Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 1138655, Japan
1 To whom correspondence should be addressed. E-mail: yutakaos-tky{at}umin.ac.jp
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Abstract |
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Key words: endometriosis/interleukin/mitogen-activated protein kinase/proliferation/protease-activated receptor 2
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Introduction |
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The concentrations of chemokines, such as interleukin (IL)-8, epithelial neutrophil-activating peptide 78, and growth-regulated , that induce the activation and chemotaxis of neutrophils, are increased in the peritoneal fluid (PF) in women with endometriosis (Kutteh et al., 1995
; Arici et al., 1996
; Oral et al., 1996
; Szamatowicz et al., 2002
; Mueller et al., 2003
). Similarly, concentrations of stem cell factor, which stimulates the activation and migration of mast cells, are increased in PF of women with endometriosis (Osuga et al., 2000
). In view of the thesis that endometriosis is characterized by recurrent ectopic bleeding (Brosens, 1997
), it is conceivable that endometriotic tissues, including ovarian, are repeatedly exposed to neutrophils in the blood. Furthermore, reduced spontaneous apoptosis of neutrophils is suggested to be involved in the development of the disease (Kwak et al., 2002
). An increase of mast cells in endometriotic lesions has been demonstrated (Matsuzaki et al., 1998
; Uchiide et al., 2002
; Fujiwara et al., 2004
). Therefore, it is reasonable to speculate that neutrophils and mast cells may play roles in the pathophysiology of the disease.
In recent years, protease-activated receptors (PAR), members of the seven-transmembrane G-protein-coupled receptor family, have been noted to be important mediators of inflammation (Cocks and Moffatt, 2000; Macfarlane et al., 2001
). To date, four PAR have been discovered and characterized. PAR2 is unique in that it is activated by various proteases but not by thrombin, while PAR1, PAR3 and PAR4 are all activated by thrombin. Interestingly, proteinase 3, human leukocyte elastase and cathepsin G, which are secreted from neutrophils, are activators for PAR2 (Uehara et al., 2002
, 2003
). In addition, tryptase from mast cells also activates PAR2 (Molino et al., 1997
).
In light of the possible implication of neutrophils and mast cells in endometriosis, we speculated that proteases from these cells function via PAR2. In particular, PAR2-mediated proliferative effects reported in certain cells (Frungieri et al., 2002; Gaca et al., 2002
) prompted us to investigate whether PAR2 activation may be involved in the development of endometriosis. In the present study, we examined the effects of PAR2 activation on endometriotic cell proliferation, and the possible involvement of mitogen-activated protein kinases (MAPK) therein.
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Materials and methods |
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Collection of endometriotic tissues
Endometriotic tissues were obtained from patients with ovarian endometriomas (n = 24) undergoing laparoscopy or laparotomy after obtaining written informed consent under a study protocol approved by the institutional review board of the University of Tokyo. These patients had not received hormones or GnRH agonist for 3 months before surgery. Endometriotic tissue samples were obtained from the cyst wall of ovarian endometrioma under sterile conditions and transported to the laboratory on ice in DMEM/F-12.
Isolation, purification, and culture of endometriotic stromal cells (ESC)
We have reported this procedure previously (Hirota et al., 2005b,c
). Briefly, endometriotic tissue was minced into small pieces, incubated in DMEM/F-12 with type I collagenase (2.5 mg/ml) and deoxyribonuclease I (15 IU/ml) for 12 h at 37°C, and filtered through nylon cell strainers with apertures of 100 µm, and then 70 µm. Stromal cells remaining in the filtrate were centrifuged at 200 g for 5 min, washed with phosphate-buffered saline (PBS), resuspended in DMEM/F-12, and plated onto 100 mm dishes and allowed to adhere at 37°C for 30 min, after which non-adhering epithelial cells and blood cells were removed with PBS rinses. ESC were cultured in DMEM/F-12 containing 10% FBS, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. When the cells became confluent in 2 or 3 days, they were dissociated with 0.25% trypsinEDTA, harvested by centrifugation at 200 g for 5 min, replated in 6-well plates at 2x105 cells/well, 48-well plates at 1x104 cells/well or 96-well plates at 0.5x104 cells/well, and incubated at 37°C in a humidified 5% CO2/95% air environment for 24 h. The complete media were then removed and replaced with fresh serum-free media containing antibiotics, and the cells were cultured for an additional 1224 h. Purity of the stromal cell population was determined by immunocytochemical staining for the following: vimentin (stromal cells), cytokeratin (epithelial cells) and CD45 (monocytes and other leukocytes). The purity of the stromal cells was >98%, as judged by positive cellular staining for vimentin and negative cellular staining for cytokeratin and CD45. More specifically, immunostaining with anti-CD10 antibody identified >95% of the cells as endometriotic stromal cells.
Isolation, purification, and culture of neutrophils
Human neutrophils (polymorphonuclear leukocytes) were isolated from freshly drawn venous blood samples from healthy volunteers. The methods of neutrophil isolation were divided by three steps, i.e. dextran sedimentation, Ficoll-Paque centrifugation and lysis of contaminated red blood cells.
All manipulations were performed in sterile conditions. Twenty millilitre of venous blood drawn into heparin-coated syringes was mixed with 10 ml of 0.9% saline with 3% Dextran 500, and the mixture was left standing for 30 min at room temperature to allow sedimentation of red cells. After the sedimentation, leukocyte-rich supernatant was recovered and centrifuged at 250 g for 10 min. The pellet was then diluted in 8 ml PBS and carefully layered over 4 ml Ficoll-Paque Plus. After centrifugation at 400 g for 15 min, the supernatant, which contains mononuclear cell layer, was discarded. To lyse contaminated red blood cells, the remaining pellet was resuspended with 0.2% sodium chloride for 30 s, and subsequently was mixed with an equal volume of 1.6% sodium chloride. The purified neutrophils were washed, pelleted and resuspended at 1x106 cells/ml in DMEM/F-12 containing 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. The cells were then plated in 6-well plates at 2x106 cells/well and incubated at 37°C in a humidified 5% CO2/95% air environment for 12 h. After the incubation, the conditioned media were collected and used for the treatment of ESC.
The isolation procedure yielded 1x107 neutrophils from a 10 ml sample of whole blood, with 99% viability by trypan blue exclusion and 98% purity by Wright stain.
Treatment of ESC
To evaluate the dose effects of PAR2AP on PCNA expression and cytokine secretion of ESC, the wells were replenished with serum-free media with different concentrations of PAR2AP and the cells were incubated for 24 h. The doses used in this study was chosen based on our previous findings about PAR2AP on eutopic endometrial cells (Hirota et al., 2005a). To assess the effects of PAR2AP on DNA synthesis and number of ESC, the cells were incubated with serum-free media with PAR2AP at 30 µmol/l for 24 or 48 h. To evaluate the effects of PAR2AP on MAPK phosphorylation of ESC, the cells were incubated with serum-free media with PAR2AP at 30 µmol/l for different durations. To evaluate the effects of three MAPK inhibitors on PAR2-induced proliferation of ESC, the cells were preincubated with SB202190 (p38 MAPK inhibitor, 10 µmol/l), PD98059 (p42/44 MAPK inhibitor, 25 µmol/l) or SP600125 (SAPK/JNK inhibitor, 10 µmol/l) for 1 h before PAR2AP treatment (30 µmol/l). To evaluate the proliferative effect of neutrophil-derived serine proteases on ESC, the cells were incubated with either serum-free media or conditioned media of neutrophil culture with or without different concentrations of serine protease inhibitor aprotinin for 24 h.
Immunocytochemical staining for PCNA
PCNA was chosen as a proliferation marker in view of our previous report that used PCNA to show thrombin-stimulated proliferation of ESC (Hirota et al., 2005b). The cells were fixed with cold methanol: acetone (1:1) at 20°C for 20 min, and washed twice with PBS. The fixed cells were treated with 3% hydrogen peroxide for 5 min to eliminate endogenous peroxidase. After blocking with 1.5% horse serum for 20 min, the cells were incubated with a mouse monoclonal antibody to PCNA (1:200) for 20 min at room temperature. Control cells were incubated with non-immune murine IgG2a, the concentration of which was adjusted to that of the primary antibody. The cells were then incubated with biotinylated horse anti-mouse IgG, followed by avidin peroxidase using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). The chromogenic reaction was performed with diaminobenzidine (Vector Laboratories).
Immunostained cells were analysed in a blinded fashion by the same person without knowledge of the treatment group. The PCNA-positive rate (PCNA-positive cells/total cells) was determined by observing >500 nuclei for each sample, and was used for evaluating the proliferating activity of the cells.
Control and PAR2AP-treated cells were also stained with primary antibodies to vimentin and cytokeratin to evaluate the effect of PAR2AP on ESC purity.
Western blot analysis
Western blot analysis was performed as we have reported previously (Yoshino et al., 2003; Hirota et al., 2005a
). Cultured cells were homogenized in a lysis buffer containing 50 mmol/l Tris/HCl (pH 6.8), 2% sodium dodecyl sulphate (SDS), 10% glycerol, 50 mmol/l dithiothreitol and 0.1% bromophenol blue, and diluted to 1 mg total protein/ml. Concentrations of total protein in the homogenized cells were measured using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Samples were resolved by 10% SDSpolyacrylamide gel electrophoresis. Proteins were blotted onto a Hybond-ECL nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK) and incubated with rabbit antibodies to total p38 MAPK (1:1000), to phospho-specific p38 MAPK (1:1000), to total p42/44 MAPK (1:1000), to phospho-specific p42/44 MAPK (1:1000), to total SAPK/JNK (1:1000), or to phospho-specific SAPK/JNK (1:1000) as primary antibodies, and anti-rabbit horseradish peroxidase antibody (1:1000) as a secondary antibody. The blotted membranes were also developed with a goat antibody to actin (1:1000) as a primary antibody and anti-goat horseradish peroxidase antibody (1:1000) as a secondary antibody. Immune complexes were visualized by the enhanced chemiluminescence western blotting system (Amersham Biosciences). Densitometric analysis of bands on developed X-ray films was performed using National Institutes of Health image software.
The 5-bromo-2' deoxyuridine (BrdU) proliferation assay
The BrdU proliferation assay was performed as we have reported previously (Tang et al., 2002; Hirota et al., 2005a
,c
) using the Biotrak cell proliferation enzyme-linked immunosorbent assay (ELISA) system (Amersham Biosciences) according to the manufacturers instructions. Briefly, ESC were treated with serum-free medium with PAR2AP for 24 h, and 100 µl BrdU solutions were added and incubated at 37°C for an additional 2 h. After removing the culture medium, the cells were fixed and the DNA denatured by the addition of 200 µl/well fixative. The peroxidase-labelled anti-BrdU bound to the BrdU incorporated in the newly synthesized, cellular DNA. The immune complexes were detected by the subsequent substrate reaction, and the resultant colour was read at 450 nm in the DigiScan Microplate Reader (ASYS Hitech GmbH, Eugendorf, Austria).
Cell counting of ESC
ESC were treated with serum-free medium with or without PAR2AP for 48 h, dissociated with 0.25% trypsinEDTA, and collected by centrifugation at 200 g for 5 min. The number of ESC, resuspended in 10 ml PBS, was determined using a Coulter Counter Z1 (Beckman Coulter, Fullerton, CA, USA).
Measurement of IL-6 and IL-8
After the treatments, the conditioned media were collected, centrifuged and stored at 80°C until assay. Concentrations of IL-8 and IL-6 were measured using specific ELISA kits (Quantikine; R&D Systems, Minneapolis, MN, USA) according to the manufacturers protocol. Absorbance was read at 450 nm with the DigiScan Microplate Reader. Cultured cells were homogenized, and total protein in the homogenized cells was measured by a protein assay kit. Data were standardized by total protein of cell lysates.
Statistical analysis
Data were evaluated using Students t-test and analysis of variance with post-hoc analysis (Fishers protected least significance). P < 0.05 was accepted as significant.
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Results |
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PAR2AP stimulated phosphorylation of p38 MAPK, p42/44 MAPK and SAPK/JNK
As demonstrated in Figure 2, PAR2AP at 30 µmol/l stimulated the phosphorylation of all three MAPK (p38 MAPK, p42/44 MAPK and SAPK/JNK) within 5 min. The phosphorylation levels of p38 MAPK significantly increased between 5 and 30 min, followed by a decrease to basal levels at 60 min. The phosphorylation levels of p42/44 MAPK significantly increased between 5 and 15 min and decreased at 60 min as compared with control, followed by recovery to basal levels at 120 min. The phosphorylation levels of SAPK/JNK significantly increased between 5 and 60 min, followed by a decrease to basal levels at 120 min. PAR2AP did not alter control actin protein levels (data not shown).
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MAPK inhibitors suppressed PAR2AP-induced PCNA expression in ESC
Inhibitors of each MAPK (SB202190, PD98059 and SP600125) significantly suppressed the PAR2AP-induced increase in the PCNA-positive rate in ESC (Figure 3).
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PAR2AP stimulated DNA synthesis and increased number of ESC
As shown in Figure 4A, 30 µmol/l PAR2AP significantly increased BrdU incorporation into DNA in ESC (139 % of control). PAR2AP (30 µmol/l) also significantly increased ESC number (124% of control, Figure 4B).
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Neutrophil-derived serine proteases promoted PCNA expression in ESC
As shown in Figure 5, the conditioned medium of neutrophil cell culture had a proliferative effect on ESC. This effect was abolished by aprotinin, a serine protease inhibitor, at a dose of 1 and 10 µg/ml.
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Effects of PAR2AP on the secretion of IL-6 and IL-8 in ESC
PAR2AP induced a dose-dependent increase in IL-6 and IL-8 secretion by ESC (Table I). The minimal effective concentrations for producing significant increases in the secretions of IL-6 and IL-8 were 30 and 300 µmol/l in ESC respectively.
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Discussion |
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A current paradigm is that local inflammation associated with endometriosis plays important roles in the pathophysiology of the disease; various proinflammatory molecules are suggested to promote the progress of the disease (Osuga et al., 2002; Wu and Ho, 2003
). In the present study, we showed that PAR2AP, like serine protease(s) secreted from neutrophils, stimulated the proliferation of ESC, suggesting another possible link between inflammation and the development of the disease. Specifically, apart from cytokines, several proteases secreted from leukocytes recruited and activated in the disease may stimulate proliferation of ESC via PAR2 activation, though a further in vivo study is needed to reinforce the thesis. We have also recently shown that PAR2 activation stimulates the proliferation of stromal cells of the eutopic endometrium (Hirota et al., 2005a
). PAR2 thus appears to participate in various physiological and pathological events in different sites.
The activation of PAR2 has been shown to increase the expression of IL-6 and IL-8 in respiratory endothelial cells and neutrophils (Asokananthan et al., 2002; Shpacovitch et al., 2004
). These molecules are also proinflammatory and have been suggested to be involved in the pathophysiology of endometriosis (Ota et al., 2001
; Chishima et al., 2002
; Berkkanoglu and Arici, 2003
). We observed that PAR2 activation induced, besides proliferation of ESC, the secretion of IL-6 and IL-8 from ESC. These findings imply that PAR2 activation may also play roles in the disease by stimulating the secretion of proinflammatory molecules. It is interesting to speculate that PAR2-induced IL-8 secretion by ESC may promote the migration of neutrophils, consequently causing self-perpetuating inflammation at endometriotic lesions.
In the pathogenesis of endometriosis, another protease system, such as the plasminogen-activating system, is suggested to be involved (Bruse et al., 2004, 2005
). Together with our findings, it implies that various proteases contribute to the pathophysiology of the disease.
The finding that PAR2AP activated all three MAPK in ESC may imply pleiotropic functions of PAR2 in endometriotic tissues, given that the activation of each MAPK can lead to the expression of various sets of molecules functioning in proliferation, differentiation, apoptosis and inflammation (Pearson et al., 2001). Our previous study showed that IL-1
-induced inflammatory responses were mediated by p38 MAPK in endometrial stromal cells (Yoshino et al., 2003
). PAR2-induced activation of MAPK may not only stimulate proliferation of ESC, but also induce various responses of the cells that contribute to the development of endometriosis.
The present finding that the stimulation of PAR2 activated all three MAPK appears to be in contrast with findings on rat aortic smooth muscle cells (Belham et al., 1996) and rat cardiac muscle cells (Sabri et al., 2000
), in which PAR2 did not activate SAPK/JNK, but activated the other two MAPK. The types of MAPK activated by PAR2 have been suggested to differ depending on the cell types.
Given that PAR2 activation stimulates the progress of endometriosis, the development of a PAR2 antagonist would be expected for anti-proliferative therapy for the disease. In fact, various chemicals that antagonize PAR1, another family member of PAR2, have been developed (Chackalamannil, 2003; Derian et al., 2003
). A PAR2 antagonist might also have direct analgesic effects for endometriosis-associated pain, as tissue inflammation has been suggested to induce pain by activation of PAR2 of afferent neurons (Dai et al., 2004
).
In summary, the present study demonstrated that PAR2 activation stimulates the proliferation of ESC and the secretion of IL-6 and IL-8 from ESC, suggesting that PAR2 might be involved in the development of endometriosis.
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Acknowledgements |
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Submitted on September 10, 2004; resubmitted on May 12, 2005; resubmitted on June 27, 2005; accepted on July 6, 2005.
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