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a Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520-8063
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Shed menstrual endometrium is viable [4], and so are the endometrial cells found in the peritoneal fluid [5]. In order to implant and grow, these cells need to establish cell-to-cell or cell-to-extracellular matrix (ECM) interactions with the peritoneal lining. Endometrial stromal cells (ESC) are relevant in stimulating and inhibiting the growth of glandular epithelium [6]. It is likely that ESC are actively involved in the adhesion of endometrial implants to the peritoneum. There is evidence suggesting that early endometriosis lesions invade the ECM of the peritoneum after the initial attachment [7]. Many of these interactions between endometrial cells and the ECM are mediated by the integrin family of cell surface receptors [8]. Integrins are able to transduce intracellular signals, although they lack the characteristics of signal-generating receptors [8]. Adhesion of endometrial cells to the peritoneal ECM may result in the transmission of the necessary signals for cell migration and invasion. Additionally, it has been shown that the mesothelial cells of the peritoneum express integrins in vitro as well as in vivo [9]. However, the precise mechanisms by which signals from ECM proteins are transduced via integrins to the intracellular machinery that controls cell growth and differentiation are not completely understood.
Activated macrophages are a dominant feature of the inflammatory reaction and frequently contribute to the pathogenesis of the underlying disease. Leukocyte chemoattraction in the inflammatory lesion is mediated by several factors. Monocyte chemotactic protein (MCP)-1 is one of the potent chemotactic factors for monocytes/macrophages. MCP-1 is secreted by a number of cell types including ESC [10], and its levels in the peritoneal fluid are elevated both in women with endometriosis [11] and in women with abdomino-pelvic adhesions [12].
We hypothesized that cell adhesion itself may enhance the expression of MCP-1, thus contributing to the recruitment of macrophages to the peritoneal cavity, and as a consequence may play a role in the pathogenesis of endometriosis and/or pelvic adhesions. In the present study, we have investigated the role that the interaction between ESC with ECM components may have in the regulation of MCP-1 gene expression and secretion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fibronectin was acquired from Becton Dickinson (Bedford, MA). Collagen IV, laminin, and poly-L-lysine (Mr 70 000–150 000) were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse anti-human integrin ß1 and ß2 monoclonal antibodies were purchased from Chemicon International (Temecula, CA). The ECM proteins were dissolved in calcium- and magnesium-free PBS to the concentration of 20 µg/ml fibronectin, 10 µg/ml type IV collagen, and 20 µg/ml laminin. For poly-L-lysine coating, a 0.1 mg/ml solution was applied. Coating of 100-mm Petri dishes and 24-well plates was carried out by overnight incubation at 4°C. Dishes were then incubated for 2 h at 37°C with 1% (w:v) BSA denatured at 70°C for 15 min, to block the nonspecific binding sites on plastic, and washed with sterile PBS before plating. As control, dishes were directly blocked with denatured BSA.
Stock cytochalasin D was prepared at a concentration of 5 mg/ml in dimethyl sulfoxide (Baker, Phillipsburg, NJ).
Tissue Collection and Cell Culture
Endometrial tissue samples were obtained from women undergoing diagnostic laparoscopy or hysterectomy for benign disease. Informed consent was obtained from each woman prior to surgery using protocols approved by the Human Investigation Committee of Yale University. Tissues were kept in Hanks' Balanced Salt Solution (HBSS) and transported to the laboratory for culture.
Endometrial cells were dispersed by incubation of minced tissue in HBSS containing Hepes (25 mM), penicillin (200 U/ml), streptomycin (200 mg/ml), collagenase (2 mg/ml; 15 U/mg), and DNase (0.2 mg/ml; 1500 U/mg) for approximately 20 min at 37°C with constant agitation. The dispersed ESC were separated from glands by filtration through a wire sieve (73-µm-diameter pore). Endometrial glands remained largely undispersed and were retained by the sieve, while stroma passed through into the filtrate.
ESC were plated in Dulbecco's Modified Eagle's medium:Ham's F-12 (DMEM/F12 1:1, v:v) containing antibiotics-antimycotics (1% v:v) and fetal bovine serum (10% v:v) in plastic flasks (75 cm2), maintained at 37°C in a humidified chamber (5% CO2 in air), and allowed to replicate until confluent in monolayer. Thereafter, the cells were passed by standard methods of trypsinization and again allowed to replicate to confluence. After cells reached confluence, cultures were treated with serum-free, phenol red-free medium for 24 h.
Northern Analysis
The ESC cultures were trypsinized, centrifuged, and resuspended in fresh serum-free, phenol red-free medium at a concentration of approximately 500 000 cells/ml. Two milliliters of the cell suspension was then seeded onto 100-mm Petri dishes precoated as indicated above, and the cells were incubated for 6 h. The medium was then aspirated to remove nonadherent cells. Total RNA was extracted from the adhering cells using Trizol (Gibco BRL, Grand Island, NY), size-fractionated (10 µg per lane) by electrophoresis on 1% formaldehyde-agarose gels, transferred electrophoretically to Hybond-N+ membrane (Amersham, Arlington Heights, IL), and cross-linked to the membrane using ultraviolet light. Prehybridization was conducted for 5 h at 65°C in a buffer composed of NaCl (0.9 M), Tris-Cl (90 mM, pH 8.3), EDTA (6 mM), 5-strength Denhardt's solution, SDS (0.1% w:v), sodium pyrophosphate (0.1% w:v), and salmon sperm DNA (0.2 mg/ml). Hybridizations were conducted for 16 h at 65°C in a buffer that contained a 30-mer oligo DNA probe radiolabeled with [
-32P]ATP complementary to MCP-1 mRNA. Thereafter, blots were washed once with 6-strength standard saline citrate and SDS (0.1% w:v) for 15 min at room temperature, once with double-strength standard saline citrate and SDS (0.1% w:v) for 15 min at room temperature, and once with double-strength saline citrate and SDS (0.1% w:v) for 20 min at 65°C. Autoradiography of the membrane was performed at -80°C using Kodak X-Omat AR film (Eastman Kodak, Rochester, NY). The amount of RNA in each lane was normalized by the analysis of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. The autoradiographic bands were quantified using a laser densitometer (Molecular Dynamics, Sunnyvale, CA). G3PDH hybridization was also used to elucidate whether the effects shown were reflective of the activation of the MCP-1 gene specifically or were a consequence of global transcriptional activation.
MCP-1 ELISA
ESC grown to confluence and serum starved for 24 h were trypsinized and resuspended in serum-free, phenol red-free medium at a concentration of approximately 100 000 cells/ml. Identical aliquots of cell suspension (400 µl) were then plated into 24-well plates precoated with the appropriate ECM component as indicated above. After 24 h the conditioned media were collected, briefly centrifuged at 4°C to remove cells, and stored at -80°C until analysis. Immunoreactive MCP-1 concentration was quantitated using a commercial ELISA kit specific for human MCP-1 (R&D Systems, Minneapolis, MN). According to the manufacturer, there is no measurable cross-reactivity with other known cytokines in this assay. The sensitivity for MCP-1 was 4.7 pg/ml in medium and 18.1 pg/ml in serum. Each experiment was done using three replicate wells for each condition, and supernatant from each well was tested in a single ELISA assay. Each experimental setup was repeated on three occasions using cells obtained from different patients. The intraassay and interassay coefficients of variation were 4.9% and 5.9%, respectively.
Statistical Analysis
The levels of MCP-1 mRNA and protein were normally distributed (tested by Kolmogorov-Smirnov test). ANOVA and Scheffe's correction for pair-wise multiple comparisons were used for statistical analysis. A value of 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 whether adhesion of ESC to various ECM proteins would affect MCP-1 expression, cells were plated on different ECM proteins. Cells plated on BSA expressed low levels of MCP-1 mRNA, whereas cells adhering to fibronectin, laminin, or collagen IV expressed markedly higher levels of MCP-1 mRNA (Fig. 1). Compared to BSA (control), fibronectin, laminin, and collagen IV induced increases to 6-, 4-, and 2-fold in MCP-1 mRNA levels (n = 5) (p < 0.05), respectively. Interestingly, MCP-1 mRNA levels in cells plated on poly-L-lysine, a non-integrin-dependent adhesion matrix, were similar to those in controls.
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Integrin-Dependent Cell Adhesion Up-Regulated MCP-1 mRNA
To establish whether the effect of ECM on the expression of MCP-1 is mediated by interactions with specific integrins, ESC were plated on dishes coated with anti-human ß1 integrin monoclonal antibody. Engagement of ß1-containing integrins was associated with ESC adhesion and resulted in up-regulation of MCP-1 secretion (13-fold that of control, p < 0.05; n = 5) (Fig. 2). This overexpression was 3-fold that induced by fibronectin. Poly-L-lysine, a non-integrin-dependent adhesion substrate, induced an MCP-1 mRNA expression similar to that in the control.
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An Intact Cytoskeleton Was Required to Stimulate MCP-1 Gene Expression in Response to Integrin Activation
In order to demonstrate that the cytoskeleton plays a role in the integrin-mediated signaling process of MCP-1 expression on adhesion, ESC were pretreated for 30 min with 5 µg/ml cytochalasin D, which disrupts the actin cytoskeleton. Then ESC were plated on fibronectin-coated plates for 6 h. Northern blot analysis showed a complete block in the increase of MCP-1 mRNA that was induced in response to integrin activation (n = 4) (Fig. 3).
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Adhesion Induced MCP-1 Secretion from ESC
To confirm that adhesion stimulated not only MCP-1 mRNA expression but also MCP-1 protein secretion, we assessed the production of MCP-1. Supernatants from ESC plated on various ECMs were collected after 6, 12, and 24 h; then MCP-1 levels from 6 replicates were measured by ELISA. Figure 4 (top panel) shows the time-dependent production of MCP-1 from ESC; differences were statistically significant at 12 and 24 h (n = 4) (p < 0.001, fibronectin vs. poly-L-lysine). These experiments also confirmed at the protein level that ESC engagement with an ECM protein or with ß1, but not ß2 integrin, significantly up-regulated MCP-1 secretion as compared to control or poly-L-lysine, a non-integrin-dependent adhesion substrate (Fig. 4, bottom panel).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adhesion of endometrial cells to the ECM is a critical process in the initial onset of endometriosis. Endometrial cells are transported through the fallopian tubes to the peritoneal cavity in 76–90% [15] of women during menses, and these cells are viable [5]. At the end of the menstrual cycle, endometrial epithelial cell proliferation ceases almost completely, while the number of proliferating cells in the stroma increases again after its postovulatory nadir [16]. If some of these cells are able to evade the defense mechanisms of the peritoneal fluid such as macrophages and lymphocytes, they will attach to the ECM and grow. We have shown how ESC adhesion to various ECM proteins induces an up-regulation in MCP-1 gene expression and protein secretion. These findings have important implications in the pathogenesis of some gynecologic diseases.
The mesothelium that covers the peritoneum is a delicate thin layer. Infections, antibodies, hypoxia, inflammatory conditions such as in endometriosis, residual blood, or toxins may produce tissue damage [17]. ECM proteins are produced by fibroblasts and other cells involved in wound healing. Any ESC that enters the peritoneal cavity under such conditions could attach to these ECM proteins, participating in the maintenance of the inflammatory condition by inducing secretion of MCP-1.
Endometrial cells may survive many days in the peritoneal fluid. The ability of endometrial fragments recovered from the peritoneal fluid to establish cell-cell or cell-ECM contacts with the peritoneal lining was demonstrated recently, as these fragments express integrins and cadherins [18]. Any condition that is likely to damage the serosal surface of the abdominal organs, such as surgery or infection, may expose the ECM proteins underneath the thin mesothelial layer. When endometrial cells attach to ECM, they secrete higher levels of MCP-1 among other substances. Macrophages are recruited, thus contributing to the inflammatory reaction. In fact, we have recently shown that MCP-1 plays a role in adhesion formation in a rodent model [19], and women with peritoneal adhesions have elevated peritoneal fluid MCP-1 levels [12]. Integrin expression in endometrial cells varies throughout the menstrual cycle [20, 21]. The only integrin constitutively expressed in ESC is the so-called classic fibronectin receptor, 
5ß1 [20]. Interestingly, only the fibronectin receptor persists in endometriotic implants, and it is expressed at a higher level than in eutopic endometrial glands [18]. Examination of the v integrins was not included in our study. Although stromal cells are known to express vß3, we focused our study on the ß subunits, since ß integrins are relevant in mediating the organization of ECM proteins [22] and are part of most of the various integrin dimers. The hypothetical role of ß integrins in connecting the ECM framework to intracellular cytoskeletal structures was our main investigative goal. We evaluated MCP-1 expression after adhesion of ESC to various ECM proteins (fibronectin, laminin, and collagen IV) that are the ligands for the ß1 subunit. They all up-regulated MCP-1 gene expression and protein secretion. Although fibronectin, collagen IV, and laminin receptors share structural components such as ß1 integrin, the -cytoplasmic domain regulates the specificity of the ligand-dependent interactions [23], and differences in the subunits may account for the observed response. In fact, fibronectin, although not the most abundant ECM protein in the mesothelium, induced a much higher MCP-1 expression in ESC than other more abundant ECM substrates such as collagen IV. Also, this variable response may be due to binding to two different regions within the ß1 subunit [24]. Interestingly, when a non-integrin-dependent cell adhesion matrix was tested (poly-L-lysine), it did not induce any change in MCP-1 mRNA or protein. The finding that ESC, when seeded on plates coated with the antibody against the ß1 subunit, induced an increase in MCP-1 levels confirmed that MCP-1 secretion on adhesion is an integrin-dependent event. When ESC were attached to anti-ß1 or anti-ß2 integrin antibody-coated plates, the rise in MCP-1 expression suggests that it is the engagement of the ß1 that induces the MCP-1 up-regulation.
Although some integrins interact with only a single ECM protein [25], most commonly an individual integrin will recognize several ECM proteins. The integrins of the ß1 subfamily recognize various ECM proteins. These integrins provide a physical linkage between cytoskeletal structure and the ECM. By organizing the cytoskeleton, integrins may transmit signals, regulating cell shape and internal cellular architecture [26]. We treated ESC with cytochalasin D just before plating the cells. Cytochalasin D disrupts actin filament organization and blocks tyrosine phosphorylation of ppl 25fak, the focal adhesion kinase. The disruption of the actin cytoskeleton blocked the increase in MCP-1 induced by fibronectin, confirming that an intact cytoskeleton is required to stimulate MCP-1 gene expression in response to integrin activation. This is in accordance with the proposed hypothesis explaining the mechanisms of integrin-mediated signal transduction, a theory based on the assumption that integrins bind directly to the cytoskeleton. In this way, reorganization of the cell architecture is promoted, and the newly formed cytoskeleton framework may influence adhesiveness and determine integrin-mediated regulatory signal propagation throughout the cell [27]. Transmission of the correct signal to endometrial cells may be an important step for determining which cells are going to adhere and develop endometriosis or pelvic adhesions.
Dou et al. [28] showed that some growth factors such as transforming growth factor ß1, a protein released by macrophages that are attracted to the site of injury, may facilitate cell-cell or cell-matrix interactions. It is known that this and other growth factors are increased in the peritoneal fluid of women with endometriosis [29]. Most cell types require adhesion to ECM in order to be stimulated to proliferate in response to growth factors or serum [30]. The synergistic interactions between growth factors and integrin signaling pathways may facilitate cell proliferation and adhesion in clinical situations with elevated growth factor levels in the peritoneal fluid, such as endometriosis and/or abdomino-pelvic adhesions [31].
In conclusion, we have shown that cell adhesion to ECM is an important event that leads to stimulation of MCP-1 expression. This process is mediated by integrins. Further investigation into the molecular mechanisms of adhesion-induced expression of inflammatory mediators should provide insight into the early events of diseases such as endometriosis and pelvic adhesions.
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FOOTNOTES |
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2 Correspondence: Aydin Arici, Yale University School of Medicine, Department of Obstetrics & Gynecology, 333 Cedar Street, New Haven, CT 06520–8063. FAX: 203 785 7134; aydin.arici{at}yale.edu
3 Current address: Juan A. Garcia-Velasco, Instituto Valenciano de Infertilidad, IVI-Madrid, Spain.
Accepted: March 23, 1999.
Received: November 6, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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