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Original Article |
Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06520-8063
Address all correspondence and requests for reprints to: Aydin Arici, M.D., Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8063. E-mail: . aydin.arici{at}yale.edu
Abstract
Numerous cytokines and growth factors are synthesized in the endometrium. IL-8 is one of these cytokines regulating endometrial function. It is a neutrophil chemoattractant/ activating factor and a potent angiogenic agent. IL-8 is elevated in the peritoneal fluid of women with endometriosis. We have previously demonstrated a direct proliferative effect of IL-8 on endometrial stromal cells. We hypothesized that increased levels of IL-8 in the endometriotic environment could up- regulate Fas ligand (FasL) expression in endometrial cells and may be relevant for the development of a relative local immunotolerance in endometriosis by inducing apoptosis of cytotoxic T lymphocytes. To test our hypothesis, we studied the in vitro regulation of FasL expression and apoptosis by IL-8 in endometrial cells. Western blot analysis in endometrial stromal, glandular, and Ishikawa cells revealed that IL-8 up- regulated FasL protein expression in these cells. By semiquantitative RT-PCR analysis, IL-8 does not alter the expression of either Fas or FasL mRNA levels in these cells. Immunocytochemistry results from endometrial stromal cells treated with IL-8 demonstrated an up-regulation of FasL protein expression. IL-8 decreased apoptosis rate in endometrial stromal cells as evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. We observed an increased apoptotic rate in Jurkat (T lymphocyte line) cells plated on endometrial stromal cells previously treated with IL-8. We speculate that increased FasL expression by IL-8 may induce apoptosis of T lymphocytes and thus produce a local immunotolerant environment for the development of ectopic implants.
APOPTOSIS, THE PROGRAMMED cell death, is an essential regulator of cell turnover in 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 that have stimulatory or inhibitory effects on cell death. Fas ligand (FasL), a mediator of apoptosis in differentiated cells and embryonic development, interacts with its receptor Fas and induces apoptosis through autocrine or paracrine signaling. FasL is expressed in nonimmune cells, mainly from immune-privileged tissues including testis, cornea, trophoblast, and cancer cells, which implies that the Fas-FasL system may be essential in the mechanism underlying this immune privileged status (2, 3, 4, 5, 6). Similarly, our recent findings on the regulation of FasL by macrophage-derived growth factors and adhesion to extracellular matrix in endometrial stromal cells suggest a role for FasL in the pathogenesis of endometriosis (7, 8).
Peritoneal fluid of women with endometriosis has increased neutrophil chemotactic activity. IL-8 is a strong candidate for this event as a chemoattractant and activating chemokine for neutrophils and as a potent angiogenic agent. IL-8 is produced by a number of cell types, including monocytes, fibroblasts, mesothelial cells, and endometrial stromal and glandular cells (9, 10, 11, 12, 13). IL-8 is elevated in the peritoneal fluid of women with endometriosis, and levels of this cytokine correlate with the severity of the disease (11, 14). Increased IL-8 concentration in the peritoneal fluid in endometriosis suggests that it may have an adverse affect on the local environment and facilitate further growth of endometriotic implants.
We have previously demonstrated the cycle-dependent IL-8 expression in endometrial cells in vivo and the direct proliferative effect of IL-8 on endometrial stromal cells (15). However, the effect of IL-8 on apoptosis by way of Fas/FasL system in human endometrial cells has not been investigated so far. We hypothesized that increased levels of IL-8 in the endometriotic environment could up-regulate FasL expression in endometrial cells and may be relevant for the development of a relative local immunotolerance in endometriosis. To test our hypothesis, we studied the in vitro regulation of FasL expression and FasL-mediated apoptosis by IL-8 in endometrial stromal cells and T lymphocytes in culture.
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. The mean age of the patients was 41.6 yr (range 34–52 yr). Diagnoses of patients were uterine fibroid (n = 3) and voluntary sterilization by tubal ligation (n = 3). None of the patients had any visual evidence of endometriosis. Tissues were placed in Hank’s balanced salt solution 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 on at least 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 (13). Ishikawa (well-differentiated endometrial adenocarcinoma cell line) cells were kindly provided to us by Dr. Richard Hochberg (Yale University). Cells were treated with various concentrations of IL-8 (R&D Systems, Minneapolis, MN) (0.001–1 ng/ml) for 24 h for protein analysis (by Western blot and immunocytochemistry) and for 2 h for mRNA analysis (by RT-PCR). FasL gene is a fast-transcribed gene, and we have previously shown that FasL mRNA peaks after 2–3 h of treatment (7).
Jurkat cells
Nonadherent human T lymphocyte cells (Jurkat cells; kindly provided to us by Dr. Gil Mor, Yale University) were maintained in continuous culture. Cells were plated in RPMI 1640 medium (Life Technologies, Inc., Rockville, MD) and fetal bovine serum (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.
FasL RT-PCR
Total RNA was extracted by Trizol reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Semiquantitative RT-PCR was performed as described previously (16). The primers used for amplification of FasL, Fas, and glycerol-3-phosphate dehydrogenase (G3PDH) have been recently described (17, 18, 19) and have the following sequences: FasL primers yielding a 311-bp reaction product (sense: 5'-ACACCTATGGAATTGTCCTGC-3'; antisense: 5'-GACCAGAGAGAGCTCAGATAC G-3'); Fas primers yielding a 266-bp reaction product (sense: 5'-CACTATTGCTGGAGTCAT G-3'; antisense: 5'-CTGAGTCACTAGTAATGTCC-3'); and G3PDH primers yielding a 788-bp product (sense: 5'-GGTCGGAGTCAACGGATTTGGTCG-3'; antisense: 5'-CTTCCGACGCCTGCTTCACCAC-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 G3PDH band to semiquantitatively compare values between samples.
FasL Western blot analysis
Protein extraction and Western blot analysis were performed as described previously (16). Incubation with mouse antihuman FasL monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY) diluted at 1:1,000 was performed for 1 h and thereafter washed with PBS-T buffer. The membrane was further incubated for 1 h with peroxidase-labeled antimouse IgG (Vector Laboratories, Inc., Burlingame, CA) diluted at 1:10,000. The immunoblot was developed using a chemiluminescent kit (NEN Life Science Products, Boston, MA).
Equal loading of proteins (10 µg) in each lane was confirmed by staining the membrane with Ponceau 2S (Sigma, St. Louis, MO). Ponceau red signals and autoradiographic bands for FasL were quantified by a digital imaging and analysis system (AlphaEase, 
Innotech Corp., San Leandro, CA) and a laser densitometer (Molecular Dynamics, Inc., Sunnyvale, CA), respectively. FasL expression was normalized by dividing the arbitrary densitometry units for FasL to the amount of Ponceau red staining for each band.
Immunocytochemistry
Endometrial stromal cells were grown to preconfluence on four-chamber slides (Falcon, Franklin Lakes, NJ). Following treatments, immunocytochemistry was performed as described previously (16). Immunocytochemical staining intensity was ranked from 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 x50 original magnification, and the percentage of the cells for 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 assay
Apoptosis in endometrial stromal cells plated on tissue chamber slides, with or without IL-8 treatment, was detected by enzymatic labeling of DNA strand breaks using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). TUNEL 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, in which 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 before permanent mounting. Quantification of apoptotic cells was accomplished by counting the number of apoptotic bodies sighted in the microscopic field and 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’s F12/DMEM (1:1 vol/vol) and FBS (10% vol/vol) and were allowed to reach confluence. Confluent cells were treated with serum-free phenol red-free media for 24 h and then treated with various concentrations of IL-8 (0.001–10 ng/ml). Following 24-h treatment with and without IL-8, the media was removed, and nonadherent human T lymphocytes (Jurkat cells; 106 cell/ml) were plated either alone or on endometrial stromal cells. After 24-h incubation, Jurkat cells in suspension were removed and labeled by TUNEL as described above. TUNEL-positive and -negative cells were quantified using flow cytometry. The flow cytometry analysis was performed using FACS IV flow cytometer (Becton Dickinson and Co., San Jose, CA) gated on lymphocytes. A minimum of 10,000 cells was analyzed in each sample. Cells were excited at 488 nm, and fluorescence above 590 nm was recorded. Apoptotic cells were considered to be those that had lower fluorescence and somewhat lower forward angle light scatter than cell debris.
Statistical analyses
Data for densitometry of FasL protein, RT-PCR bands, and immunocytochemistry and TUNEL scores were normally distributed (as determined by Kolmogorov-Smirnov test). Thus, ANOVA and post hoc Tukey test for pair-wise multiple comparisons were used for statistical analysis. P value less than 0.05 was considered to be significant. Statistical calculations were performed using Sigmastat for Windows, version 2.0 (Jandel Scientific Corp., San Rafael, CA).
Results
Regulation of FasL protein expression by IL-8 in endometrial cells in culture
To investigate the regulation of FasL protein expression by IL-8, endometrial stromal cells were incubated with various concentrations of IL-8 (0.001–1 ng/ml) for 24 h, and FasL protein levels were analyzed by Western analysis. Untreated endometrial stromal cells expressed FasL protein (Fig. 1
). IL-8 treatment stimulated the FasL expression in a concentration-dependent manner. IL-8 treatments, 0.001, 0.01, 0.1, and 1 ng/ml, induced 17%, 61%, 70%, and 103% increase, respectively, in FasL protein levels (P < 0.05 between control and all IL-8 concentrations) (Fig. 1).
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) and Ishikawa cells (Fig. 2B) expressed FasL protein. IL-8 treatment (1 ng/ml) up-regulated the FasL protein expression by 33% and 27%, compared with the control in glandular and Ishikawa cells, respectively (P < 0.05).
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, a and b). IL-8 induced an increase in both intensity and distribution of FasL immunoreactivity in endometrial stromal cells, compared with untreated cells (P < 0.05) (Fig. 3b vs. 3a).
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To evaluate whether the up-regulatory effect of IL-8 on FasL protein expression was secondary to an increase in FasL mRNA levels, we measured FasL mRNA levels by RT-PCR analysis. We also investigated FasL receptor (Fas) mRNA expression in human endometrial cells. Endometrial stromal cells were incubated with various concentrations of IL-8 (0.001–1 ng/ml) for 2 h. IL-8 did not induce any concentration-dependent alterations in FasL mRNA expression in human endometrial stromal cells (Fig. 4A). Untreated endometrial stromal cells express Fas mRNA (Fig. 4, B and C). We did not observe any variation in Fas levels by increasing concentrations of IL-8 (Fig. 4B). Fas mRNA expression did not change with IL-8 treatment (1 ng/ml) at various time intervals between 0 and 24 h (Fig. 4C).
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). Similarly, we did not observe any changes in Fas mRNA levels with IL-8 treatment (1 ng/ml) in both cell types (Fig 5B).
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Endometrial stromal cells plated in tissue chamber slides were incubated with IL-8 (1 ng/ml) for 24 h, and cells were analyzed by TUNEL assay for apoptosis. Following IL-8 treatment, the ratio of apoptotic endometrial stromal cells was decreased (Fig. 6b), compared with untreated cells (P < 0.05) (Fig. 6a).
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).
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Endometriosis is defined as the presence of viable endometrial glandular and stromal cells outside the uterine cavity. Dysfunctional immune response is postulated in the pathogenesis of endometriosis (20). Apoptosis, as a regulator of cellular turnover in the immune system, may represent one of the modulatory factors for the survival of ectopic implants. We have previously shown that endometrial glandular and stromal cells express FasL both in vivo and in vitro (16). Besides FasL expression, we have also observed the expression of its receptor, Fas, in endometrial stromal and glandular cells (16). Peritoneal natural killer cells and cytotoxic T lymphocytes are suppressed in women with endometriosis (21, 22). Spontaneous apoptosis in ectopic and eutopic endometrium in endometriosis is less than in the eutopic endometrium from the healthy controls (1). Relative immunotolerance around the ectopic implants is an important step in the pathogenesis of endometriosis (20). Local immunotolerance as evidenced by depressed T lymphocyte activity may explain the decrease in spontaneous apoptosis of eutopic and ectopic endometrium in this disorder (1, 23).
The Fas/FasL system has been shown to have an essential role in the development of an immune-privileged environment in Sertoli cells (2), the cornea (3), and at the decidua-trophoblast interface (5, 24). Tumor escape from immunologic rejection in colon cancer (25), hepatocellular carcinoma (26), and melanoma (27) is also attributable to the FasL-induced apoptosis of activated T lymphocytes. Fas/FasL-dependent immune regulatory pathways are also reported to down-regulate the early processes of neutrophil-binding activity that precede apoptosis in human retinal pigment epithelial cells infected by human cytomegalovirus (28). Considering that FasL expression in normal endometrial tissue is 9- to 12-fold higher, compared with the normal ovarian and cervical tissues, Fas/FasL system contributes to immune-privileged status in both physiologic and pathologic events in human endometrium (17).
Cytokines and growth factors are proteins with paracrine and autocrine effects on cell growth, differentiation, and cell functions. IL-8, a potent mediator of angiogenesis, induces chemotaxis and activation of granulocytes. It also stimulates proliferation of epidermal, melanoma, smooth muscle, and endometrial stromal cells (15, 29, 30, 31).
IL-8 is elevated in the peritoneal fluid of women with endometriosis, compared with those without endometriosis, and these levels correlate with the severity of the disease (11, 14). In the peritoneal cavity, mesothelial cells, peritoneal macrophages, follicular fluid, and endometrial cells are potential sources of this cytokine (11, 13, 32). Elevated levels of peritoneal fluid IL-8 are suggested to have a role in the growth and maintenance of ectopic endometrial tissue not only by the stimulation of leukocytes to secrete growth factors and cytokines but also by a direct proliferative effect on endometrial stromal cells (15). IL-8 acts as an autocrine/paracrine growth factor in the endometrium and may also play a role in the development or propagation of endometriosis.
The mean concentration of IL-8 in the peritoneal fluid of women with moderate or severe endometriosis is 0.5 ng/ml (11). We have observed significant increases in FasL protein expression in endometrial stromal cell cultures at IL-8 concentrations compatible with the ones observed in vivo. On the basis of the proliferative effect of IL-8 on endometrial cells, we have previously suggested that elevation of IL-8 levels in peritoneal fluid might create an environment favorable for the implantation and/or growth of ectopic menstrual debris (15). In the present study, we did not observe any change in the expression of Fas mRNA in endometrial stromal, glandular, and Ishikawa cells. TUNEL assay results demonstrated that the increase in FasL expression with IL-8 treatment in endometrial stromal cells corresponded to a decreased apoptosis in these cells. We have observed an increased apoptotic rate in Jurkat cells plated on endometrial stromal cells pretreated with IL-8. Interestingly, untreated endometrial stromal cells decreased the apoptotic rate of T lymphocytes, suggesting a paracrine antiapoptotic effect of endometrial cells for T lymphocytes. IL-8 pretreatment of stromal cells increased the apoptotic cell death of Jurkat T lymphocytes. This observation supports the hypothesis that induction of FasL protein expression by IL-8 leads to the development of local immune tolerance in endometriosis.
We have demonstrated a concentration-dependent increase in the protein expression of FasL by IL-8; however, we did not observe any increase in FasL mRNA levels. Immunoelectron microscopy study of Fas and FasL proteins in human endometrium revealed 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 (33). These discrepant results suggest that IL-8 may regulate FasL by increasing the rate of its translation and/or decreasing FasL degradation in the proteosome.
In conclusion, we have shown that IL-8 up-regulates the FasL protein in human endometrial cells. Soluble components of the peritoneal fluid, including cytokines and growth factors are involved in the pathogenesis of endometriosis. We speculate that elevated peritoneal fluid IL-8 levels, by stimulating FasL-induced apoptosis in activated T lymphocytes, contribute to an immune-privileged environment around the endometriosis implants supporting their survival.
Acknowledgments
We thank Ozlem G. Kayisli, M.Sc., and Nathalie Bonafe, Ph.D., for their technical support.
Footnotes
Present address for J.A.G.-V.: IVI-Madrid, C/Santiago de Compostela 88, 28035 Madrid, Spain.
Abbreviations: FasL, Fas ligand; G3PDH, glycerol-3-phosphate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling.
Received January 7, 2002.
Accepted April 18, 2002.
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