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Regular Article |
From the Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, Centre Hospitalier Universitaire de Québec, Université Laval, Québec, Canada
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interleukin-1 (IL-1) is one of the major pro-inflammatory cytokines found to act on and to be produced by endometrial tissue.2-5 Circulating levels of IL-1 were shown to be variable during the menstrual cycle and to reach maximal levels during the secretory phase (after ovulation).6 The cytokine is produced by trophoblastic cells, and is believed to act as an embryonic signal and to play an important role during the implantation process.2,7,8 IL-1 is produced locally in endometrial tissue as well, mainly in the late secretory phase,3,9 suggesting that beside its potential role in implantation and embryonic development, this cytokine may be involved in the inflammatory-like process that takes place in the endometrium at the end of each menstrual cycle.
Based on the above evidence, it is reasonable to believe that endometrial tissue possesses the appropriate regulatory mechanisms that can operate locally and maintain tight control on the local level of pro-inflammatory cytokines. This is critical for maintaining the inflammatory-like process within safe physiological limits. Any defect in such mechanisms may lead to endometrial dysfunction and consequently to endometrium-related disorders affecting the reproductive function (ie, infertility, endometriosis, dysfunctional bleeding, and neoplasia).
Little is known about the mechanisms that modulate the expression and the action of pro-inflammatory cytokines such as IL-1, in the endometrium. Cell activation by IL-1 results from its binding to cell surface IL-1 receptor type-1 (IL-1RI) that in concert with IL-1 receptor accessory protein (IL-1RAcP) is capable of transducing the activation signal.10,11 Type II IL-1 receptor (IL-1RII) has, in contrast to the type I receptor, no signaling properties, but has recently been described as a "decoy receptor." The extracellular domain of the receptor can be shed from the cell surface as a soluble molecule that is capable of capturing IL-1, thus preventing its interaction with the functional receptor. These studies suggest that IL-1RII play an important physiological role in the regulation of IL-1 action in the inflammation sites.12-16
In the present study, we investigated the expression of IL-1 RII in the endometria of healthy women, and women with endometriosis, a very frequent endometrium-dependent gynecological disorder. The disease is characterized by an abnormal development of endometrial tissue outside the uterus, mainly in the peritoneal cavity, and associated with an immuno-inflammatory process that has been described in the both ectopic and eutopic endometrial sites.17-22
Our study revealed that IL-1RII is indeed expressed in endometrial tissue and in a cycle-dependent manner. The expression was omnipresent in both epithelial and stromal compartments, and was more conspicuous in the secretory phase of the menstrual cycle. The most intense immunostaining was, however, located in the luminal side of endometrial glands and surface epithelium. Interestingly, we found out that such expression was strikingly deficient in women with endometriosis, particularly in the secretory phase of the menstrual cycle.
The study provides for the first time evidence for the local expression in human endometrial tissue of the IL-1 decoy receptor, one of the most specific down-regulators of IL-1 action. Furthermore, it reveals a defect in that expression in the intrauterine endometrium of women suffering from endometriosis, that is, in the tissue where the disease is believed to take origin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Women were recruited into the study after they provided informed
consent for a protocol approved by the Saint-François d’Assise
Hospital Ethics Committee on Human Research. Women included in the
study (Table 1)
had no signs of endometrial hyperplasia
or neoplasia and were not receiving any anti-inflammatory or hormonal
medication at least 3 months before laparoscopy. Endometriosis was
diagnosed during investigative laparoscopy for infertility and/or
pelvic pain, or at tubal ligation. The stage of endometriosis was
determined according to the revised classification of the American
Fertility Society.23
Patients with endometriosis
(n = 54) otherwise had no other pelvic
pathology. Normal women (n = 39) were fertile,
requesting tubal ligation, and having no visible evidence of
endometriosis at laparoscopy. Menstrual cycle dating was determined by
menstrual history and confirmed by histological examination using the
criteria of Noyes and colleagues.24
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Endometrial biopsies were obtained during laparoscopy with the use of a Pipelle (Unimar Inc., Prodimed, Neuilly-En-Tchelle, France). Specimens were placed at 4°C in sterile Hanks’ balanced salt solution containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin, immediately transported to the laboratory, snap-frozen in liquid nitrogen or embedded in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN), and stored at -70°C until analyzed.
Immunohistochemistry
Serial 4-µm cryosections were placed on
poly-L-lysine-coated glass microscope slides and fixed for
20 minutes in formaldehyde [4% in phosphate-buffered saline (PBS)]
(Fisher Scientific, Montreal, Quebec, Canada). All incubations were
performed at room temperature in a humidified chamber. Sections were
rinsed in PBS, immersed in PBS-1% Triton X-100 for 20 minutes at room
temperature, rinsed again in PBS, and treated for 20 minutes with
hydrogen peroxide (H2O2)
(0.3% in absolute methanol) to eliminate endogenous peroxidase. After
a PBS rinse, immunostaining was performed using a mouse monoclonal
anti-human IL-1RII antibody (R and D Systems, Minneapolis, MN) (primary
antibody), a Vectastain Elite ABC kit (Vector Laboratories, Burlingame,
CA) and diaminobenzidine (Sigma Chemical Co., St. Louis, MO) as
chromogen. Briefly, after incubation with blocking serum for 30
minutes, sections were rinsed in PBS, incubated for 90 minutes with an
appropriate and predetermined dilution of primary antibody (15 µg/ml
of PBS containing 1% bovine serum albumin), rinsed in PBS, and
incubated for 60 minutes with the secondary antibody consisting of
biotinylated goat anti-mouse polyclonal antibody. Sections were then
rinsed in PBS and the avidin-biotinylated horseradish peroxidase
complex was applied for 45 minutes. After a PBS rinse followed by a
10-minute incubation with
diaminobenzidine:H2O2 (0.5
mg/0.03% H2O2 in PBS)
sections were washed in tap water, counterstained with hematoxylin, and
mounted with Mowiol (Calbiochem-Novabiochem Corp., La Jolla, CA).
Sections incubated without the primary antibody or with nonimmune mouse
serum were included as negative controls in all experiments. Slides
were viewed using a Leica microscope (Leica mikroskopie und
systeme GmbH, Model DMRB; Postfach, Wetzlar, Germany) and
photomicrographs were taken with Kodak 100 ASA film (Kodak, Toronto,
Ontario, Canada). IL-1RII immunostaining was evaluated in a blinded
manner by two independent observers having no knowledge of laparoscopic
findings. The intensity of staining was evaluated three times in three
different areas randomly selected in the section and a mean score was
given using an arbitrary scale (0, absent; 1, light; 2, moderate; and
3, intense). High concordance between the two observers was found as
determined by the 
measure of agreement ( = 0.89).
Dual Immunofluorescent Staining
Tissue sections were treated and incubated at room temperature with the mouse monoclonal anti-IL-1RII antibody as described earlier. After a PBS rinse, the sections were incubated for 60 minutes with a rabbit polyclonal anti-IL-1{beta} antibody diluted 8:1,000 in PBS-1% bovine serum albumin (R and D Systems), washed in PBS, incubated for 60 minutes with a biotinylated goat anti-rabbit antibody (Vector Laboratories) diluted 1:100 in PBS-1% bovine serum albumin, washed again in PBS, and finally incubated simultaneously for 60 minutes in the dark with fluorescein isothiocyanate-conjugated streptavidin and a rhodamine-conjugated goat anti-mouse antibody (Sigma), which were used at a final dilution of 1:100 and 1:10 in PBS-1% bovine serum albumin, respectively. Slides were then mounted with Mowiol to which p-phenylenediamine (Sigma), an anti-fading agent, was added at a final concentration of 1 mg/ml, then observed under the Leica microscope equipped for fluorescence with a 100 watt UV lamp and photomicrographs were made with Kodak 400 ASA film. In every experiment, sections from each endometrial tissue incubated with normal mouse and normal rabbit IgGs (used at concentrations equivalent to those of the primary antibodies) were included as negative controls.
Western Blot Analysis
Frozen endometrial tissues were directly homogenized with a microscale tissue grinder (Kontes, Vineland, NJ) in a buffer containing 0.5% Triton X-100, 10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 2 mmol/L ethyleneglycoltetraacetic acid, 2 mmol/L ethylenediaminetetraacetic acid, 0.02% NaN3, and a mix of anti-proteases composed by 5 µmol/L aprotinin, 63 µmol/L leupeptin, and 3 mmol/L phenylmethylsulfonyl fluoride. Tissue homogenate was then incubated at 4°C for 45 minutes under gentle shaking, and centrifuged at 11,000 x g for 30 minutes to recover the soluble extract, whose total protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada). Proteins (100 µg) from each extract were then heat-denatured in a boiling bath for 3 minutes in 5x sodium dodecyl sulfate sample buffer (1.25 mol/L Tris-HCl, pH 6.8, 50% glycerol, 25% {beta}-mercaptoethanol, 10% sodium dodecyl sulfate, and 0.01% bromophenol blue), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% acrylamide linear gradient gel slabs, and transferred onto 0.45-µm nitrocellulose membranes using electrophoretic transfer cell (trans-Blot, Bio-Rad). Nitrocellulose membranes were then immersed in PBS containing 5% skimmed milk and 0.1% Tween 20 (blocking solution) for 1 hour at 37°C, cut into strips, and incubated overnight at 4°C with a monoclonal mouse anti-human IL-1RII antibody (2 µg/ml of blocking solution) (R and D Systems) or with normal mouse immunoglobulins (IgGs) of the same immunoglobulin class and concentration as the primary antibody (R and D Systems). The specificity of the immunoreaction was also verified by pre-absorption of the antibody with an excess of IL-1RII (20 µg/ml).Thereafter, the strips were incubated for 1 hour at 37°C with Fc-specific peroxidase-labeled goat anti-mouse antibody (1:3000 dilution in the blocking solution) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), washed three times in PBS/0.1% Tween 20, incubated with chemiluminescence reagent (Amersham, Oakville, Ontario, Canada) for 1 minute, air-dried, wrapped in a plastic bag, and exposed to a Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) for 1 minute.
Statistical Analysis
IL-1RII staining scores follow an ordinal scale. Statistical analysis was performed using Fisher’s exact test,25 and the Bonferroni procedure was applied when more than two groups were compared. Comparison of patient’s age was performed using one-way analysis of variance. All analyses were performed using the statistical analyses system (SAS Institute Inc., Cary, North Carolina). Differences were considered as statistically significant for P values <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, the most marked staining was primarily located in epithelial
cells. Immunostaining was observed all around cells (cellular
staining), but also had the appearance of an intense brown
extracellular deposit that was predominantly located within the lumen
of the glands and the apical side of surface epithelium (luminal
staining) (Figure 1)
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. However, only a weak tendency
for an increased luminal staining in glandular and surface epithelium
was noted in the secretory phase of the cycle as compared to the
proliferative phase (P = 0.1370) (Table 3)
. Scattered brown deposits, which were
encountered in the stroma, albeit less markedly than in luminal and
glandular epithelium, were also more frequent in the secretory phase
than in the proliferative phase, but the difference did not reach the
level of statistical significance (P = 0.0930).
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Scattered brown deposits were also observed in the stroma of women with
endometriosis. As in normal women, they were more obvious in the
secretory phase of the menstrual cycle and showed a comparable level of
staining. The most striking difference between women with endometriosis
and normal women was however detected at the level of IL-1RII staining
in the lumen of endometrial glands and the apical side of surface
epithelium. In fact, statistical analysis of the data (Table 3)
showed
a considerable lack of staining in women with endometriosis compared to
normal controls (P =
10-6). Endometriosis patients were then
stratified by severity of disease (stage I, II, and III–IV).
Comparison of individual groups using the Fisher’s exact test and the
procedure of Bonferroni showed that the intensity of staining was
significantly lower in each endometriosis stage compared to controls,
but the most significant decrease in IL-1RII immunostaining was found
in the milder stages (I and II) (P = 0.0006 and
0.0015, respectively). Furthermore, statistical analysis of the data
taking into account the phase of the menstrual cycle revealed that the
most marked drop in IL-1RII luminal staining in endometriosis
occurred in the secretory phase (P = 8 x
10-7). This was also observed in all
stages of endometriosis, but was more pronounced in the early
(I and II) (P = 0.0020) than in the late
stages of the disease (III–IV) (P = 0.0060). In
contrast, during the proliferative phase of the menstrual cycle, the
difference in IL-1RII luminal immunostaining between women with and
without endometriosis was perceptible, but did not reach the level of
statistical significance (P = 0.0760). The 54
patients with endometriosis were also stratified for infertility and
IL-1RII luminal immunostaining scores were compared. Using the
Fisher’s exact test, both fertile and infertile patients with
endometriosis had decreased levels of immunostaining compared with
control women (P = 0.0010 and 4 x
10-5, respectively), but more significant
differences in infertile women with endometriosis was noted.
Representative examples of IL-1RII immunostaining in the endometrium of
women with and without endometriosis are shown in Figure 3
(A, normal secretory, day 24; B,
endometriosis secretory, day 26). Note the fine brown immunostaining
around cells both in the stroma and glandular epithelium, and the brown
deposit in the lumen of glands in normal women. No immunoreaction was
observed in negative controls in which the anti-IL-1RII antibody was
replaced by an equal concentration of mouse immunoglobulins of the same
isotype or pre-absorbed with an excess of IL-1RII before incubation
with endometrial tissue sections (data not shown).
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Figure 4
shows that monoclonal
anti-IL-1RII antibody reacted primarily with a 68-kd band and a doublet
of 45- and 48-kd molecular weight bands. Sixty-eight and 45 kd
correspond to the reported molecular weights of the membrane-bound and
the soluble forms of the IL-1RII receptor,
respectively.11-13
Minor bands of lower molecular weights
recognized specifically by the antibody have not been reported
previously and may presumably correspond to degradation products.
However, for the same amount of total endometrial proteins, the
intensity of IL-1RII bands was clearly lower in biopsies from women
with endometriosis included within the same experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of IL-1RII in endometrium-related disorders, we assessed its expression in the endometrium of women suffering from endometriosis. The disease is associated with an immuno-inflammatory process observed consistently in the peritoneal cavity where endometrial tissue abnormally develops,9,17,27 and recently noticed in the eutopic intrauterine endometrium of patients as well.21,28,29 According to our data, the eutopic endometrium of women with endometriosis expresses in situ increased levels of MCP-1,22 a chemokine endowed with the potent ability of inducing monocyte/macrophage chemoattraction and activation.30 Furthermore, cultured endometrial epithelial cells from women with endometriosis displayed an increased responsiveness to IL-1 in vitro by secreting higher amounts of MCP-1 than cells from normal women after exposure to the same concentrations of the proinflammatory cytokine.31 However, the cause(s) of such an exaggerated inflammatory reaction remain unknown. The present study shows a dramatic lack in IL-1RII expression in endometrial tissue of women with endometriosis. This was particularly obvious at the level of IL-1RII luminal secretion, but was also noticeable at the level of the cellular expression both in epithelial and stromal cells. Western blot analysis of IL-1RII expression in endometrial tissue confirmed immunohistochemical data as it showed that the intensity of the 68-kd and lower molecular weight bands recognized specifically by the anti-IL-1RII antibody, was markedly lower in women with endometriosis as compared to normal controls.
The most striking lack in IL-1RII luminal expression was observed in the earliest and initial stages of the disease (stages I and II). On one hand, this in keeping with numerous studies indicating that endometriosis is more active in the initial stages32-34 and is consistent with the pattern of MCP-1 expression that we observed in the endometrium of endometriosis patients that increased in initial and decreased in late endometriosis stages.22 On the other hand, these results suggest that abnormal IL-IRII expression may be involved in the initiation of the inflammatory process in the intrauterine endometrial tissue where the disease is believed to take origin. Interestingly, our study also showed that defective IL-1RII expression was more significant in infertile than in fertile women having endometriosis, which suggests an involvement in endometriosis-associated infertility.
The mechanisms underlying the decreased expression of IL-1RII in women
with endometriosis remain to be further elucidated. The most
significant deficiency occurred at the level of IL-1RII luminal
secretion in epithelial cells, in particular in the secretory phase of
the menstrual cycle. This would suggest an inhibited shedding of the
receptor in endometriosis occurring throughout the cycle, but to a
greater extent in the second phase. At the present time, it is still
unclear what molecular and biochemical pathways could be involved in
the generation of soluble IL-RII. According to recent data, matrix
metalloproteases rather than differential splicing, play a key role in
the production of soluble decoy RII by enzymatic cleavage from the cell
surface receptor.15
The observation of higher levels of
cellular staining in epithelial cells of women with endometriosis in
the secretory phase of the menstrual cycle as compared to the
proliferative phase (P = 0.00037, Table 2
),
makes plausible a potential inhibition of IL-1RII release from the cell
surface in the secretory phase. However, our results also show that
either cellular staining in epithelial cells or the intensity of the
68-kd band corresponding to the reported membrane-bound form of IL-1RII
receptor was reduced in women with endometriosis. This suggests that
beyond a potential aberrant release of sIL-1RII from endometrial cells
of women with endometriosis, a deficiency in IL-1RII protein synthesis
and/or a reduced IL-1RII mRNA levels or gene transcription might be
involved. In fact, our preliminary analyses of IL-1RII mRNA levels in
the endometria of women with and without endometriosis tend to support
such a hypothesis (data not shown).
In conclusion, this is the first study to show the expression in endometrial tissue of the decoy IL-1 receptor type II, a specific natural inhibitor of IL-1 that plays an important role in the regulation of IL-1{beta} activity in the uterine environment. Furthermore, our study revealed a striking lack in IL-1RII expression in women suffering from endometriosis. This may represent a plausible mechanism underlying immuno-inflammatory changes observed in the eutopic endometrium of women with endometriosis as well as in endometrial tissue abnormally implanted in ectopic sites.
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Acknowledgements |
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Footnotes |
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Supported by grant MT-14638 from the Medical Research Council of Canada (to A. A.). A. A. is a Chercheur-Boursier Senior of the Fonds de la Recherche en Santé du Québec (FRSQ).
Accepted for publication November 13, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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