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Regular Article |
a Yale University School of Medicine, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, New Haven, Connecticut 06520
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, gene regulation, immunology, implantation, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eutopic endometrium from women with endometriosis displays significantly reduced apoptosis compared with eutopic endometrium from healthy controls [1]. In addition, spontaneous apoptosis in ectopic endometrium is also less than what occurs in eutopic endometrium from the same patient. GnRH analogues, on the other hand, increase the apoptotic rate of endometrial cells in endometriosis [2]. The Fas-Fas ligand (FasL) system has been suggested as the mediator of the action of GnRH on endometriosis.
Fas, also called APO-1 or CD95, is a type I membrane protein of 45 kDa that belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family [3]. FasL, a type II membrane protein of 37 kDa, belongs to the TNF superfamily [4]. FasL expression has been reported in nonimmune cells, mainly from immune-privileged tissues such as testis, cornea, trophoblast, and cancer cells, which suggests that the Fas-FasL system may play an important role in the mechanism underlying this immune-privileged status [5–9]. We recently described the FasL modulation by macrophage-derived growth factors in endometrial stromal cells, thereby establishing a role for FasL in the pathogenesis of endometriosis [10]. FasL expression in human endometrium by means of interaction between endometrial stromal cells and extracellular matrix proteins has not been investigated so far.
The extracellular matrix (ECM), a protein network in the intercellular spaces, has profound interactions with both stromal and glandular cells in human endometrium. The ECM regulates cell proliferation, differentiation, and apoptosis by means of integrins, a family of cell surface receptors that attach cells to the matrix. Signal transduction pathways from the extracellular matrix into the cell through integrins trigger changes in gene expression, regulate activities of cytoplasmic kinases, growth factor receptors, ion channels, and control the organization of the intracellular actin cytoskeleton [11]. The composition of the ECM is a significant regulatory factor in the control of cell growth and differentiation. In many cell types, loss of attachment to the ECM may lead to apoptosis [12], and the ECM seems to control apoptosis in an integrin-specific manner.
Implantation theory proposes adhesion of retrogradely menstruated viable endometrial cells into the peritoneal cavity, as a consequential step in the pathogenesis of endometriosis [13]. It is well established that endometrial stromal cells are involved in the initial steps of attachment to the mesothelial surface of the peritoneum [14]. These endometrial cells establish cell-cell and cell-ECM interactions with the peritoneal lining by means of adhesion molecules and integrins during implantation and continued growth. Alterations in the interactions between endometrial cells and the ECM could be one of the factors in the proliferation and invasion of endometriotic cells. We hypothesized that interaction between endometrial cells and the ECM proteins laminin, fibronectin, and collagen IV could up-regulate FasL expression and may be crucial for the development of a relative local immunotolerance in endometriosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endometrial tissues were obtained from human uteri after diagnostic laparoscopy or hysterectomy conducted for benign diseases. Informed consent in writing was obtained from each patient before surgery; consent forms and protocols were approved by the Human Investigation Committee of Yale University. Mean age of the patients was 35 (range 32–36). A diagnosis of normal endometrium included uterine fibroid and voluntary sterilization by tubal ligation (n = 3).
Endometrial tissues were also obtained by endometrial biopsy from patients with endometriosis (n = 3). Cells obtained from each patient were considered as separate experiments. Each experimental setup was repeated at least on three occasions using cells obtained from different patients. Endometrial tissues were placed in Hanks balanced salt solution (HBSS) and transported to the laboratory for separation and culture of endometrial stromal cells.
Isolation and Culture of Human Endometrial Stromal Cells
Endometrial stromal cells were separated and maintained in monolayer culture, as described previously [15]. Briefly, endometrial tissue was digested by incubating tissue minces in HBSS that contained Hepes (25 mmol), penicillin (200 U/ml), streptomycin (200 mg/ml), collagenase (1 mg/ml, 15 U/mg), and deoxyribonuclease (0.1 mg/ml, 1500 U/mg) for 30 min at 37°C with agitation. The dispersed endometrial cells were separated by filtration through a wire sieve (diameter pore 73 µm). Endometrial glands (largely undispersed) were retained by the sieve, whereas the dispersed stromal cells passed through the sieve into the filtrate.
The stromal cells were plated in Hams F12/Dulbeccos Minimal Essential Medium (DMEM; 1:1 vol/vol) containing fetal bovine serum (FBS; 10% vol/vol) and antibiotics-antimycotics (1% vol/vol). Cells were plated in plastic flasks (75 cm2), maintained at 37°C in a humidified atmosphere (5% CO2 in air), and allowed to replicate to confluence. Thereafter, the stromal cells were passed by standard methods of trypsinization and plated in culture dishes (100 mm diameter) and were allowed to replicate to confluence. Endometrial stromal cells after first passage were characterized as described previously [15], and were found to contain 0%–7% epithelial cells, no endothelial cells, and 0.2% macrophages. Experiments were commenced 1–3 days after confluence was attained. The confluent cells were treated with serum-free media for 24 h before treatment with test agents was initiated.
Endometrial stromal cells were treated with trypsin, centrifuged, and resuspended in fresh serum-free, phenol red-free Hams F12/DMEM at a concentration of 250 000 cells/ml. Cell suspension was then seeded onto 100-mm culture dishes precoated as indicated below, and the cells were incubated for 24 or 48 h. The medium was then aspirated to remove nonadherent cells prior to protein analysis by Western blot and immunocytochemistry.
Chemicals and Extracellular Matrix Preparation
Fibronectin was acquired from Becton Dickinson(Bedford, MA). Collagen IV and laminin (molecular weight 70–150 kDa) were purchased from Sigma Chemical Co. (St. Louis, MO). The ECM proteins were dissolved in calcium-free and magnesium-free PBS to the concentration of 20 µg/ml fibronectin, 10 µg/ml type IV collagen, and 20 µ/ml laminin. Coating of 100-mm culture dishes and tissue culture slides was carried out by overnight incubation at 4°C for 15 min to block the nonspecific binding sites on plastic, and washed with sterile PBS before plating. For controls, dishes and slides were directly blocked with denatured BSA. Stock cytochalasin D was prepared at a concentration of 5 µg/ml in dimethyl sulfoxide (DMSO; Baker Inc., Phillipsburg, NJ).
FasL Western Blot Analysis
Total protein from the cells was extracted by lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2-6H2O, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, and protease inhibitors (1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 4 mM eCG). The protein concentration was determined by a detergent compatible protein assay (Bio-Rad Laboratories, Hercules, CA). Protein (10 µg) was loaded into each lane, separated by SDS-PAGE using 7.5% Tris-HCl Ready Gels (Bio-Rad) and electroblotted onto Hybond+ ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Equal loading of proteins in each lane was confirmed by staining the membrane with Ponceau 2S (Sigma). The membrane was blocked with 5% nonfat dry milk in PBS-T (Dulbeccos PBS and 0.05 % Tween-20) buffer for 1 h to inhibit the nonspecific binding. The membrane was incubated for 1 h with mouse anti-human FasL monoclonal antibody (Transduction Laboratories, Lexington, KY) diluted at 1:1000 and further incubated for 1 h with peroxidase-labeled anti-mouse immunoglobulin G (IgG; Vector Laboratories, Burlingame, CA) diluted at 1:10 000. The specificity of the FasL monoclonal antibody that we used has been described by other authors [16, 17]. The immunoblot was developed using 3,'3,'5,'5'-tetramethylbenzidine (TMB) peroxidase substrate kit (Vector Laboratories) and chemiluminescent kit (NEN Life Science, Boston, MA). The FasL signal was normalized using the amount of total protein in each lane, measured by Ponceau red to confirm that the protein amount was similar in each band. The Ponceau red and FasL signals were quantified with a digital imaging and analysis system (AlphaEase, Alpha Innotech Corporation; San Leandro, CA), which was also used for the bands developed by the TMB peroxidase substrate kit, and by using a laser densitometer (Molecular Dynamics, Sunnyvale, CA) for the autoradiographic bands. FasL expression was normalized by dividing the arbitrary densitometry units for FasL protein to the amount of Ponceau red staining of total protein for each band.
Immunocytochemistry
The endometrial stromal cell cultures were treated with trypsin, centrifuged, resuspended, and plated on four-chamber slides precoated as indicated above. Following 24 h of incubation, chamber slides were fixed for 10 min in methanol and dried for 20 min at room temperature. Endogenous peroxide activity was quenched by 3% H2O2 (0.6 ml H2O2 and 5.4 ml methanol) for 10 min in both frozen sections and tissue culture slides and rinsed in PBS-T. Sections were then incubated with the mouse anti-FasL monoclonal antibody (Transduction Laboratories; diluted at 1:100) for 30 min at room temperature. Normal mouse IgG was applied for negative control staining instead of primary antibody. After several rinses in PBS-T, biotinylated secondary antibody (goat anti-multispecimens immunoglobulin; Biogenex, San Ramon, CA) was applied for 30 min. Following several PBS-T rinses, culture slides were incubated with streptavidin-peroxidase complex for 30 min (Biogenex). Subsequently, slides were rinsed several times in PBS-T and then incubated with aminoethyl carbazole (AEC; Biogenex). Slides were lightly counterstained with hematoxylin prior to permanent mounting. Endometrial stromal cells on tissue culture slides were evaluated in a semiquantitative fashion [0 (absent) to 3 (most intense)]. For each slide, an HSCORE value was derived by summing the percentages of cells staining at each intensity multiplied by the weighted intensity of the staining [HSCORE = 
Pi (i + 1), where i is the intensity scores and Pi is the corresponding percentage of the cells]. In each slide, five different areas were evaluated under microscope with 50x original magnification and the percentage of the cells for each intensity within these areas were determined by two investigators at different times. An average score of the two was used.
Statistical Analysis
Levels of FasL protein were normally distributed (tested by the Kolmogorov-Smirnov test). Because we compared the expression of FasL in endometrial stromal cells from two different patient groups (endometriosis vs. nonendometriosis) at 24 h vs. 48 h and assessed the effect of the ECM on this expression using an in vitro adhesion model, we used ANOVA and the post hoc Bonferoni test for pair-wise multiple comparisons for statistical analysis. P < 0.05 was considered significant. Statistical calculations were performed using Sigmastat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the regulation of FasL expression in endometrial stromal cells by their attachment to various ECM proteins, cells were plated onto Petri dishes that had been coated previously with laminin, fibronectin, or collagen IV and onto uncoated dishes as controls. Following 24-h and 48-h incubations, FasL protein expression was analyzed by Western blot (Fig. 1). Cells plated on BSA (control) for 24 h expressed low levels of FasL protein, which gradually increased by 48 h. Adherence of stromal cells to an integrin-dependent matrix (i.e., laminin, fibronectin, and collagen IV) up-regulated FasL expression. Compared with BSA (control), fibronectin, laminin, and collagen IV induced increases in FasL protein levels by 34%, 65%, and 56%, respectively at 24 h; and by 38%, 78%, and 20% at 48 h, respectively (P < 0.05 between fibronectin, laminin, and collagen IV vs. the control at both 24 h and 48 h).
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To identify whether the up-regulatory effects of ECM proteins on FasL expression were brought about through an increase in cytoplasmic synthesis or membrane expression, endometrial stromal cells were plated onto tissue chamber slides that were previously coated with laminin, fibronectin, or collagen IV, and BSA as a control. Immunocytochemical staining was analyzed after a 24-h incubation (Fig. 2). We observed an increase in both intensity and distribution of FasL immunoreactivity by adherence of endometrial stromal cells to ECM proteins. FasL immunoreactivity revealed that the up-regulatory effects of ECM on FasL protein level was by increased membranous and cytoplasmic expression. FasL immunoreactivity was observed weakly in the control cells. The strongest expression of FasL was observed in cells plated on laminin and collagen IV (P < 0.05).
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Endometrial Cells from Women with Endometriosis Express Higher Level of FasL Protein
To investigate whether there were any changes of FasL protein expression in endometrial stromal cells of women with endometriosis by adhesion to ECM, eutopic endometrial stromal cells from women with endometriosis were plated onto dishes coated with fibronectin, laminin, or collagen IV, and incubated for 24 h (Fig. 3). Western blot analysis demonstrated that eutopic endometrial stromal cells of women with endometriosis expressed 20% higher levels of FasL protein compared to those of women without endometriosis, even when they were plated onto dishes coated with BSA (P < 0.05). Levels of FasL protein in endometrial stromal cells of women with endometriosis cultured on laminin, fibronectin, and collagen IV were 69%, 60%, and 40% higher, respectively, compared with those observed in cells of women without endometriosis (Fig. 3; P < 0.05 between fibronectin, laminin, and collagen IV in endometrial stromal cells of women with endometriosis vs. in endometrial stromal cells of women without endometriosis).
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Cytochalasin D Prevents FasL Gene Expression in Response to Integrin Activation
In order to demonstrate the role of the intact cytoskeleton in the intracellular signal pathways of FasL expression by means of adhesion, endometrial stromal cells were pretreated for 30 min with 5 µg/ml cytochalasin D, which disrupts actin cytoskeleton. We investigated the effect of cytochalasin D treatment on FasL expression in control cells plated on BSA. We did not observe any significant change in the FasL protein expression between untreated cells and those treated with cytochalasin D (Fig. 4, left panel). Endometrial stromal cells that had been pretreated with cytochalasin D were plated on fibronectin, laminin, and collagen IV coated plates for 24 h. Western blot analysis demonstrated that cytochalasin D treatment blocked the increase in FasL protein expression that was induced in response to integrin activation (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunotolerance that predisposes ectopic implants to survive is one of the theories suggested in the pathogenesis of endometriosis [18]. Peritoneal natural killer cells and cytotoxic T lymphocytes are suppressed in women with endometriosis [19, 20]. A local immunotolerance mechanism by depressed T lymphocyte activity may explain the reduction in spontaneous apoptosis of eutopic and ectopic endometrium in this disorder [1, 2]. In addition to various growth factors and cytokines, we suggest that adhesion to ECM components may alter the apoptotic milieu by means of FasL expression in endometriosis.
ECM proteins have a direct relationship with apoptosis of different cell types in both physiologic and pathologic events. Adhesion of small-cell lung cancer cells to the ECM enhances tumorigenicity and confers resistance to chemotherapeutic agents as the result of ß1 integrin-stimulated tyrosine kinase activation, which in turn suppresses chemotherapy-induced apoptosis [21]. Normal mesangial ECM proteins, collagen IV, and laminin protect rat mesangial cells from serum starvation-induced apoptosis by a ß1 integrin-mediated mechanism [22]. In human fetal and neonatal melanocytes, attachment to fibronectin suppresses apoptosis through an integrin-dependent pathway [23]. In the present study, we demonstrated that adhesion of endometrial stromal cells to the ECM proteins laminin, fibronectin, and collagen IV up-regulates FasL expression in these cells. Up-regulation of FasL expression may induce apoptosis of the local immune cells including activated T lymphocytes, thereby promoting the survival of endometrial stromal cells by adhesion to the ECM during the initial attachment of endometrial implants. Early lesions of endometriosis invade the ECM of the peritoneum [24]. FasL expression that occurs when endometrial stromal cells attach to the ECM may be one of the critical events in the development of endometriosis.
ECM and endometrial stromal cell interaction may also have a role in maternal immune tolerance during decidualization and implantation. Elementary remodeling of the ECM is observed during decidualization. Large decidual cells develop from endometrial stromal cells, and synthesize and deposit type IV collagen, laminin, and fibronectin into their external lamina during differentiation [25]. External lamina around the decidual cells is suggested to function as a cell to cell recognition barrier between the trophoblast and maternal cells [25]. Deposition of a thick ECM is suggested to be primary to the functions of decidua, such as regulation of trophoblast invasion by providing the appropriate ECM ligand domains for blastocyst adhesion and invasiveness, as well as immune protection of the fetal allograft. Immune protective function seems to be performed by the decidual ECM acting as the groundwork for infiltration and proliferation of suppressor lymphocytes. ECM proteins secreted by the decidua are proposed to have a masking effect on the fetal antigens presented by the trophoblast. Decidual cell and external lamina connection may induce FasL expression similar to the interaction between the ECM and endometrial stromal cells reported in the current study, and thereby may function in maternal immune tolerance by inducing apoptosis in the activated T lymphocytes of maternal origin.
Integrin cell surface adhesion molecules mediate cell-to-ECM interactions. Integrin-mediated cell adhesion co-clusters focal adhesion kinase (FAK), and stimulates tyrosine phosphorylation [26]. Integrins bound to their ECM ligands are coupled to actin microfilaments. Disruption of the actin cytoskeleton by treatment with cytochalasin D inhibits the tyrosine phosphorylation of FAK and numerous downstream signaling pathways [27]. Treatment of endometrial stromal cells with cytochalasin D before plating them on plates coated with ECM proteins blocks the increase in FasL expression induced by laminin, collagen IV, and fibronectin. Results of the cytochalasin D experiment suggest that actin microfilaments could be important in FasL production and/or its transport to the cell surface. Alternative possibilities such as the presence of different growth factors in the ECM preparations that affect FasL expression may also exist. Studies using anti-integrin antibodies should be performed to verify whether the response of FasL to ECM proteins is mediated by integrin.
We have previously demonstrated the stimulatory effect of cell adhesion to ECM on IL-8 and MCP-1 expression in human endometrium [28, 29]. The current study further emphasizes the role of adhesion to the ECM in the pathogenesis of endometriosis. Adhesion of endometrial stromal cells to the ECM components fibronectin, laminin, and collagen IV up-regulate FasL expression, which may lead to apoptosis of activated T lymphocytes and development of immune tolerance in endometriosis.
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FOOTNOTES |
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1 Correspondence: Aydin Arici, Yale University School of Medicine, Department of Obstetrics and Gynecology, 333 Cedar Street, New Haven, CT 06520-8063. FAX: 203 785 7134; aydin.arici{at}yale.edu 
2 Current address: IVI-Madrid, C/Santiago de Compostela 88, 28035 Madrid, Spain.
Accepted: August 3, 2001.
Received: January 3, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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