Hum. Reprod. Advance Access originally published online on October 27, 2005
Human Reproduction 2006 21(3):605-609; doi:10.1093/humrep/dei375
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Tumour necrosis factor-
, interleukin-6 and interleukin-8 do not promote adhesion of human endometrial epithelial cells to mesothelial cells in a quantitative in vitro model*
1 Leuven University Fertility Center, UZ Gasthuisberg, KU Leuven, Belgium, 2 Center for Human Genetics KU Leuven, Belgium and 3 The Fertility Centers of New England, Reading, MA, USA.
4 To whom correspondence should be addressed: Leuven University Fertility Center, UZ Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium E-mail: sophie.debrock{at}uz.kuleuven.ac.be
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Abstract |
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BACKGROUND: A key factor in the pathogenesis of endometriosis is the endometrial–peritoneal adhesion. To study the pathogenesis of endometriosis, a quantitative in vitro assay (QIVA) was developed to measure in vitro adhesion between human endometrial epithelial cells and mesothelial cells using commercially available cell lines. Using the QIVA, the hypothesis was tested that tumour necrosis factor-
(TNF-), interleukin-6 (IL-6) and interleukin-8 (IL-8) promote adhesion of endometrial epithelial cells to mesothelial cells. METHODS: Mesothelial cells were pre-treated with TNF-, IL-6 or IL-8 in various concentrations (ranging from 0 to 1000 IU/ml) for 24 h. Confluent endometrial epithelial cells were labelled with [35S]methionine, added to the confluent mesothelial cells and incubated for 1 h. After incubation, non-adhering cells were removed and adherent cells were solubilized and their [35S]methionine radioactivity was counted to quantify the adherence of endometrial epithelial cells to mesothelial cells. RESULTS: The in vitro adhesion of human endometrial epithelial cells to human mesothelial cells was inhibited in a dose-dependent manner by TNF- (P = 0.0007), IL-6 (P < 0.0001) and IL-8 (P = 0.0004). CONCLUSIONS: Using a quantitative in vitro adhesion assay, we were unable to confirm our hypothesis that TNF-, IL-6 and IL-8 promote the in vitro adhesion between endometrial epithelial cells and mesothelial cells.
Key words: cytokines/endometriosis/endometrium/in vitro adhesion assay/mesothelium
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Introduction |
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The pathogenesis of endometriosis is not completely understood. According to the implantation theory (Sampson, 1927
) endometriosis arises as a result of retrograde menstruation of endometrial fragments through the Fallopian tubes into the peritoneal cavity with subsequent implantation and growth of these endometrial cells onto the peritoneum. However, the exact mechanism of endometrial implantation on peritoneum is not known and cannot be studied in vivo in women.
Therefore, the interaction between endometrium and human peritoneum has been studied in various in vitro models using autologous monolayered mesothelial cells (Zhang et al., 1993; Wild et al., 1994), autologous peritoneum (Groothuis et al., 1999; Witz et al., 1999, 2001; Debrock et al., 2002) or amniotic membranes (Van der Linden et al., 1996; Groothuis et al., 1998) as a model for pelvic peritoneum. Recently, Lucidi et al. (2005) reported an in vitro model using peritoneal mesothelial cells of different sources including a commercially available cell line, normal ovarian surface epithelium and the anterior abdominal wall. Some investigators reported that mesothelium acts as a barrier to the attachment of ectopic endometrium and suggested that peritoneal damage is required for the adhesion of endometrial fragments (Van der Linden et al., 1996; Groothuis et al., 1998), whereas others observed that endometrium can attach to the intact mesothelial surface of the peritoneum and concluded that peritoneal damage is not required before endometrial implantation (Witz et al., 1999, 2001).
Endometriosis is associated with signs of pelviperitoneal inflammation (D’Hooghe and Debrock, 2002) including increased volume of peritoneal fluid (PF), increased concentration of PF macrophages, and increased PF concentrations of TNF-, IL-6, IL-8 and other cytokines and growth factors. Indeed, these cytokines have been reported to increase the endometrial–peritoneal adhesion in vitro. The in vitro adhesion of human endometrial stromal cells to mesothelial cells has been reported to be increased by pre-treatment of mesothelial cells with TNF- (Zhang et al., 1993). Similarly, in vitro incubation of endometrial stromal cells with increasing concentrations of IL-8 has been reported to stimulate their adhesion to fibronectin, an extracellular matrix (ECM) protein (Garcia-Velasco and Arici, 1999). Recently, it has been reported that in vivo adhesion of human endometrial cells to mouse peritoneum was moderately increased by treatment with TNF- and IL-6 (Beliard et al., 2003).
However, there is a need to develop a quantitative reproducible in vitro assay to measure adhesion between well-defined endometrial cells (epithelial cells, stromal cells), mesothelial cells and extracellular matrix. Such an assay could have the potential to measure the effect of various compounds on the adhesion process, as a model to study the pathogenesis of endometriosis and to test new molecules that could be useful for prevention or treatment of endometriosis.
The first aim of this study was to develop a quantitative in vitro assay (QIVA) to measure the adhesion between human endometrial epithelial cells and human mesothelial cells using existing commercially available cell lines. The second aim was to test the hypothesis that TNF-, IL-6 and IL-8 promote the adhesion of endometrial epithelial cells onto mesothelial cells in this quantitative in vitro adhesion model.
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Materials and methods |
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Quantitative in vitro adhesion (QIVA) assay
Human mesothelial cells (CRL-9444; epithelial, virus transformed cell type) and human endometrial epithelial carcinoma cells (CRL-1671) were obtained from ATCC (Manassas, USA). Human mesothelial cells were cultured in 4-well dishes (Nunc, VWR, Leuven, Belgium) in M199 culture medium (Gibco BRL, Belgium) containing 5% fetal calf serum (FCS) for 24 h at 37°C in 5 % CO2 in a concentration of 2x105 cells/well that resulted in confluency the next morning. The total volume of culture medium in each well was 500 µl. The next morning, confluent mesothelial cells were washed three times with FCS-free culture medium. Human endometrial epithelial carcinoma cells were cultured in a 1:1 mixture of Ham’s F-12/Dulbecco’s modified Eagle’s medium (Invitrogen, Merelbeke, Belgium) supplemented with glucose (4.5 g/l) and 10% FCS. Confluent endometrial epithelial cells were labelled for 18 h at 37°C in medium supplemented with [35S]methionine (20 µCi/ml). The next morning, the labelled endometrial epithelial cells were trypsinized, centrifuged at 700 g for 10 min, washed three times with FCS-free culture medium to remove non-bound radioactivity and then resuspended in FCS-free culture medium. Subsequently, 100 000 labelled endometrial epithelial cells (300 µl) were added to the confluent mesothelial cells (300 µl) in a total volume of 600 µl. Adhesion was studied after variable periods on incubation (10, 30, 60, 90 and 270 min). After incubation, non-adhering cells were removed by washing three times with phosphate-buffered saline (PBS, at 37°C) by adding and removing the PBS carefully at the side of the wells; adherent cells were solubilized in 1% sodium dodecyl sulphate and their [35S]methionine radioactivity was counted (counts per minute, c.p.m.) to quantify the adherence of endometrial epithelial cells to mesothelial cells. Each assay was performed in quadruplicate. Percentage binding was expressed as the rate of the c.p.m. count after each incubation period (10, 30, 60, 90 or 270 min) over the maximal c.p.m. count after 270 min.
Effect of TNF-, IL-6 and IL-8 on the QIVA assay
The effect of TNF-, IL-6 and IL-8 on the adhesion between mesothelial and endometrial cells was studied using the QIVA assay as follows (Figure 1). The mesothelial cells were cultured for 24 h to confluency as described above in 4-well dishes at least in duplicate. The next morning (time 24 h), when a subconfluent monolayer was obtained, mesothelial cells were pre-treated with TNF- (Invitrogen, Merelbeke, Belgium), IL-6 (Calbiochem, VWR, Leuven, Belgium) or IL-8 (Calbiochem, VWR, Leuven, Belgium) in various concentrations (ranging from 0 to 1000 U/ml) for 24 h. The confluent mesothelial cells, pre-treated with the respective cytokines, were then washed three times with FCS-free culture medium. Subsequently, prior to the addition of the endometrial epithelial cells, the pre-treated mesothelial cells were incubated with 1% bovine serum albumin (BSA) in PBS for 30 min at 37°C to prevent adhesion of endometrial epithelial cells to non-specific binding sites. [35S]Methionine-labelled endometrial epithelial cells were processed as described above and added to the pre-treated mesothelial cells. Adhesion was studied after 1 h of incubation. After incubation, non-adhering cells were removed, adherent cells were solubilized and their [35S]methionine radioactivity was counted to quantify the adhesion of endometrial epithelial cells to mesothelial cells. Percentage binding was expressed as the rate of the c.p.m. count in the presence of different concentrations of TNF-, IL-6 and IL-8 over the maximal c.p.m. count in the absence of TNF-, IL-6 and IL-8 respectively.
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Morphology and identity of cells
Morphology, confluency and condition of the cells were evaluated under a phase contrast microscope. Endometrial epithelial cells showed a characteristic clustering of small round cells, whereas mesothelial cells were flat and polygonal. The identity of the cells was regularly evaluated with immunohistochemistry using monoclonal antibodies against vimentin (Clone V9; Dako, Prosan, Merelbeke, Belgium) and cytokeratin (MNF clone 116; Dako) in a double-staining procedure (Pijnenborg et al., 2000). Mesothelial cells expressed both vimentin and cytokeratin, whereas endometrial epithelial cells only expressed cytokeratin.
MTT test
To measure the viability of human mesothelial cells in the presence of TNF-, IL-6 or IL-8, the MTT assay was performed (Mosmann, 1983). Mesothelial cells were plated (120 000 cells/500 µl) in 96-well plates, pre-treated with different concentrations of TNF-, IL-6 and IL-8 (ranging from 0 to 1000 IU/ml), and cultured for 24 h at 37°C in 5% CO2. The assay depends on the reduction of the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma Chemical Co.] by functional mitochondria to formazan. After 2 h incubation at 37°C of the cells with MTT, the cells were lysed with dimethyl sulphoxide in Sorensen’s glycine buffer and the formazan crystals solubilized. Absorbance was read at 550 nm using a spectrophotometric microplate reader.
Statistical analysis
Binding assays in the presence of the cytokines were performed 10–16 times (see legend to Figure 3). Analysis of variance analysis was performed to detect statistically significant effects. P < 0.05 was considered to be statistically significant.
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Results |
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QIVA assay
Binding of labelled human endometrial epithelial cells to human mesothelial cells occurred in a time-dependent manner with an initial exponential fast phase (up to 90 min) and a secondary slower phase (between 90 and 270 min) (Figure 2). Fifty-eight per cent binding was obtained after 30 min. Therefore, we decided that the binding assays in the presence of the cytokines would be performed during 1 h of incubation, i.e. during the exponential phase of the time–response course.
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Effect of TNF-, IL-6 and IL-8 on QIVA assay (Figure 1)
Mesothelial cells were cultured to confluency before the treatment with cytokines and after incubation with the respective cytokines, the mesothelial cells were incubated with 1% BSA. Treatment of the mesothelial cells with the different cytokines (Figure 3) resulted in a significantly decreased binding between human endometrial epithelial cells and human mesothelial cells. In vitro adhesion was inhibited in a dose-dependent manner by TNF- (P < 0.0001), IL-6 (P < 0.0001) and IL-8 (P = 0.0004) (Figure 3).
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MTT tests showed that increasing concentrations of TNF-, IL-6 and IL-8 had no effect on the viability of the mesothelial cells 24 h after treatment (results not shown). Using this test, it could be excluded that an effect of cell death was measured.
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Discussion |
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To the best of our knowledge, this is the first report of a QIVA assay measuring adhesion between human endometrial epithelial and human mesothelial cells. In this model, adhesion occurred rapidly within 1 h of incubation. These results are in agreement with those from other in vitro studies using explants of peritoneum and endometrium and reporting that both endometrial stromal and endometrial epithelial cells adhere to intact mesothelium within 1 h, followed by transmesothelial invasion of endometrial cells within 1 and 18–24 h (Witz et al., 1999
, 2001). Other investigators using in vitro co-culture of endometrial stromal and endometrial epithelial cells also observed adhesion of endometrial stromal cells to monolayers of cultured mesothelial cells within 1 h (Zhang et al., 1993; Dechaud et al., 2001; Witz et al., 2002). Recently, Lucidi et al. (2005) also reported that the difference in the adhesion of endometrial cells to mesothelial cells is dependent on the source of endometrial cells.
In this study, TNF-, IL-6 and IL-8 did not stimulate, and even inhibited to a certain degree, in vitro adhesion between endometrial epithelial cells and mesothelial cells in a dose-dependent fashion. Our data are in disagreement with results from other reports describing a dose-dependent stimulation of in vitro adhesion of endometrial stromal cells to peritoneal mesothelial cells and to fibronectin by TNF- (Zhang et al., 1993) and IL-8 (Garcia-Velasco and Arici, 1999) respectively, and with the results of in vitro adhesion of endometrial cells to mouse peritoneum by TNF- and IL-6 (Beliard et al., 2003). However, several differences in study design between our study and these studies may explain this discrepancy. Firstly, in our study endometrial epithelial cells were used, whereas endometrial stromal cells were used in two other studies (Zhang et al., 1993; Garcia-Velasco and Arici, 1999b) and a mixture of endometrial stromal and epithelial cells (in 
80/20 ratio) were used in a third study (Beliard et al., 2003). Secondly, the origin of cells was different in our study when compared to the other studies. Indeed, we used a virus-transformed mesothelial cell line and an endometrial epithelial cancer cell line for our QIVA assay, whereas in the other studies (Zhang et al., 1993; Garcia-Velasco and Arici, 1999a; Beliard et al., 2003), the adhesion assay was performed with endometrial cells isolated from endometrium of women without endometriosis who were undergoing surgery for benign indications. We made this choice since we wanted to develop a QIVA assay in a standardized way without having to deal with inter-patient variability related to genetics, cycle phase, pelvic pathology, etc. Thirdly, different methods were used in the studies mentioned to quantify endometrial–peritoneal adhesion. In our study, human endometrial cells were labelled with [35S]methionine, whereas other researchers studied adhesion by pre-labelling cells with the radioisotope 51Cr (Zhang et al., 1993; Dechaud et al., 2001; Witz et al., 2002), with a fluorescent dye (Garcia-Velasco and Arici, 1999), with 111Indium (Beliard et al., 2003) or with the fluorophore Calcein-AM (Lucidi et al., 2005). Fourthly, pre-treatment with TNF- and IL-6 did also not promote in vitro adhesion between colon epithelial carcinoma cells and mesothelial cells in a recent publication (van Rossen et al., 2001). It is possible that these cytokines have differential effects on different cell types.
It is tempting to speculate that the stimulating effect of TNF-, IL-6 and IL-8 on in vitro adhesion between mesothelial cells and endometrial stromal cells but not endometrial epithelial cells may explain why endometrial stromal cells are more involved in the initial attachment process than endometrial epithelial cells. Indeed, previous studies on endometrial–peritoneal adhesion have demonstrated that especially endometrial stromal cells are involved in the initial attachment process (Witz et al., 2001; Debrock et al., 2002; Lucidi et al., 2005), although adhesion of endometrial epithelium to mesothelium was also identified after 18–24 h of culture (Witz et al., 2001). Clearly, further studies are necessary in monolayer cultures to evaluate the interaction between endometrial stromal and epithelial cells in the adhesion process to mesothelium.
The exact cellular mechanisms or structural and/or functional changes that are responsible for the effect of the cytokines on the adhesion of endometrial to mesothelial cells are unclear and should be subject to further research. Cell adhesion molecules are involved in the adhesion process to establish cell–cell and cell–extracellular matrix interactions. Zhang et al. (1993) hypothesized that TNF- induced the expression of cell adhesion molecules in mesothelial cells in vitro. Most of the interactions in the adhesion process will be regulated by the integrin family of cell surface receptors. Garcia-Velasco and Arici (1999b) showed that the adhesion of endometrial stromal cells to different ECM proteins induced variable levels of IL-8 gene expression and protein expression and that this event is integrin-mediated. However, to elucidate the mechanisms of the functional and structural changes during cell–cell adhesion, more detailed research is necessary.
In this study, we decided to develop in the first place a new in vitro assay model using existing cell lines in order to achieve the highest possible reproducibility. Our in vitro assay can only give a quantitative estimate of cell–cell adhesion, but cannot give an explanation of the formation of endometriotic lesions in the pathogenesis of endometriosis. More experiments using primary cells would obviously strengthen the obtained results. In addition, primary cell culture models are not representative for the in vivo situation since structural and functional integrity of tissue may have been affected by the isolation process of the different cell types. Furthermore, working with primary tissues and cells may be more difficult because of the variability within and between patients (women with or without endometriosis, cycle stage), which may impair reproducibility of results. These disadvantages may have been overcome using a reproducible system with existing cell lines.
In conclusion, this QIVA assay may be used to quantitatively determine inhibition and/or stimulation of endometrial–mesothelial adhesion by several cytokines, growth factors and proteinases. Using autologous endometrial cells from women with endometriosis, perhaps this assay may also have the potential to be developed as a screening test for women at risk for the development of endometriosis. Moreover, the possibility to stimulate or block in vitro adhesion between endometrial cells and mesothelial cells can be exploited to develop new therapeutic approaches for the treatment of endometriosis. Our next research efforts will also test the adhesion of endometrial stromal cells (immortalized cell lines, cells from women with and without endometriosis) on mesothelial cell lines and extracellular matrix (fibronectin) in this QIVA assay. As a result, it will be possible to study adhesion between endometrial epithelial and stromal cell lines from women with or without endometriosis on the one hand, and mesothelial cell lines, mesothelium from women with or without endometriosis, or extracellular matrix on the other hand. Collectively, these studies will provide fundamental insight into the interaction between endometrial and mesothelial cells.
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Financial support from KULeuven, Bijzonder Onderzoeksfonds OT/99/30 and from FWO, Fundamental Clinical Investigator Program.
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Notes |
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* In part presented at the VII World Congress on Endometriosis, San Diego, California, February 24–27, 2002 and at the 58th Annual Meeting of the ASRM Conference, Seattle, Washington, October 12–17, 2002.
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Submitted on July 5, 2005; resubmitted on September 2, 2005; accepted on September 21, 2005.
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