Molecular Human Reproduction, Vol. 6, No. 9, 829-834,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Increased concentrations of secretory leukocyte protease inhibitor in peritoneal fluid of women with endometriosis
1 Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University, 2-2 Yamada-oka, Suita City, Osaka 565-0871, and 2 Department of Obstetrics and Gynecology, Osaka Police Hospital, 10–31 Kitayama-cho, Tennouji-ku, Osaka 543-8502, Japan
Abstract
Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of human leukocyte elastase. We investigated whether SLPI was present in the peritoneal fluid of women with endometriosis and to clarify the role of SLPI in the pathogenesis of endometriosis. Western blot analyses revealed that SLPI protein was detected as a 12 kDa band in peritoneal fluid. The peritoneal fluid concentrations of SLPI, elastase and interleukin-6 were assayed by enzyme-linked immunosorbent assays (ELISA). SLPI concentrations and the SLPI/elastase ratio in the peritoneal fluid of women with endometriosis were higher than in samples from women without endometriosis. There was no significant correlation between concentrations of SLPI and interleukin-6 in the peritoneal fluid. Immunohistochemistry using an anti-SLPI polyclonal antibody revealed positive staining in peritoneal macrophages, but not lymphocytes. The present findings suggest that SLPI found in the peritoneal fluid of patients with endometriosis may contribute to the pathogenesis of endometriosis.
elastase/endometriosis/peritoneal fluid/peritoneal macrophage/secretory leukocyte protease inhibitor (SLPI)
Introduction
Endometriosis is estimated to occur in 7% of reproductive age women and may be associated with pelvic pain and infertility. The incidence of endometriosis among infertile women may be as high as 30–40%. In severe endometriosis, one cause may be mechanical obstructions of the reproductive organs, but the mechanisms which cause infertility in mild or moderate endometriosis are still unclear (Fazleabas et al., 1987
). Although the pathogenesis of endometriosis is poorly understood, the peritoneal fluid from women with endometriosis contains higher concentrations of macrophages and monocytes (Halme et al., 1983) which are responsible for the secretion of a variety of substances, e.g. cytokines, chemokines and prostaglandins. Activated macrophages are a source of inflammatory mediators. A number of studies have shown the presence of macrophage-derived substances and demonstrated their activity in the peritoneal fluid of such women. These substances are assumed to be involved in the pathogenetic cascade of this disease. It has also been widely suggested that the peritoneal fluid microenvironment may contribute to endometriosis and/or endometriosis-associated infertility (Ramey and Archer, 1993).
Secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor found concentrated in secretory fluids, has been postulated to participate in the body's natural defence mechanisms. SLPI is a potent inhibitor of human leukocyte elastase, human cathepsin G and human trypsin (Thompson and Ohlsson, 1986). Concentrations of SLPI in biological samples have been monitored in order to relate these concentrations to pathological conditions (Kida et al., 1992; Sluis et al., 1994). SLPI is found in various fluids, including parotid secretions (Thompson and Ohlsson, 1986), bronchial mucus, nasal mucus (Fryksmark et al., 1989), cervical mucus (Casslen et al., 1981; Helmig et al., 1995; Moriyama et al., 1999) and seminal plasma (Ohlsson et al., 1995; Moriyama et al., 1998). However, it is not clear whether SLPI is found in the peritoneal fluid of women with endometriosis. In the present study, we used enzyme-linked immunosorbent assays (ELISA) to quantify the concentrations of SLPI, elastase and interleukin-6 (IL-6) in the peritoneal fluid of women both with and without endometriosis in order to understand better the role of SLPI in the pathogenesis of endometriosis. We also examined the localization of SLPI expression in the peritoneal fluid.
Materials and methods
Samples
Peritoneal fluids were obtained from 44 women of reproductive age who were undergoing laparascopy for infertility work-up or laparoscopic cystectomy for ovarian chocolate cysts. The laparoscopic findings of the 44 patients in the study group were as follows: 11 had no endometriosis and 33 had pelvic endometriosis (19 mild, stages I and II; and 14 severe, stages III and IV). All women had regular menstrual cycles of 25–35 days and had not taken corticosteroids, hormones, or gonadotrophin-releasing hormone (GnRH) agonists. This study was approved by the local ethics committee of the Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University, Japan. Informed consent was obtained from each patient. Endometriosis was categorized according to the criteria of the revised American Fertility Society (AFS) classification system, by visual inspection of the pelvis. Peritoneal fluids were aspirated from the Pouch of Douglas at the time of laparoscopy. The samples were centrifuged (700 g for 10 min) to separate the cell pellet and the supernatants were collected. The supernatant was then stored at –20°C until assayed. Peritoneal fluid concentrations of SLPI, elastase and IL-6 were measured using commercially available ELISA kits.
Reagents
Goat anti-SLPI polyclonal antibodies, recombinant SLPI (Escherichia coli-expressed) and ELISA kits specific for SLPI were purchased from R&D Systems (Minneapolis, MN, USA). A control goat immunoglobulin (Ig)G for the control of histochemical analysis was purchased from Zymed Laboratories (San Francisco, CA, USA). ELISA kits specific for elastane were purchased from E.Merck (Darmstadt, Germany).
Western blot analysis
The homogenizing buffer for protein extraction from peritoneal fluid consisted of 0.5 mol/l Tris–HCl (pH 6.8), 10% sodium dodecyl sulphate (SDS), 6% ß-mercaptoethanol, and 1% Bromophenol Blue (BPB). To determine the presence of SLPI protein in the peritoneal fluid, we performed Western blotting analysis using an anti-human SLPI polyclonal antibody. Peritoneal fluid (20 µl) was electrophoresed on a 15% SDS–polyacrylamide gel and transferred onto nitrocellulose membranes (0.45 µm; Schleicher and Schuell, Dassel, Germany). The membrane was incubated with 5% dried milk protein followed by anti-human SLPI polyclonal antibody. The primary antibody was used at a final concentration of 1.0 µg/ml. SLPI immunoreactivity was visualized using an enhanced chemiluminescence Western blotting analysis system.
Protein assay
Protein concentrations were determined with Bio-Rad protein determination reagent (Bio-Rad, Hercules, CA, USA), according to the method of Bradford (Bradford, 1976).
Cell cultures
Samples of peritoneal fluid from women with or without endometriosis were centrifuged at 200 g for 10 min. The supernatant was decanted and cells were washed once with PBS, layered on to 50% Ficoll–Hypaque, Necalai tesque, Japan, and re-centrifuged at 200 g for 20 min to remove most of the red blood cells. Primary lymphocytes and monocyte/macrophages were collected from the peritoneal fluid of women with endometriosis. Peritoneal macrophages were isolated via selective adherence to 8-well chamber slides (Lag-Tec Nunc. IL, USA). Briefly, the cell supernatant was centrifuged at 200 g for 10 min and the supernatant was discarded to remove residual peritoneal fluid. After resuspension in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS), 2 mmol/l glutamin, 100 IU/ml penicillin and 100 µg/ml streptomycin, macrophages were cultured in 8-well culture plates for 2 h at 37°C. Non-adherent cells were removed by rinsing with warmed RPMI 1640. Adhered macrophages were incubated in fresh media for 24 h.
Immunocytochemical staining of SLPI in peritoneal fluid cells
To determine the localization of SLPI in the peritoneal fluid, we performed immunocytochemical staining using an avidin–biotin peroxidase complex method kit (OminiTags Universal Streptavidin/Biotin Affinity Immunostaining Systems, Lipshaw, Pittsburg, PA, USA). On each chamber slide, most macrophages and lymphocytes adhered to the plastic substrate. Lymphocytes and monocyte/macrophages were bleached in 0.3% hydrogen peroxide to block endogenous peroxidase and covered with 2% goat IgG to minimize non-specific binding. The appropriately diluted goat polyclonal anti-SLPI antibody (R&D Systems) or control goat IgG for the control was applied at room temperature (RT) and left for 1 h. After rinsing with phosphate-buffered saline (PBS) solution, the sections were further incubated for 30 min with biotin-labelled goat anti-mouse IgG, followed by the addition of avidin–peroxidase complex at 4°C. Peroxidase activity in the sections was visualized with 0.1% 3,3-diaminobenzidinine-tetrahydrochloride containing 0.02% hydrogen peroxide in 0.1 mol/l Tris buffer (pH 7.2). The slides were counterstained with Mayer's haematoxylin.
Determination of SLPI in peritoneal fluid
To determine concentrations of SLPI in the peritoneal fluid, ELISA kits specific for SLPI (R&D Systems) were used. The SLPI concentration detection limit of this kit was 62.5 pg/ml. No cross-reactivity with cytokines, growth factors, elastase, trypsin or chymotrypsin could be found in this kit. The intra-assay variability of the SLPI kit was 4.2–8.0%, and the inter-assay variability was 4.9–8.0%.
Determination of elastase in peritoneal fluid
To measure the titres of elastase in the peritoneal fluid, ELISA kits specific for elastase (E.Merck) were used. Peritoneal fluid titres of elastase which the kit detected were >1.0 µg/l. The intra- and inter-assay variabilities of the elastase kit were 2.7–5.2 and 4.9–9.5% respectively.
Determination of IL-6 in peritoneal fluid
To determine concentrations of IL-6 in the peritoneal fluid, ELISA kits specific for IL-6 (R&D Systems) were used. The IL-6 concentration detection limit of this kit was 0.70 pg/ml. The intra-assay variability of the IL-6 kit was 2.0–4.2%, and its inter-assay variability was 3.3–6.4%.
Statistical analysis
Statistical analyses of SLPI, elastase and IL-6 concentrations in the peritoneal fluid were conducted using a non-parametric test; P < 0.05 was considered to be statistically significant. The relationships between SLPI and IL-6 and between SLPI and elastase in the peritoneal fluid were examined by a simple linear analysis.
Results
To quantify SLPI protein in peritoneal fluid, we performed Western blot analysis. As shown in Figure 1, SLPI protein was detected as a 12 kDa band in peritoneal fluid. These findings show the presence of SLPI in peritoneal fluid.
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To compare the SLPI concentrations in normal women and women with endometriosis, we measured peritoneal fluid concentrations of SLPI, elastase and IL-6 for different grades according to the revised AFS classification of endometriosis (American Fertility Society, 1985
). As shown in Figure 2, the SLPI titres of peritoneal fluid from women without endometriosis were 12.1–50.5 ng/ml (median, 36.4 ng/ml); from women with mild endometriosis, 16.2–166 ng/ml (median, 50.5 ng/ml), and from women with severe endometriosis, 31.3–131 ng/ml (median, 80.8 ng/ml). SLPI concentrations were significantly higher in the peritoneal fluid of women with moderate or severe endometriosis compared with the controls. However, no significant differences between SLPI concentrations in mild and severe endometriosis were observed.
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We also determined the elastase and IL-6 concentrations in the peritoneal fluid of women using specific ELISAs. No significant differences in elastase titres in peritoneal fluid among the three groups were demonstrated (control: median 92 ng/ml, range 46–480 ng/ml; mild endometriosis: median 80 ng/ml, range 44–240 ng/ml; severe endometriosis: median 104 ng/ml, range 54–260 ng/ml). The IL-6 titres were higher in women with severe endometriosis (median 127 pg/ml, range 49.5–506 pg/ml) than in controls (median 57.2 pg/ml, range 49.5–82.5 pg/ml; P < 0.05). There was no significant difference in IL-6 concentrations in peritoneal fluid between the controls and those with mild endometriosis (median 49.5 pg/ml, range 41.8–77.0 pg/ml).
As shown in Figure 3, the SLPI/elastase ratio in peritoneal fluid of the controls ranged from 0.11 to 0.85 (median: 0.33), while in those with mild endometriosis, it ranged from 0.14 to 1.67 (median: 0.64) and in those with severe endometriosis it ranged from 0.12 to 2.24 (median: 0.56). The SLPI/elastase ratio was significantly higher in the peritoneal fluid of women with mild or severe endometriosis, compared with the controls (P < 0.05). We examined the correlation between SLPI and IL-6 in the peritoneal fluid by a simple linear analysis. There was no significant correlation between concentrations of SLPI and IL-6 in the peritoneal fluid. It was also demonstrated that there was no significant correlation between SLPI and elastase in the peritoneal fluid.
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To identify the origin of SLPI in peritoneal fluid, primary lymphocytes and monocyte/macrophages were cultured from the peritoneal fluid of women with or without endometriosis. On each chamber slide, peritoneal macrophages were distinguishable from the other cells, as judged from morphological features and adherence to the plastic substrate. Peritoneal macrophages of endometriosis patients were intensely stained but lymphocytes were not stained (Figure 4
). Macrophages in peritoneal fluid from women without endometriosis were also stained (data not shown).
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Discussion
In the present study, Western blot analysis revealed that SLPI protein is present in the peritoneal fluid of women both with and without endometriosis. These findings suggested that the SLPI molecule was constitutively present in the peritoneal fluid. Proteases and protease inhibitors play essential interactive roles during the inflammatory process. SLPI, which is a potent inhibitor of human leukocyte elastase, human cathepsin G and human trypsin (Thompson and Ohlsson, 1986) participates in the body's natural defence mechanisms. This molecule might contribute to homeostasis in the peritoneal cavity.
Recent studies suggest that the symptoms associated with endometriosis are the result of local peritoneal inflammation. We demonstrated that the SLPI concentrations in the peritoneal fluid of women with mild or severe endometriosis were significantly higher compared with those without endometriosis. Immunocytochemical analysis using anti-SLPI polyclonal antibody showed that peritoneal macrophages were intensely stained, indicating that macrophages might be one of the main sources of SLPI in peritoneal fluids. The peritoneal fluids of women with endometriosis contain an increased concentration of macrophages which are related to the progression of endometriosis. However, since the peritoneum has a very large epithelial surface area, it is possible that this is another source of SLPI. Peritoneal fluid circulation around the abdominal cavity may be important for the spread of pelvic infections or for post-operative adhesion formation, but this has not been investigated in relation to reproductive biology and endometriosis (Dizerega and Rodgers, 1992). Therefore, the mechanism resulting in the elevation of the SLPI concentration has not been clarified. One possibility is that an increased concentration of macrophages might cause an increase in SLPI and the SLPI/elastase ratio. Another possibility is that there might be a selective expression of protease inhibitors into the peritoneal cavity from the circulation in response to the inflammatory reaction caused by this disease.
Proteases and protease inhibitors, that have been implicated to be associated with inflammation and proteolytic equilibrium, play essential interactive roles during the inflammatory process. Women with endometriosis have higher concentrations of a protease inhibitor (Fazleabas et al., 1987), possibly related to an altered fibrinolytic system (Pattinson et al., 1981). A higher basal activation status of peritoneal macrophages in women with endometriosis may impair fertility by reducing sperm motility, increasing sperm phagocytosis or interfering with fertilization (Halme et al., 1987; Zeller et al., 1987). Haney et al. reported the presence of peritoneal macrophages that can phagocytize and destroy normal human sperm in vitro (Haney et al., 1981; Muscato et al., 1982). Peritoneal macrophages from infertile patients with endometriosis showed greater sperm phagocytosis than those from normal fertile women or from infertile women without endometriosis (Martinez et al., 1997). It has been suggested that oviductal macrophages may arise from peritoneal macrophages that migrate into the oviducts (Haney et al., 1983). Thus macrophages associated with endometriosis are likely to affect fertility. We have previously reported beneficial effects on fertility by SLPI in the cervical mucus during the menstrual cycle and on sperm motility damaged by elastase (Moriyama et al., 1998, 1999). It has been suggested that SLPI might constitute the major defence of cervical tissues against inflammation and may also have important effects on the sperm penetrability of human cervical mucus. Therefore, it may be more appropriate to see SLPI as having a protective role in preventing the further development of endometriosis through various anti-inflammatory mechanisms, e.g. macrophage digestion and the release of inhibitory factors. A monkey model of endometriosis demonstrated that treatment with a proteinase inhibitor impaired ectopic growth of endometrium (Sillem et al., 1996). Further investigations will be necessary to examine the relationship between SLPI concentrations and endometriosis-associated infertility.
Various studies have demonstrated that the peritoneal fluid of women with endometriosis contains a higher amount of cytokines, e.g. IL-6, IL-8, monocyte chemotactic factor, growth factors, and other proteins (Overton, 1996; Murphy et al., 1998). In agreement with the current study, Rier et al. showed that elevated concentrations of IL-6 were present in peritoneal fluid of patients with severe (stages III or IV) endometriosis (Rier et al., 1995). It has been suggested that IL-6 concentrations in the peritoneal fluid are increased with active endometriosis (Harada, 1997). In the present study, there was no significant relationship between the concentrations of SLPI and IL-6 in the peritoneal fluid. From these findings, other pathogenetic mechanisms of endometriosis might exist in both mild and severe endometriosis. However, SLPI may play an important role in endometriosis-associated infertility. SLPI itself has been considered exclusively an epithelial cell product (Abe et al., 1991). In mice, the SLPI transcript and protein were detected in peritoneal macrophages, bone marrow-derived macrophage cell lines and peritoneal polymorphonuclear leukocyte (Jin et al., 1997) and an anti-inflammatory role for macrophage-derived SLPI appears probable based on the slow production of SLPI in response to constituents of gram-negative and gram-positive bacteria (Jin et al., 1997). Recently, it was demonstrated that cytokine-induced neutrophil chemoattractant (CINC) production by peritoneal macrophages was increased by neutrophil elastase in a dose-dependent fashion and ONO-5046, which is a specific synthetic inhibitor of neutrophil elastase, decreased CINC production by peritoneal macrophages in response to neutrophil elastase, suggesting that a elastase inhibitor can down-regulate the function of elastase in the peritoneal cavity (Wang et al., 1996). In the present study, the SLPI/elastase ratio was significantly higher in the peritoneal fluid of women with mild or severe endometriosis compared with the controls. The increased SLPI concentration in the peritoneal fluid of women with endometriosis may have an anti-inflammatory effect. A possible mechanism for this effect may be through the inhibition of elastase-induced cytokine production by peritoneal macrophages by the consequent prevention of neutrophil accumulation in the peritoneal fluid.
Notes
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University, 2–2 Yamada-oka, Suita City, Osaka 565-0871, Japan. E-mail: shimoya{at}gyne.med.osaka-u.ac.jp 
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