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BOR - Papers in Press, published online ahead of print May 10, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.045716
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BIOLOGY OF REPRODUCTION 75, 203–209 (2006)
DOI: 10.1095/biolreprod.105.045716
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Regulation of FAS Ligand Expression by Chemokine Ligand 2 in Human Endometrial Cells

Belgin Selam 23 , Umit A. Kayisli 2, G. Eda Akbas 2, Murat Basar 2, and Aydin Arici 1 23 

Yale University School of Medicine,2 Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, New Haven, Connecticut 06520-8063 Yeditepe University,3 Faculty of Medicine, Department of Obstetrics and Gynecology, 34752 Istanbul, Turkey

ABSTRACT

Human endometrium is a dynamic tissue under the influence of numerous hormones, growth factors, and cytokines interacting to maintain a balance of cellular growth, differentiation, and apoptosis. We have previously demonstrated that several factors including interleukin-8, extracellular matrix, and steroid hormones modulate FASLG, one of the apoptotic molecules, in human endometrium. Chemokine ligand 2 (CCL2), a monocyte chemoattractant and activating factor, is a cytokine involved in endometrial function. CCL2 is elevated in the peritoneal fluid of women with endometriosis. We hypothesize that increased levels of CCL2 in the endometriotic environment may upregulate FASLG expression in human endometrial stromal cells and induce a local immunotolerance in endometriosis. To test our hypothesis, we studied the in vitro regulation of FASLG expression and apoptosis by CCL2 in endometrial stromal cells. Western blot analysis revealed that CCL2 upregulated FASLG protein expression in cultured endometrial stromal cells. Based on semiquantitative RT-PCR analysis, CCL2 did not alter either FAS or FASLG mRNA expression in endometrial stromal cells. Immunocytochemistry results from the same cells treated with CCL2 demonstrated upregulation of FASLG protein expression. CCL2 did not change rate of apoptosis in endometrial stromal cells as evaluated by TUNEL assay. However, an increased apoptotic rate was detected in Jurkat (T lymphocytes) cells cocultured with endometrial stromal cells previously treated with CCL2. We speculate that increased FASLG expression by CCL2 may induce apoptosis of T lymphocytes and thus produce an immunotolerant environment for the development of ectopic implants.

apoptosis, CCL2, cytokines, endometriosis, endometrium, FASLG, female reproductive tract, gene regulation, immunology

INTRODUCTION

Apoptosis, the programmed cell death, regulates cell turnover in both physiologic and pathologic events in the human endometrium. Ectopic endometrial tissue is relatively resistant to macrophage-mediated cytotoxicity, and a decrease in spontaneous apoptosis of endometrial cells is one of the proposed factors in the pathogenesis of endometriosis [1]. Regulation of apoptosis involves interaction of several genes with stimulatory or inhibitory effects on cell death. FAS ligand (FASLG), a mediator of apoptosis in differentiated cells and in embryonic development, interacts with its receptor FAS and induces apoptosis through autocrine or paracrine signaling. Our recent findings on the regulation of FASLG by macrophage-derived growth factors and adhesion to extracellular matrix in endometrial stromal cells suggest a role for FASLG in the pathogenesis of endometriosis [2, 3].

The peritoneal fluid of women with endometriosis has increased monocyte and macrophage chemotactic activity [4, 5]. Chemokine ligand 2 (CCL2), as a chemoattractant and activating chemokine for monocytes and macrophages, is one of the candidates for this activity [6, 7]. The level of CCL2 is elevated in the peritoneal fluid of women with endometriosis and in women with abdomino-pelvic adhesions [8–11]. Medical treatment suppresses inflammatory cytokine levels and eliminates the embryo toxicity of the peritoneal fluid in endometriosis [8]. CCL2 is secreted by a number of cell types, including endothelial cells, fibroblasts, leukocytes, peritoneal fluid mesothelial cells, and endometrial cells [9, 12, 13]. Adhesion of endometrial stromal cells to extracellular matrix proteins induces CCL2 gene expression through integrin signaling in human endometrium [14]. Increased CCL2 concentration of the peritoneal fluid in endometriosis may have an adverse affect on the local environment and facilitate further growth of endometriotic implants.

Endometrial stromal cells play an essential role in the pathogenesis of endometriosis. They are involved in both adhesion to peritoneum and dynamics of immune system in human endometrium. Adhesion of retrogradely menstruated viable endometrial cells into the peritoneal cavity is a consequential step in the development of endometriosis according to implantation theory [15]. Endometrial stromal cells are involved in the initial steps of attachment to the mesothelial surface of the peritoneum [15]. T lymphocytes, macrophages, and granulated lymphocytes account for a substantial proportion of endometrial stromal cells and constitute the immune milieu of human endometrium [16].

We have previously demonstrated CCL2 expression in human endometrial and peritoneal cells and its in vitro modulation by macrophage-derived cytokines [9, 13]. In vivo CCL2 expression of human endometrium is also upregulated in endometriosis. [17]. However, the effect of CCL2 on apoptosis through FAS/FASLG system in human endometrium has not been investigated so far. We hypothesized that increased levels of CCL2 in the endometriotic environment could upregulate FASLG expression in endometrial stromal cells and may be relevant in the development of a relative local immunotolerance in endometriosis. To test our hypothesis, we studied the regulation of FASLG expression and FASLG-mediated apoptosis by CCL2 in endometrial stromal cells and T lymphocytes.

MATERIALS AND METHODS

Tissue Collection

Endometrial tissue was 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 40 (range 32–50). Diagnosis of patients were uterine fibroid (n = 3) and voluntary sterilization by tubal ligation (n = 3). Tissues were placed in Hank balanced salt solution (HBSS) and transported to the laboratory for separation and culture of endometrial cells. 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.

Isolation and Culture of Human Endometrial Stromal and Glandular Cells

Endometrial stromal and glandular cells were separated and maintained in monolayer culture, as described previously [18]. Endometrial stromal cells were treated with various concentrations of CCL2 (R&D, Minneapolis, MN) (0.001–1 ng/ml) and with CCL2 neutralizing antibody (R&D) (0.01–10 µg/ml) for 24 h for protein analysis (by Western blot and immunocytochemistry) and for 2 h for mRNA analysis (by RT-PCR). Endometrial glandular cells and Ishikawa cells (well-differentiated endometrial adenocarcinoma cell line, kindly provided by Dr. Richard Hochberg, Yale University) were treated with CCL2 for 2 h for mRNA analysis. FASLG gene is a fast-transcribed gene, and levels of FASLG mRNA peak after 2–3 h of treatment [2]. Untreated endometrial stromal cells express FAS mRNA and the expression FAS mRNA does not change with CCL2 treatment at various time intervals between 0 and 24 h. We used 2 h as the optimal incubation time for expressions of FASLG and FAS mRNA.

Jurkat Cells

Nonadherent human T-lymphocyte cells (Jurkat cells; kindly provided by Dr. Gil Mor, Yale University) were maintained in continuous culture. The cells were plated in RPMI 1640 medium (Gibco BRL) with FBS (10% vol/vol). Cells were plated in plastic flasks, maintained at 37°C in a humidified atmosphere (5% CO2 in air), and allowed to replicate to confluence.

FASLG RT-PCR

A semiquantitative RT-PCR was performed. The RT-PCR protocol was optimized for temperature, amount of RNA, and number of cycles in order to ascertain linear amplification range where the density of bands correlates with the amount of gene expression. Total RNA was extracted by Trizol Reagent (Gibco BRL) according to manufacturer's instructions. Semiquantitative RT-PCR was performed as described previously [19]. The primers used for amplification of FASLG, FAS, and glycerol-3-phosphate dehydrogenase 1 (GPD1) have been previously described [20–22] and have the following sequences:

  1. 1. FASLG primers yielding 311-bp reaction product:
  2. 2. Sense: 5'-ACA CCT ATG GAA TTG TCC TGC-3'
  3. 3. Antisense: 5'-GAC CAG AGA GAG CTC AGA TAC G-3'
  4. 4. FAS primers yielding 266-bp reaction product:
  5. 5. Sense: 5'-CAC TAT TGC TGG AGT CAT G-3'
  6. 6. Antisense: 5'-CTG AGT CAC TAG TAA TGT CC-3'
  7. 7. GPD1 primers yielding 788-bp product:
  8. 8. Sense: 5'-GGT CGG AGT CAA CGG ATT TGG TCG-3'
  9. 9. Antisense: 5'-CTT CCG ACG CCT GCT TCA CCA C-3'

PCR products and molecular weight markers were separated in agarose gels containing ethidium bromide (10 mg/ml) and visualized by UV light. The intensity of each band was normalized to its corresponding GPD1 band to semiquantitatively compare values between samples.

FASLG Western Blot Analysis

Protein extraction and Western blot analysis were performed as described previously [19]. Incubation with mouse anti-human FASLG monoclonal antibody (Transduction Laboratories, Lexington, KY) diluted at 1:1000 was performed for 1 h and thereafter washed with PBS-T buffer. The membrane was further incubated for 1 h with peroxidase-labeled anti-mouse IgG (Vector Laboratories, Burlingame, CA) diluted at 1:10 000. The immunoblot was developed using chemiluminescent kit (NEN Life Science, Boston, MA).

Equal loading of proteins in each lane was confirmed by staining the membrane with Ponceau 2S (Sigma Chemical Co., St. Louis, MO). The Ponceau red signals and autoradiographic bands for FASLG were quantified by a digital imaging and analysis system (AlphaEase, Alpha Innotech Corporation; San Leandro, CA) and a laser densitometer (Molecular Dynamics, Sunnyvale, CA), respectively. FASLG expression was normalized by dividing the arbitrary densitometry units for FASLG to the amount of Ponceau red staining for each band.

Immunocytochemistry

Endometrial stromal cells were grown to preconfluence on four-chamber slides (Falcon). Following treatments, immunocytochemistry was performed as described previously [19]. Immunocytochemical staining intensity was ranked between 0 (absent) and 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 the each intensity within these areas was determined by two investigators blinded to treatments. Average scoring of the investigators was used.

TUNEL In Situ Apoptosis Detection

Apoptosis in endometrial stromal cells plated on tissue chamber slides, with or without CCL2 treatment, was detected by enzymatic labeling of DNA strand breaks using TUNEL. TUNEL labeling was carried out using a Cell Death Detection Kit (Roche, Mannheim, Germany) and performed according to the manufacturer's instructions. Briefly, chamber slides were fixed 20 min in 4% paraformaldehyde (at room temperature). Samples were washed in PBS and treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 5 min on ice. After washing with PBS, the labeling reaction was performed using 50 µl TUNEL reagent to each sample, except the negative control, where reagent without enzyme was added and incubated for 1 h at 37°C. Following PBS washing, slides were incubated with converter reagent for 30 min at 37°C. After washing, color development for localization of cells containing labeled DNA strand breaks was performed by incubating the chambers with Fast Red substrate solution for 10 min. Slides were lightly counterstained with hematoxylin prior to permanent mounting. Quantitation of apoptotic cells was accomplished by counting the number of apoptotic bodies sighted in the microscopic fields while counting 500 cells for determination of the labeling index. Labeling indices were calculated as the number of labeled cells divided by total cells counted (labeled cells/500 cells).

Coculture of Endometrial Stromal Cells and Jurkat Cells and Analysis by Flow Cytometry

Endometrial stromal cells were plated in 12-well plates in Ham F12/DMEM (1:1 vol/vol) and FBS (10% vol/vol) and were allowed to reach confluence. The confluent cells were treated with serum- and phenol red-free media for 24 h and then treated with various concentrations of CCL2 (0.001–1 ng/ml). Following 24 h treatment with and without CCL2, the media was removed, and nonadherent human T lymphocytes (Jurkat cells; 106 cell/ml) were plated either alone or on the stromal cells. After 24 h incubation, Jurkat cells in suspension were removed and labeled by TUNEL as described previously. TUNEL-positive and -negative cells were quantified using flow cytometry.

Statistical Analysis

The densitometry of FASLG protein, RT-PCR bands, and immunocytochemistry and TUNEL scores were normally distributed as tested by Kolmogorov-Smirnov test. Thus, ANOVA and post hoc Tukey test for pairwise multiple comparisons were used for statistical analysis (P < 0.05 was considered to be significant). Statistical calculations were performed using Sigmastat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA).

RESULTS

Regulation of FASLG Protein Expression by CCL2 in Endometrial Cells in Culture

According to time-course experiments for expression of FASLG protein in endometrial stromal cells, we used 24 h as the optimal incubation time (Fig. 1a). To investigate the regulation of FASLG protein expression by CCL2, endometrial stromal cells were incubated with various concentrations of CCL2 (0.001–1 ng/ml) for 24 h, and FASLG protein levels were analyzed by Western analysis. Untreated endometrial stromal cells expressed FASLG protein (Fig. 1b). CCL2 treatment stimulated FASLG protein expression in a concentration-dependent manner. CCL2 treatments of 0.01, 0.1, and 1 ng/ml induced 3.2-, 3.7-, and 4-fold increases, respectively, in FASLG protein levels (P < 0.01 between control and CCL2 concentrations of 0.01–1 ng/ml) (Fig. 1b). Similarly, untreated endometrial epithelial and Ishikawa cells expressed FASLG protein, and CCL2 treatment stimulated FASLG protein expression in both cells (Fig. 1c). To assess the specificity of the induction of FASLG protein by CCL2 in endometrial stromal cells, we used a blocking polyclonal antibody to human CCL2. We incubated endometrial stromal cells with 1 ng/ml CCL2 and added various concentrations of blocking antibody (0.01, 0.1, 1, 10 µg/ml). CCL2 blocking antibody treatment blocked the induction of FASLG protein expression by CCL2 treatment (P < .01 between control, CCL2 [1 ng/ml] and CCL2 blocking Ab [0.01, 0.1 µg/ml] and P < 0.05 between control and CCL2 blocking antibody [10 µg/ml]) (Fig. 2).


Figure 1
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  		FIG. 1. a) Time course experiment with CCL2 for FASLG protein expression in endometrial stromal cells. FASLG protein expressions at 0, 12, 24, and 48 h after CCL2 treatment. b) FASLG protein expression in endometrial stromal cells: concentration-dependent effect of CCL2. Cultured cells were treated with 0.001, 0.01, 0.1, and 1 ng/ml CCL2 for 24 h. Total protein was extracted, and FASLG protein expression was analyzed by Western blot. +, recombinant FASLG as positive control; Ponceau, Ponceau red signals used for equal protein loading. The graph demonstrating the FASLG/total protein as arbitrary densitometric unit for 0.001-, 0.01-, 0.1-, and 1-ng/ml CCL2 treatments compared with control (bars represent mean ± SEM from different experiments, P < 0.01 between 0.01, 0.1, and 1 ng/ml CCL2 vs. control). c) FASLG protein expression in endometrial epithelial and Ishikawa cells following treatment with CCL2 for 24 h


Figure 2
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  		FIG. 2. FASLG protein expression in endometrial stromal cells: concentration-dependent effect of CCL2 and CCL2 neutralizing antibody. Cultured cells were treated with 1 ng/ml CCL2 and 0.01, 0.1, 1, and 10 µg CCL2 neutralizing antibody for 24 h. Total protein was extracted, and FASLG protein expression was analyzed by Western blot (n = 3) (bars represent mean ± SEM, P < 0.01 between control, CCL2 [1 ng/ml] and CCL2 blocking Ab [0.01, 0.1 µg/ml] and P < 0.05 between control and CCL2 blocking antibody [10 µg/ml])

Regulation of FAS and FASLG mRNA Expression by CCL2 in Endometrial Cells in Culture

To evaluate whether the upregulatory effect of CCL2 on FASLG protein expression was secondary to an increase in FASLG mRNA levels, we measured FASLG mRNA levels by RT-PCR analysis. We also investigated FASLG receptor (FAS) mRNA expression in human endometrial cells. Endometrial stromal cells were incubated with various concentrations of CCL2 (0.001–1 ng/ml) for 2 h. CCL2 did not induce any concentration-dependent alterations in FASLG mRNA expression in human endometrial stromal cells (Fig. 3a). Untreated endometrial stromal cells express FAS mRNA (Fig. 3b). We did not observe any variation in FAS mRNA levels by increasing concentrations of CCL2 (Fig. 3b).


Figure 3
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  		FIG. 3. Effect of CCL2 on FAS and FASLG mRNA expressions in cultured human endometrial stromal cells. Cells were treated with CCL2 (0.001–1 ng/ml) for 2 h. Total RNA was extracted, and FASLG and FAS mRNA expressions were analyzed by RT-PCR. Concentration-dependent FASLG (a) and FAS (b) mRNA expressions are shown. FASLG and FAS expressions were normalized to the amount of glycerol-3-phosphate dehydrogenase 1 (GPD1) for each band. C, Control; M, molecular weight marker

We also evaluated the FAS and FASLG mRNA expressions in endometrial glandular and Ishikawa cells. Both cell types expressed FAS and FASLG mRNA. CCL2 treatment (1 ng/ml) induced 3.5- and 3.8-fold increases in FASLG mRNA expression in endometrial glandular and Ishikawa cells, respectively (P < 0.01) (Fig. 4a). Similar to endometrial stromal cells, we did not observe any changes in FAS mRNA levels with CCL2 treatment (1 ng/ml) in either cell types (Fig. 4b).


Figure 4
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  		FIG. 4. Effect of CCL2 on FAS and FASLG mRNA expressions in cultured human endometrial glandular cells and Ishikawa cells. Total RNA was extracted, and FAS and FASLG mRNA expressions were analyzed using RT-PCR. a) FASLG mRNA expression in endometrial glandular and Ishikawa cells following 2 h of CCL2 (1 ng/ml) treatment. b) FAS mRNA expression in endometrial glandular and Ishikawa cells treated with CCL2 (1 ng/ml) for 2 h. FASLG and FAS expressions were normalized to the amount of glycerol-3-phosphate dehydrogenase 1 (GPD1) for each band. C, Control; M, molecular weight marker (bars represent mean ± SEM from different experiments)

Regulation of FASLG Expression by CCL2 in Endometrial Stromal and Glandular Cells: Immunocytochemistry

Endometrial stromal and glandular cells plated onto tissue chamber slides were incubated with CCL2 (1 ng/ml) for 24 h, and cells were analyzed by immunocytochemistry. FASLG immunoreactivity was observed as membranous and cytoplasmic (Fig. 5, a and b). In untreated endometrial stromal cells, FASLG immunoreactivity was weak (Fig. 5a). CCL2 induced an increase in both intensity and distribution of FASLG immunoreactivity in endometrial stromal cells (P < 0.05) (Fig. 5, b and g). Similar to stromal cells, CCL2 induced FASLG immunoreactivity in endometrial glandular cells when compared to control in culture (P < 0.05) (Fig. 5, c, d, and g).


Figure 5
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  		FIG. 5. Immunocytochemistry of FASLG expression in endometrial stromal (a, b) and glandular (c, d) cells in culture. Endometrial cells plated on chamber slides were incubated with or without CCL2 (1 ng/ml) for 24 h. a, c) Untreated cells (control). b, d) Cells treated with CCL2 (1 ng/ml). TUNEL assay for the assessment of apoptosis in endometrial stromal cells in culture. Endometrial stromal cells plated on chamber slides were incubated with vehicle only (control) or with CCL2 (1 ng/ml) for 24 h. e) Untreated cells (control). f) Cells treated with CCL2 (1 ng/ml). TUNEL-positive cells are seen with red nuclei. g) The distribution of immunostaining intensity (HSCORE) in endometrial stromal and glandular cells treated with or without CCL2. * represents a significant increase in FASLG expression when compared to control (bars represent mean ± SEM, P < 0.05). Original magnifications: a, b: x100; c, d: x25; e, f: x50

Effect of CCL2 on Endometrial Cell and T-Lymphocyte Apoptosis

Endometrial stromal cells plated onto tissue chamber slides were incubated with CCL2 (1 ng/ml) for 24 h, and cells were analyzed by TUNEL assay for apoptosis. Following CCL2 treatment, relative proportion of apoptotic cells in endometrial stromal cells did not change significantly compared to control cells (Fig. 5, e and f).

Endometrial stromal cells were plated on 12-well plates and treated with various concentrations of CCL2 (0.001, 0.01, 0.1, 1 ng/ml). Following 24 h treatment, supernatant was aspirated, and nonadherent human T lymphocytes (Jurkat cells; 106cell/ml) were added over the stromal cell culture and incubated together for another 24 h without any CCL2. The percentage of apoptotic Jurkat cells labeled with TUNEL was quantified using flow cytometry and compared between Jurkat cells plated alone or plated on endometrial stromal cells with and without CCL2 pretreatment. Percentage of apoptotic Jurkat cells decreased 60% by incubation with endometrial stromal cells compared to Jurkat cells plated alone (P < 0.05) (Fig. 6). Pretreatment of endometrial stromal cells with various concentrations of CCL2 including 0.001, 0.01, 0.1, and 1 ng/ml induced 2.3-, 2.4-, 2.6-, and 2.7-fold increases, respectively, in Jurkat cell apoptosis compared to control (P < 0.05) (Fig. 6).


Figure 6
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  		FIG. 6. Endometrial stromal cells were plated on 12-well plates and treated with various concentrations of CCL2 (0.001, 0.01, 0.1, 1 ng/ml). Following 24 h of CCL2 treatment, supernatant was removed, and Jurkat T-lymphocyte cells were added on the stromal cell culture and incubated with them for another 24 h. Jurkat cells were labeled with TUNEL, and apoptotic cells were quantified by flow cytometry. The percentage of apoptotic Jurkat cells was compared between Jurkat cells plated alone (first bar on the left) or plated on endometrial stromal cells (remaining bars) with and without CCL2 pretreatment. Pretreatment of endometrial stromal cells with CCL2 (0.01, 0.1, 1 ng/ml) induced significant increase in Jurkat cell apoptosis compared to control (P < 0.05) (bars represent mean ± SEM from different experiments; C, Control)

DISCUSSION

Endometriosis is a common gynecologic disorder defined by the presence of viable endometrial glandular and stromal cells outside the uterine cavity. Retrograde menstruation is a universally accepted etiologic factor in most cases. Genetic predisposition, amount of retrograde flow, and dysfunctional immune response are also suggested to enhance subsequent endometrial implantation, invasion, and propagation of endometriosis [23]. Peritoneal natural killer cells and cytotoxic T lymphocytes are suppressed in women with endometriosis [24, 25]. Spontaneous apoptosis in ectopic and eutopic endometrium in endometriosis is less than what is occurring in eutopic endometrium from the healthy controls [1]. Local immunotolerance mechanism by depressed T-lymphocyte activity might be an explanation for decreased spontaneous apoptosis of eutopic and ectopic endometrium in this disorder [1, 26].

FAS/FASLG apoptotic pathway has an essential role in the development of an immune-privileged environment in Sertoli cells [27], the cornea [28], and decidua-trophoblast interface [29, 30]. Tumor escape from immunologic rejection in colon cancer [31], hepatocellular carcinoma [32], and melanoma [33] are also attributable to FASLG-induced apoptosis of activated T lymphocytes. Human endometrial glandular and stromal cells express FASLG and its receptor, FAS, both in vivo and in vitro [19]. Considering 9–12-fold higher levels of FASLG expression in normal endometrial tissue compared to the normal ovarian and cervical tissues, FAS/FASLG system in reproductive system most likely contributes to immune-privileged status in both physiologic and pathologic events in human endometrium [20].

Cytokines and growth factors are proteins with local paracrine and autocrine effects on modulation of regulatory genes involved in various cell functions including apoptosis. We have recently observed upregulation of FASLG protein expression by interleukin-8 (IL8) in human endometrium [34]. Interestingly, IL8 pretreatment of endometrial stromal cells upregulates apoptosis of T lymphocytes plated on these cells [34]. In the present study, CCL2, a monocyte chemotactic and activating factor, demonstrates similar findings with IL8.

We observed significant increase in FASLG protein expression in endometrial stromal cell cultures with CCL2 in a concentration-dependent manner. We did not observe any change in the expression of FAS mRNA in endometrial stromal, glandular, and Ishikawa cells. FASLG mRNA expression did not change with various concentrations of CCL2 in endometrial stromal cells. On the other hand, CCL2 induced FASLG mRNA expression in both endometrial glandular and Ishikawa cells. We have detected similar findings with IL8 in endometrial stromal cells [34]. Immunoelectron microscopy study of FAS and FASLG proteins in human endometrium reveals that these proteins are incorporated into the cell membrane from their localization in Golgi apparatus during the secretory phase, during which they are found to be expressed at higher levels, compared with the proliferative phase [35]. These discrepant results suggest that CCL2 as well as IL8 may regulate FASLG by increasing the rate of its translation and/or decreasing FASLG degradation in endometrial stromal cells. However, the mechanism in endometrial stromal cells may not be the same with endometrial epithelial and Ishikawa cells, as they display separate type of receptors and different signaling activation compared to stromal cells.

We have previously detected the median peritoneal fluid CCL2 levels as 138 pg/ml (range 27–1173) and 352 pg/ml (range 75–6000) in women with minimal to mild endometriosis and with moderate to severe endometriosis, respectively [36]. Thus, significant increases in FASLG protein expression observed in endometrial stromal cell cultures at CCL2 levels are compatible with the ones observed in vivo. In the present study, regulation of FASLG gene by CCL2 is evaluated at both the mRNA and the protein level in endometrial stromal cells in correlation with in vivo CCL2 level. FASLG gene is a fast-transcribed gene, and we have previously shown that FASLG mRNA peaks after 2–3 h of treatment [2]. Moreover, as demonstrated previously, FASLG mRNA in endometrial stromal cells demonstrates a cyclic expression pattern similar to its protein expression [37]. Therefore, we preferred a 2-h incubation period as the optimal incubation time for FASLG mRNA expression.

We observed an increased apoptotic rate in Jurkat cells plated on endometrial stromal cells pretreated with CCL2. Interestingly, endometrial stromal cells without any treatment decreased the apoptotic rate of T lymphocytes suggesting a paracrine mechanism for the antiapoptotic effect of endometrial cells for T lymphocytes. However, CCL2 pretreatment of stromal cells upregulated the apoptotic cell death of T lymphocytes. Induction of FASLG expression by CCL2 may be the one of the reasons inducing apoptosis in activated T lymphocytes. However, CCL2-induced FASLG expression did not increase the apoptosis of endometrial cells suggesting that increased FASLG in endometrial stromal cells does not activate apoptotic signaling in an autocrine manner. It may rather cause development of immunotolerance by increasing apoptosis of leukocytes in a paracrine manner.

There are various data proposing CCL2 as one of the key factors mediating cellular events in the pathogenesis of endometriosis. Circulating levels and activity of CCL2 increase in the plasma of women with endometriosis compared with normal fertile women [38]. CCL2 is elevated in the peritoneal fluid of women with endometriosis compared to those without endometriosis, and medical treatment with GnRH analogues suppresses its levels [9]. Furthermore, medical treatment eliminates the embryo toxicity of the peritoneal fluid from women with endometriosis [8]. CCL2 secretion by eutopic endometrial cells is upregulated in endometriosis [39], and ectopic endometrial cells isolated from endometriotic implants also secrete CCL2 in vitro in response to macrophage-derived cytokines, which are elevated in the peritoneal fluid of patients with endometriosis [38]. Elevated levels of CCL2 in circulation, peritoneal fluid, and endometrium may have a role in the growth and maintenance of ectopic endometrial tissue not only by recruiting leukocytes but also by demonstrating an autocrine/paracrine effect in the development or propagation of endometriosis.

In the current study, we have demonstrated that CCL2 upregulates the FASLG protein in human endometrial cells and induces apoptosis of T lymphocytes. Considering induced FASLG mRNA expression in endometrial glandular and Ishikawa cells, similar effects of CCL2 on these cells remain to be further elucidated. Since soluble components of the peritoneal fluid, including cytokines and growth factors, may be mediators of the pathogenesis of endometriosis, we speculate that elevated peritoneal fluid CCL2 levels may contribute to a relative local immune tolerance around the endometriosis implants by stimulating FASLG-induced apoptosis in activated T lymphocytes and thereby support the survival of ectopic implants.

FOOTNOTES

1 Correspondence: Aydin Arici, Yale University School of Medicine, Department of Obstetrics and Gynecology, 333 Cedar Street, New Haven, CT 06520-8063. FAX: 203 7857134; aydin.arici{at}yale.edu Back

Received: 18 July 2005.

First decision: 15 August 2005.

Accepted: 8 May 2006.

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C. Bredhult, L. Sahlin, and M. Olovsson
Gene expression analysis of human endometrial endothelial cells exposed to op'-DDT
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