase-conjugated rabbit anti-goat antibody (1:20 000 dilution in PBS, 0.1% Tween-20) for 1 h at room temperature, PBS, Tween 20, an enhanced chemiluminescence reagent (Roche Diagnostic, Canada) for 1 min at room temperature and exposed to BioMax film for 1–2 min for an optimal detection (all bands visible but not overexposed).

Dual immunofluorescent staining
Cryosections (5 µm) of Optimal Cutting Temperature (OCT)-frozen endometrial tissue were mounted on poly-L-lysine-coated microscope glass slides, fixed for 20 min in a 10% buffered formalin phosphate solution (Fisher Scientific, Canada), and washed in PBS. After permeabilization with Triton X-100 (1% in PBS) and elimination of endogenous peroxidase with H2O2 (0.3% in absolute methanol), sections were incubated at room temperature for 90 min with a mouse monoclonal anti-hMMP-9 antibody (1:20 dilution in PBS–BSA 1%). Sections were then incubated at room temperature for 60 min with a goat polyclonal anti-shIL-1RII antibody (1:7 dilution in PBS–BSA 1%) followed by a 60 min incubation with biotinylated rabbit anti-goat antibody (1:100 in PBS–BSA 1%). After a subsequent wash in PBS, tissue sections were incubated simultaneously for 60 min at room temperature in the dark with fluorescein isothiocyanate-conjugated streptavidin (diluted 1:100 in PBS–BSA 1%) and rhodamine-conjugated sheep antimouse antibody (diluted 1:10 in PBS–BSA 1%). After a final wash in PBS, slides were mounted in Mowiol containing 10% p-phenylenediamine (Sigma, USA), an antifading agent, and observed under the microscope (Olympus) equipped for fluorescence with a 100 W UV lamp. Images were taken using a M300 digital camera (JAI) and analysed with Isis 4.4.25 software.

Statistical analysis
Data followed a parametric distribution, and were therefore expressed as mean±SEM. An unpaired t-test was used for comparing those means and analysis of correlation was carried out using the Pearson correlation coefficient. All analyses were performed using GraphPad Software, Prism 3.0 (GraphPad Software, USA). Differences were considered as statistically significant for P<0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Soluble IL-1RII release by the endometrial tissue of normal and endometriosis women
Fresh explants of endometrial tissue from normal and endometriosis women were cultured for 24 h and the amount of sIL-1RII released in the culture medium was determined by ELISA. Data shown in Figure 1A show that sIL-1RII concentrations were significantly lower in women with endometriosis than in normal women (P<0.01). Western blot analysis of sIL-1RII in endometrial tissue culture supernatant showed a major 45 kDa band whose mol. wt is equivalent to the known mol. wt of the soluble receptor and a series of lower mol. wt bands which may correspond to degraded sIL-1RII. Furthermore, sIL-1RII bands were generally less intense in women with endometriosis than in normal women. A representative western blot is shown in Figure 1B.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 1. Soluble IL-1RII secretion in endometrial tissue conditioned medium. Explants of endometrial tissue from patients with endometriosis and normal controls were incubated for 24 h with serum-free media and then collected for analysis. (A) Soluble IL-1RII concentration as measured by ELISA (ng/µg of tissue proteins). E: endometriosis women (n=14); N: normal women (n=9). Values are means±SEM of duplicate measurements from two different assays. **Significant difference between endometriosis patients and normal controls (P<0.01). (B) Representative western blot of IL-1RII in endometrial tissue conditioned media from normal (lanes 1 and 3) and endometriosis (lanes 2 and 4) women. Lanes 1 and 2: incubation with polyclonal goat anti-shIL-1RII antibody; lanes 3 and 4: incubation with normal goat IgG; lane 5: rhIL-1RII. Note the detection of a series of low mol. wt sIL-1RII bands (~40.8, 36.8 and 33.4 kDa) in normal and endometriosis women, the reduced intensity of these bands in endometriosis and the appearance of additional smaller sIL-1RII bands (~33.4 and 31 kDa) resulting possibly from increased degradation.

 
Soluble IL-1RII release from the cell surface is known to result from proteolytic cleavage of the membrane-bound receptor (Orlando et al., 1997Go; Cui et al., 2003Go). Our data described above suggest that proteases may have the capability of degrading the soluble receptor following its release, thereby contributing to down-regulating its extracellular levels. Furthermore, our previous data showed that endometrial tissue from women with endometriosis produces and releases higher amounts of proteases than tissue from normal women, and that MMP-9 is one of the main overproduced proteases (Collette et al., 2004). Therefore, in parallel to sIL-1RII concentrations in endometrial tissue culture supernatants, we quantified the proteolytic activity and MMP-9 concentrations and found a significant increase in women with endometriosis compared to normal women (P<0.05 and P<0.05, respectively) (Figure 2), which is in keeping with our previous data (Collette et al., 2004). Interestingly, sIL-1RII concentrations showed a significant inverse correlation with the proteolytic activity (P<0.05; r=–0.47) and a trend for an inverse correlation with MMP-9 concentrations (P=0.08; r=–0.40). Thus, these data suggest an endometriosis-associated diminution of sIL-1RII stability due to increased proteolysis and possible involvement of MMP-9.



View larger version (12K):
[in this window]
[in a new window]
 
Figure 2. Proteolytic activity and MMP-9 concentration in endometrial tissue conditioned medium. Explants of endometrial tissue from patients with endometriosis and normal controls were incubated for 24 h with serum-free medium and then collected for analysis. The proteolytic activity (A) was assessed using a quantitative modified azocasein-based protease assay and was expressed in USP units/µg of tissue proteins taking trypsin activity as reference. MMP-9 concentration (B) in the culture medium was measured by ELISA. Values are means±SEM of duplicate measurements from two different assays. E: endometriosis women (n=13); N: normal women (n=7). *Significant difference between endometriosis patients and normal controls (P<0.05).

 
Soluble IL-1RII stability
Based on the above data, we further assessed sIL-1RII stability in the culture supernatants of endometrial tissues from women with and without endometriosis. Soluble rhIL-1RII was labelled with [125I]Na and incubated at 37 °C with culture supernatants for a 3 h period. This time was required for conspicuous degradation of [125I]srhIL-1RII, and was selected on the basis of degradation kinetics carried out with different culture supernatants. Figure 3A shows a representative autoradiogram of [125I]srhIL-1RII degradation in endometrial tissue culture supernatants. Data were expressed as the percentage of [125I]srhIL-1RII degradation in tissue culture supernatants, taking the basic culture medium as reference (0% degradation). Statistical analysis of the data showed an increased degradation of [125I]srhIL-1RII in the culture supernatants of tissues from women with endometriosis as compared to normal women (P<0.05) (Figure 3B). Statistical analysis of data using the Pearson correlation coefficient showed that [125I]srhIL-1RII degradation in women with endometriosis significantly correlated with the increased proteolytic activity observed in these women (P<0.05; r=0.57). Furthermore, a trend for a correlation between [125I]srhIL-1RII degradation and increased MMP-9 concentrations was observed (P=0.056; r=0.44).



View larger version (29K):
[in this window]
[in a new window]
 
Figure 3. Measurement of srhIL-1RII degradation in endometrial tissue conditioned medium. (A) Representative autoradiogram of soluble [125I]srhIL-1RII protein degradation. Lanes 1 and 2: [125I]srhIL-1RII incubation with DMEM for 1 and 3 h respectively. Lanes 3 and 4: [125I]srhIL-1RII incubation with tissue conditioned medium from a patient with endometriosis for 1 and 3 h respectively. Lanes 5 and 6: [125I]srhIL-1RII incubation with tissue conditioned from a normal patient for 1 and 3 h respectively. (B) Densitometric analysis of [125I]srhIL-1RII bands. Data are expressed as the percentage of [125I]srhIL-1RII degradation in tissue culture supernatants taking the basic culture medium as reference. Values are means±SEM of duplicate measurements from two different assays. E: endometriosis women (n=12); N: normal women (n=10). *Significant difference between endometriosis patients and normal controls (P<0.05).

 
Involvement of MMP-9 in sIL-1RII degradation
Considering these data, we then assessed whether MMP-9 can effectively degrade sIL-1RII and be involved in the increased degradation of the soluble receptor in women with endometriosis. In fact, available literature indicates the presence of different putative cleavage sites for MMP in sIL-1RII protein sequence. In particular, IL-1RII appears to have two different cleavage sites for MMP-9 situated in positions 139–143 and 237–241 (McMahan et al., 1991Go; Kridel et al., 2001Go). To achieve this, we first incubated endometrial tissue culture supernatants with rhTIMP-1, a natural specific inhibitor of several MMP including MMP-9, or with a sheep polyclonal antibody specific to hMMP-9. A representative autoradiogram illustrated in Figure 4A shows that both rhTIMP-1 and anti-hMMP-9 antibody partially blocked [125I]srhIL-1RII degradation in the culture supernatants, although a more marked blockade was observed with rhTIMP-1. Furthermore, incubation of srhIL-1RII with different concentrations of active rhMMP-9 resulted in a dose-dependent degradation of shrIL-1RII as analysed by western blotting (Figure 4B). Proteolysis of srhIL-1RII was inhibited following preincubation of rhMMP-9 with rhTIMP-1 or with anti-hMMP-9 antibody (Figure 4C).



View larger version (84K):
[in this window]
[in a new window]
 
Figure 4. Effect of MMP-9 on srhIL-1RII protein degradation. (A) Representative autoradiogram of [125I]srhIL-1RII incubated with tissue culture supernatants from women with endometriosis in the presence of rhTIMP-1 (lane 1), polyclonal anti-hMMP-9 antibody (lane 2) or in the absence of inhibitors (lane 3). [125I]srhIL-1RII incubated with DMEM was taken as control (lane 4). (B) Representative western blot of srhIL-1RII (10 ng) incubated with different amounts of rhMMP-9 (0, 100, 200, 400 and 600 ng) (lanes 1–5). (C) Representative western blot of srhIL-1RII (5 ng) incubated with the vehicle buffer alone (lane 1), rhMMP-9 (200 ng) and rhTIMP-1 (40 ng) (lane 2), rhMMP-9 (200 ng) and polyclonal anti-hMMP-9 antibody (4 µg) (lane 3) or rhMMP-9 alone (200 ng) (lane 4).

 
Co-localization of IL-1RII and MMP-9 in the endometrial tissue
Dual immunofluorescent staining of IL-1RII and MMP-9 in the endometrial tissue showed that IL-1RII immunostaining was remarkably lower in women with endometriosis compared to normal women, and coincided with an increased immunostaining for MMP-9 both in the stroma and epithelial glands (Figure 5).



View larger version (78K):
[in this window]
[in a new window]
 
Figure 5. Dual immunofluorescent staining of IL-1RII and MMP-9. Dual immunofluorescent staining of IL-1RII (A and D) and MMP-9 (B and E) in the endometrial tissue of normal women (A, B and C) and women with endometriosis (D, E and F). Tissue sections were incubated with goat polyclonal anti-shIL-1RII antibody and with mouse anti-hMMP-9 antibody. Sections were then incubated with rabbit anti-goat biotinylated antibody and then simultaneously with fluorescein isothiocyanate-conjugated streptavidin and rhodamine-conjugated sheep anti-mouse antibody to detect IL-1RII and MMP-9 respectively. Superimposing fluorescein (green) and rhodamine (red) shows co-expression of IL-1RII and MMP-9 (C and F). Scale bars =20 mm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study showed that human endometrial tissue releases sIL-1RII. Western blot analysis showed a major 45 kDa band and a number of lower mol. wt bands which may correspond to degraded sIL-1RII. The study further showed that sIL-1RII concentrations released by the endometrial tissue were markedly lower in women with endometriosis than in normal women, which is in keeping with our previous data showing a marked decrease in IL-1RII immunostaining in situ in the endometrial tissue of women with endometriosis (Akoum et al., 2001aGo) and a significant diminution in IL-1RII mRNA levels (Kharfi et al., 2002Go).

Soluble IL-1RII is found in the general circulation under physiological conditions (Juffermans et al., 1998Go; Laurincova, 2000Go). Elevated levels of sIL-1RII are detectable in biological fluids under conditions of immunological disorders (Van Deuren et al., 1997Go; Juffermans et al., 1998Go; Laurincova, 2000Go). The released sIL-1RII keeps its ability to bind with high affinity to IL-1, particularly the circulating form (IL-1{beta}), and to neutralize its biological effects (Colotta et al., 1994Go; Laurincova, 2000Go). Soluble IL-1RII appears to act by sequestering IL-1{beta} within the extracellular compartment, thus restricting its availability and interaction with IL-1RI. In addition, sIL-1RII binds and blocks processing of IL-1{beta} precursor, inhibits its maturation, loses affinity for IL-1 receptor antagonist (IL-1ra) and does not therefore interfere with IL-1ra-mediated inhibition of IL-1 effects (Dunne and O'Neill, 2003Go). Such a release of the soluble decoy receptor has been proposed to be an early event in the inflammatory cascade that acts to limit its severity. Therefore, the reduced release of sIL-1RII by the endometrial tissue of women with endometriosis indicates a deficiency in the capability of this tissue to down-regulate IL-1-mediated effects.

It is well documented that proteases such as MMP contribute to sIL-1RII shedding from the cell surface (Orlando et al., 1997Go; Cui et al., 2003Go). Numerous MMP were reported to be expressed in the normal human endometrial tissue, where they appear to play a significant role in normal tissue remodelling during the sequential phases of proliferation, differentiation and tissue breakdown during menstruation (Curry and Osteen, 2003Go). MMP may be therefore involved in the cleavage of the membrane-bound IL-1RII and normal shedding of sIL-1RII from endometrial cells. In endometriosis, several proteases including MMP were found to have an increased expression in ectopic and eutopic endometrial tissues and to be involved in the invasive establishment of the disease (Osteen et al., 2003Go Liu et al., 2002Go; Gilabert Estelles et al., 2003Go). Our previous studies revealed an increased release of proteolytic activity by the eutopic endometrium in women with endometriosis as compared to normal women and identified MMP-9 as one of the overproduced proteases by this tissue (Collette et al., 2004). Data of the present study corroborated these findings and further showed a significant correlation between increased proteolysis and decreased sIL-1RII levels. A trend for a correlation between MMP-9 secretion and sIL-1RII concentrations was also noted. Furthermore, our data showed a marked degradation of [125I]srhIL-1RII in the culture medium of endometriosis women-derived endometrial tissue, which significantly correlated with the proteolytic activity and showed a tendency for a correlation with MMP-9 concentrations. Therefore, while proteases contribute to sIL-1RII shedding (Orlando et al., 1997Go; Cui et al., 2003Go), our data suggest that their elevated concentrations in the endometrium of endometriosis patients may amplify the degradation of the soluble receptor and further reduce its availability. Furthermore, TIMP-1, a natural specific inhibitor of several MMP including MMP-9, partially inhibited [125I]srhIL-1RII degradation. The degradation of [125I]srhIL-1RII was also inhibited, although less markedly, by anti-hMMP-9 antibody, which indicates that MMP may contribute to sIL-1RII instability and suggests a role for MMP-9. Incubation of srhIL-1RII with active rhMMP-9 resulted in a dose-dependent degradation of the soluble receptor as shown by western blot analysis, and preincubation of rhMMP-9 with anti-hMMP-9 antibody or rhTIMP-1 inhibited srhIL-1RII degradation. This is in agreement with the presence of at least two putative cleavage sites for MMP-9 in sIL-1RII amino acid sequence as reported previously (McMahan et al., 1991Go; Kridel et al., 2001Go). Moreover, dual immunofluorescence showed an increased immunostaining for MMP-9 in situ in the endometrial tissue of women with endometriosis compared to normal women and a decreased immunostaining for IL-1RII. It is not documented yet whether sIL-1RII and MMP-9 form part of the same complex. Proteases other than MMP were shown to be involved in sIL-1RII shedding (Cui et al., 2003Go). These proteases might also be implicated in sIL-1RII protein degradation since TIMP-1 alone was unable to completely block the degradation of [125I]rhsIL-1RII. Further studies will be needed to investigate sIL-1RII–MMP-9 interaction and the involvement of other possible proteases. Proteolysis-mediated regulation of inflammation is a well-documented regulatory mechanism and described in a number of immuno-inflammatory disorders (Hiemstra, 2002Go). Several MMP were reported to cleave adhesion molecules, cytokines, chemokines, growth factors and binding proteins and to play an important role in positive or negative regulation of inflammation (Mohammed et al., 2003Go).

The present study reveals a post-translational mechanism by which proteases may contribute to down-regulating IL-1RII protein levels in the endometrial tissue of endometriosis women. Indeed, it is quite possible that decreased IL-1RII levels observed in the endometrial tissue of endometriosis patients (Kharfi et al., 2002Go) may by itself contribute to increased protease secretion as a result of an increased cell responsiveness to IL-1. It is well known that protease release by endometrial cells can be stimulated by IL-1 (Rawdanowicz et al., 1994Go) and that endometrial cells from endometriosis patients secrete more MMP or proteases in response to IL-1 (Sillem et al., 2001Go). Nevertheless, the fact that increased protease release accelerates sIL-1RII degradation makes this latter less available for binding to IL-1. This may further amplify IL-1-mediated cell activation and accentuate the immuno-inflammatory process observed locally in the eutopic endometrium of patients, as well as in the ectopic locations where this tissue can migrate, implant and develop into endometriosis lesions.

In conclusion, the present study showed for the first time a reduced release of sIL-1RII by the endometrial tissue of women with endometriosis and revealed a proteolytic post-translational mechanism which may be involved in the down-regulation of IL-1RII levels. Furthermore, it suggests that MMP and in particular MMP-9 may contribute to endometriosis-associated diminution of sIL-1RII stability. Such mechanisms may reduce the availability of the decoy sIL-1RII for IL-1 binding, thereby enhancing IL-1-mediated cell activation and accentuating the local immuno-inflammatory process observed in the eutopic endometrium of endometriosis patients.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work is supported by grant MOP-14638 to Ali Akoum from The Canadian Institutes for Health Research. A.A. is Chercheur National from the Fonds de la Recherche en Santédu Québec (FRSQ). The authors would like 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, Philippe Laberge and André Lemay for patient evaluation and providing peritoneal fluid samples, Madeleine Desaulniers, Rouslan Kats, Monique Longpré and Johanne Pelletier for technical assistance, and Dr Lucile Turcot-Lemay for her assistance with the statistical analyses.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Akoum A, Lemay A, Brunet C and Hebert J (1995a) Cytokine-induced secretion of monocyte chemotactic protein-1 by human endometriotic cells in culture. The Groupe d'Investigation en Gynecologie. Am J Obstet Gynecol 172, 594–600.[CrossRef][ISI][Medline]

Akoum A, Lemay A, Brunet C and Hebert J (1995b) Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis. Le Groupe d'Investigation en Gynecologie. Fertil Steril 63, 322–328.[ISI][Medline]

Akoum A, Jolicoeur C, Kharfi A and Aube M (2001a) Decreased expression of the decoy interleukin-1 receptor type II in human endometriosis. Am J Pathol 158, 481–489.[Abstract/Free Full Text]

Akoum A, Lawson C, McColl S and Villeneuve M (2001b) Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol Hum Reprod 7, 859–866.[Abstract/Free Full Text]

Akoum A, Lemay A and Maheux R (2002) Estradiol and interleukin-1beta 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 87, 5785–5792.[Abstract/Free Full Text]

American Society for Reproductive Medicine (1997) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 67, 817–821.[CrossRef][ISI][Medline]

Bergqvist A, Bruse C, Carlberg M and Carlstrom K (2001) Interleukin 1beta, interleukin-6, and tumor necrosis factor-alpha in endometriotic tissue and in endometrium. Fertil Steril 75, 489–495.[CrossRef][ISI][Medline]

Bigonnesse F, Marois M, Maheux R and Akoum A (2001) Interleukin-1 receptor accessory protein is constitutively expressed in human endometrium throughout the menstrual cycle. Mol Hum Reprod 7, 333–339.[Abstract/Free Full Text]

Collette T, Bellehumeur C, Kats, Maheux R, Mailloux J, Villeneuve M and Akoum A (2004) Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9. Hum Reprod 19, 1257–1264.[Abstract/Free Full Text]

Colotta F, Re F, Muzio M, Bertini R, Polentarutti N, Sironi M, Giri JG, Dower SK, Sims JE and Mantovani A (1993) Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 261, 472–475.[ISI][Medline]

Colotta F, Dower SK, Sims JE and Mantovani A (1994) The type II ‘decoy’ receptor: a novel regulatory pathway for interleukin 1. Immunol Today 15, 562–566.[CrossRef][ISI][Medline]

Cui X, Rouhani FN, Hawari F and Levine SJ (2003) Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding. J Immunol 171, 6814–6819.[Abstract/Free Full Text]

Curry TE Jr and Osteen KG (2003) The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle. Endocr Rev 24, 428–465.[Abstract/Free Full Text]

Dunne A and O'Neill LA (2003) The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci STKE 171, 3–17. re3.

Fakih H, Baggett B, Holtz G, Tsang KY, Lee JC and Williamson HO (1987) Interleukin-1: a possible role in the infertility associated with endometriosis. Fertil Steril 47, 213–217.[ISI][Medline]

Gilabert-Estelles J, Estelles A, Gilabert J, Castello R, Espana F, Falco C, Romeu A, Chirivella M, Zorio E and Aznar J (2003) Expression of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis. Hum Reprod 18, 1516–1522.[Abstract/Free Full Text]

Goldman MB and Cramer DW (1990) The epidemiology of endometriosis. Prog Clin Biol Res 323, 15–31.[Medline]

Greenfeder SA, Nunes P, Kwee L, Labow M, Chizzonite RA and Ju G (1995) Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex. J Biol Chem 270, 13757–13765.[Abstract/Free Full Text]

Hiemstra PS (2002) Novel roles of protease inhibitors in infection and inflammation. Biochem Soc Trans 30, 116–120.[CrossRef][ISI][Medline]

Hunter WM and Greenwood RC (1962) Preparation of iodine-131 labelled human growth hormone of high specific radioactivity. Nature 194, 495–496.[ISI][Medline]

Juffermans NP, Verbon A, Van Deventer SJ, Van Deutekom H, Speelman P and Van Der Poll T (1998) Tumor necrosis factor and interleukin-1 inhibitors as markers of disease activity of tuberculosis. Am J Respir Crit Care Med 157, 1328–1331.[Abstract/Free Full Text]

Kharfi A and Akoum A (2001) Correlation between decreased type-II interleukin-1 receptor and increased monocyte chemotactic protein-1 expression in the endometrium of women with endometriosis. Am J Reprod Immunol 45, 193–199.[CrossRef][ISI][Medline]

Kharfi A, Boucher A and Akoum A (2002) Abnormal interleukin-1 receptor type II gene expression in the endometrium of women with endometriosis. Biol Reprod 66, 401–406.[Abstract/Free Full Text]

Kridel SJ, Chen E, Kotra LP, Howard EW, Mobashery S and Smith JW (2001) Substrate hydrolysis by matrix metalloproteinase-9. J Biol Chem 276, 20572–20578.[Abstract/Free Full Text]

Laurincova B (2000) Interleukin-1 family: from genes to human disease. Acta Univ Palacki Olomuc Fac Med 143, 19–29.[Medline]

Lebovic DI, Bentzien F, Chao VA, Garrett EN, Meng YG and Taylor RN (2000) Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta. Mol Hum Reprod 6, 269–275.[Abstract/Free Full Text]

Liu XJ, He YL and Peng DX (2002) Expression of metalloproteinase-9 in ectopic endometrium in women with endometriosis. Di Yi Jun Yi Da Xue Xue Bao 22, 467–469.[Medline]

McMahan CJ, Slack JL, Mosley B, Cosman D, Lupton SD, Brunton LL, Grubin CE, Wignall JM, Jenkins NA, Brannan CI et al. (1991) A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J 10, 2821–2832.[Abstract]

McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I and Overall CM (2000) Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 289, 1202–1206.[Abstract/Free Full Text]

McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I and Overall CM (2002) Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo. Blood 100, 1160–1167.[Abstract/Free Full Text]

Millet J (1977) Characterization of a protein inhibitor of intracellular protease from Bacillus subtilis. FEBS Lett. 74, 59–61.[CrossRef][ISI][Medline]

Mohammed FF, Smookler DS and Khokha R (2003) Metalloproteinases, inflammation, and rheumatoid arthritis. Ann Rheum Dis 62 (Suppl 2), ii43–ii47.[Abstract/Free Full Text]

Mori H, Sawairi M, Nakagawa M, Itoh N, Wada K and Tamaya T (1992) Expression of interleukin-1 (IL-1) beta messenger ribonucleic acid (mRNA) and IL-1 receptor antagonist mRNA in peritoneal macrophages from patients with endometriosis. Fertil Steril 57, 535–542.[ISI][Medline]

Nelissen I, Martens E, Van Den Steen PE, Proost P, Ronsse I and Opdenakker G (2003) Gelatinase B/matrix metalloproteinase-9 cleaves interferon-beta and is a target for immunotherapy. Brain 126, 1371–1381.[Abstract/Free Full Text]

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

Orlando S, Sironi M, Bianchi G, Drummond AH, Boraschi D, Yabes D and Mantovani A (1997) Role of metalloproteases in the release of the IL-1 type II decoy receptor. J Biol Chem 272, 31764–31769.[Abstract/Free Full Text]

Osteen KG, Yeaman GR and Bruner-Tran KL (2003) Matrix metalloproteinases and endometriosis. Semin Reprod Med 21, 155–164.[CrossRef][ISI][Medline]

Rawdanowicz TJ, Hampton AL, Nagase H, Woolley DE and Salamonsen LA (1994) Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha. J Clin Endocrinol Metab 79, 530–536.[Abstract]

Sharpe-Timms KL (2001) Endometrial anomalies in women with endometriosis. Ann NY Acad Sci 943, 131–147.[Abstract/Free Full Text]

Sillem M, Prifti S, Koch A, Neher M, Jauckus J and Runnebaum B (2001) Regulation of matrix metalloproteinases and their inhibitors in uterine endometrial cells of patients with and without endometriosis. Eur J Obstet Gynecol Reprod Biol 95, 167–174.[CrossRef][ISI][Medline]

Sims JE, Gayle MA, Slack JL, Alderson MR, Bird TA, Giri JG, Colotta F, Re F, Mantovani A, Shanebeck K et al. (1993) Interleukin 1 signaling occurs exclusively via the type I receptor. Proc Natl Acad Sci USA 90, 6155–6159.[Abstract/Free Full Text]

Strathy JH, Molgaard CA, Coulam CB and Melton LJ, 3rd (1982) Endometriosis and infertility: a laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril 38, 667–672.[ISI][Medline]

Van Den Steen PE, Proost P, Wuyts A, Van Damme J and Opdenakker G (2000) Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact. Blood 96, 2673–2681.[Abstract/Free Full Text]

Van Den Steen PE, Wuyts A, Husson SJ, Proost P, Van Damme J and Opdenakker G (2003) Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process the chemokines human GCP-2/CXCL6, ENA-78/CXCL5 and mouse GCP-2/LIX and modulate their physiological activities. Eur J Biochem 270, 3739–3749.[Abstract/Free Full Text]

Van Deuren M, Van Der Ven-Jongekrijg J, Vannier E, Van Dalen R, Pesman G, Bartelink AK, Dinarello CA and Van Der Meer JW (1997) The pattern of interleukin-1beta (IL-1beta) and its modulating agents IL-1 receptor antagonist and IL-1 soluble receptor type II in acute meningococcal infections. Blood 90, 1101–1108.[Abstract/Free Full Text]

Zeller JM, Henig I, Radwanska E and Dmowski WP (1987) Enhancement of human monocyte and peritoneal macrophage chemiluminescence activities in women with endometriosis. Am J Reprod Immunol Microbiol 13, 78–82.[Medline]

Submitted on November 1, 2004; resubmitted on December 16, 2004; accepted on December 20, 2004.





This Article
Abstract
Full Text (PDF )
All Versions of this Article:
20/5/1177    most recent
deh749v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Bellehumeur, C.
Articles by Akoum, A.
PubMed
PubMed Citation
Articles by Bellehumeur, C.
Articles by Akoum, A.