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Tumor Necrosis Factor- Promotes Proliferation of Endometriotic Stromal Cells by Inducing Interleukin-8 Gene and Protein Expression

Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan

Address all correspondence and requests for reprints to: Tomio Iwabe, M.D, Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan.

	Abstract

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Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. We and others showed that several cytokine levels, including interleukin-8 (IL-8) and tumor necrosis factor- (TNF), are elevated in the peritoneal fluid of women with endometriosis compared with those in women without endometriosis. We also demonstrated that the addition of IL-8 to the culture medium stimulated the proliferation of cultured endometriotic stromal cells. TNF is a multipotent cytokine that induces IL-8 production in various cell types. Therefore, we hypothesized that TNF may also contribute to the pathogenesis of endometriosis by inducing the production of IL-8. To test this hypothesis, we analyzed the peritoneal fluid concentrations of IL-8 and TNF using enzyme-linked immunosorbent assay (ELISA). We observed a significant correlation between the levels of TNF and IL-8 in the peritoneal fluid of endometriosis patients. We also obtained the endometriotic stromal cells from chocolate cyst linings of the ovary. The expression of the receptors for TNF (TNFR) was examined by RT-PCR. We observed the expression of both TNFR-I and TNFR-II genes in endometriotic stromal cells. The expression of IL-8 gene and protein was analyzed by Northern blot hybridization and enzyme-linked immunosorbent assay, respectively. TNF induced the gene and protein expression of IL-8 in endometriotic stromal cells in a dose-dependent fashion. The addition of TNF promoted the proliferation of the endometriotic stromal cells, and the stimulatory effects of TNF were abolished by adding anti-IL-8 antibody. We demonstrated for the first time that TNF stimulated proliferation of endometriotic stromal cells through induction of IL-8 gene and protein expression. We concluded that the TNF may be one of the essential factors for the pathogenesis of endometriosis.

	Introduction

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ENDOMETRIOSIS, which occurs in 10% of reproductive age women, is characterized by the presence and growth of endometrial-like tissue (gland and stroma) outside the uterus (1). The pathogenesis of endometriosis is poorly understood despite a long history of study of this disease. Recent studies suggest that activated macrophages increase in the peritoneal fluid of patients with this disease. These activated macrophages secrete numerous cytokines that may contribute to the implantation of endometrial cells and the progression of endometriosis (2).

We previously showed that interleukin-6 (IL-6), IL-8, and tumor necrosis factor- (TNF) are significantly elevated in the peritoneal fluid (PF) of patients with endometriosis compared with those in women without endometriosis (2, 3). We also showed the positive correlation between the size and number of red peritoneal endometriotic lesions and the concentrations of IL-6, IL-8, and TNF. Red endometriosis is considered to be the early and active lesions, because vascularization and mitotic activity are shown to be most prominent in red lesions (4, 5). Those findings suggest that cytokines may be involved in the neovascularization of the early stage of endometriosis.

IL-8, which is a chemoattractant for neutrophils and an angiogenic agent, induces the proliferation of human melanoma and glioma cells (6, 7). In our previous studies, IL-8 significantly increased the number of cells and promoted DNA synthesis in endometrial and endometriotic stromal cells, suggesting that IL-8 may promote the progression of endometriosis (3). Other investigators reported that the levels of IL-8 messenger ribonucleic acid (mRNA) and IL-8 protein in the endometrial stromal cells in culture increased in a time- and concentration-dependent manner when the cells were treated with IL-1 and TNF (8). It has also been suggested that IL-8 may act as an autocrine growth factor of endometrial stromal cells (9). Although the researchers proposed a similar regulatory mechanism by which IL-8 promotes ectopic endometrial cell proliferation in endometriosis, these findings were obtained using only endometrial stromal cells. TNF, which is a 17.3-kDa peptide, was first identified as a cytokine secreted by endotoxin-activated macrophages that induced necrosis of tumors (10). TNF is now known as a pluripotent mediator and angiogenic cytokine that promotes the production of other cytokines in various cells. Recently, TNF was shown to enhance the production of IL-8 in human microvascular endothelial cells (11). The pathogenetic significance of TNF and IL-8 in endometriosis has not been fully elucidated.

We conducted this study to test the hypothesis that elevated TNF in PF of patients with endometriosis may contribute to the progression of endometriosis by inducing the production of IL-8. We, therefore, investigated whether TNF induces the expression and production of IL-8 in endometriotic stromal cells. The gene expression of the receptors for TNF (TNFR) was examined by RT-PCR. We also examined the effect of TNF on the proliferation of endometriotic stromal cells in the presence or absence of anti-TNF and anti-IL-8 antibodies.

	Materials and Methods

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Collection of PF

PF was obtained from 93 Japanese women of reproductive age who underwent either laparoscopy during an infertility work-up or laparoscopic cystectomy for ovarian chocolate cysts. Of the 93 patients studied, 61 had pelvic endometriosis, and 32 were free of endometriosis. There were no significant differences in clinical characteristics between patients with and without endometriosis (Table 1). PF was collected with a laparoscopic cannula immediately after the introduction of the laparoscope. Aspiration was performed under direct visualization from the posterior cul-de-sac and anterior vesicouterine fold. Fluid samples were centrifuged at 800 x g for 10 min at 4 C to separate the cell pellet and the supernatant. The cell-free supernatant was then stored at -70 C until assayed.

Chocolate cyst linings of the ovaries in patients with endometriosis [n = 19; obtained in the proliferative phase (n = 10) and luteal phase (n = 9)] were a source of endometriotic tissue. Informed consent for the use of these tissues was obtained from each woman before surgery.

We previously established and employed stromal cell monolayer cultures from ovarian chocolate cysts (3). Briefly, the tissues were minced in Hanks’ Balanced Salt Solution and digested with 0.5% collagenase in DMEM/Ham’s F-12 (DMEM/F-12; 1:1, vol/vol) at 37 C for 60 min. The dispersed cells were filtered through a 70-µm nylon mesh to remove the undigested tissue pieces containing the glandular epithelium. The filtered fraction was separated further from epithelial cell clumps by differential sedimentation at unit gravity, as follows. Cells were resuspended in 2 mL culture medium and layered slowly over 10 mL of the medium in a centrifuge tube. Sealed tubes were placed in an upright position at 37 C in 5% CO₂ in air for 30 min. After sedimentation, the top 8 mL medium were collected. Lastly, the medium containing stromal cells was filtered through 40-µm pore size nylon mesh. Final purification was achieved by allowing stromal cells (which attach rapidly to plates) to adhere selectively to culture dishes for 30 min at 37 C in 5% CO₂ in air. Nonadhering epithelial cells were removed.

Stromal cells were cultured in DMEM/F-12 supplemented with 100 IU/mL penicillin G, 50 mg/mL streptomycin, 2.5 µg/mL amphotericin B, and 10% FBS (vol/vol) at 37 C in 5% CO₂ in air. We used stromal cells in monolayer culture after the first passage.

To confirm the purification of the stromal cells, immunohistochemical analysis of isolated endometriotic stromal cells was performed using cytokeratin (DAKO Corp., Kyoto, Japan) as a marker of epithelial cells, vimentin (DAKO Corp.) as a marker of stromal cells, CD14 (Nichirei, Tokyo, Japan) as a marker of activated macrophages, and factor VIII (DAKO Corp.) as a marker of endothelial cells. The results showed that the purity of stromal was more than 98%.

Proliferation of the endometriotic stromal cells

Proliferation of the cells was determined spectrophotometrically by measuring the incorporation of tetrazolium dye [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay]. The MTT assay used in this study was described previously (3). Briefly, endometriotic stromal cells were diluted with culture medium (DMEM/F-12 with 10% FBS) to a seeding density of 2–3 x 10³/well, suspended in 96-well tissue culture plates (120 µL/well), and preincubated at 37 C for 12 h. The medium was changed to a serum-free medium and contained with 1 mg/mL BSA. Cells were treated continuously with 80 µL of various concentrations of different preparations of TNF (0–100 pg/mL; Genzyme Corp., Cambridge, MA). A monoclonal antibody against TNF (monoclonal mouse antihuman TNF, Genzyme Corp.) or anti-IL-8 antibody (Genzyme Corp.) was used to neutralize the specific effects of TNF. Monoclonal antibody mouse IgG1k (COSMO BIO Co. Ltd, Tokyo, Japan) was used as control. Each plate had one control column (six wells) containing IL-8-free medium. After cells were incubated for 72 or 120 h, 20 µL MTT solution (2.5 mg/mL) were added to each well, and the plates were incubated for another 4 h. Dimethylsulfoxide (150 µL) was added, and the plates were vigorously shaken on a plate shaker to solubilize the MTT-formazan product. Absorbance was measured at 590 nm with a microplate reader (model 450, Bio-Rad Laboratories, Inc., Richmond, CA).

The activity of DNA synthesis was determined by tritiated thymidine incorporation into the cells. Cells (2 x 10⁴/well) were cultured in 24-well dishes for 12 h. The medium was then replaced by DMEM/F-12 without FBS for 12 h, and TNF (0–100 pg/mL) and antibodies were added to the medium. After 48 h, 2 µCi [methyl-³H]thymidine (TRK120, Amersham Pharmacia Biotech, Arlington Heights, IL; SA, 3.77 gigabecquerels/mL) were added and cultured for an additional 24 h. DNA was extracted by cell lysis with sodium hydroxide, and tritiated incorporation was measured by scintillation counting (3).

Collection of supernatant of endometriotic stromal cells

Endometriotic stromal cells were diluted with culture medium (DMEM/F-12 with 10% FBS) to a seeding density of 4 x 10⁴/well and suspended in 24-well tissue culture plates (1000 µL/well). The cells were preincubated at 37 C for 48 h and allowed to reach subconfluence, and the culture medium was exchanged to the serum-free Eagle’s MEM for 24 h. Then TNF (0–100 pg/mL) was added to the medium for 24 h in dose-response experiments. TNF (100 pg/mL) was added for 0–48 h in time-course experiments to evaluate the production of IL-8 in the supernatant. The supernatant was stored at -70 C until assayed by ELISA.

RT-PCR

Total RNA was extracted from the cultured endometriotic stromal cells by the guanidium thiocyanate method according to the manufacturer’s instructions (Isogen, Nippon Gene Co. Ltd., Tokyo, Japan). RT of RNA from cultured endometriotic stromal cells into complementary DNA and PCR amplification was performed using the Gene Amp RNA PCR Core Kit (Perkin-Elmer Corp., Branchburg, NJ) as detailed previously (3). Samples were amplified for 30 cycles of denaturation (30 s at 94 C), annealing (30 s at 60 C), synthesis (1.5 min at 72 C), and primer extension of 5 min at 75 C after each cycle.

For PCR analysis, specific primers and probes for human TNF receptors (TNFR-I and -II) and glycerol-3-phosphate dehydrogenase (as positive control) were used as shown in Table 2. PCR products were resolved on 2% agarose gel with a small molecular weight DNA marker (X174 digested with HaeIII).

Northern blot analysis

After treatment with various concentrations of TNF or exposure times to TNF, total RNA was extracted from the cultured endometriotic stromal cells as described above. The total RNA (20 µg/lane) was size-fractionated by electrophoresis on 1% formaldehyde-agarose gels and transferred to nitrocellulose membrane. The membranes were baked at 80 C for 90 min. Prehybridization was performed for 6 h at 65 C in 0.9 mol/L NaCl, 90 mmol/L Tris (pH 8.3), 6 mmol/L ethylenediamine tetraacetate, 5 x Denhardt’s solution [0.1% polyvinylpyrrolidone, 0.1% BSA, and 0.1% Ficoll 400; Wako, Osaka, Japan], 0.1% SDS, and 0.2 mg/mL salmon sperm DNA. Hybridization was conducted for 12 h at 65 C in buffer that contained a commercial IL-8-specific oligonucleotide probe (40 mer, ON413, Calbiochem, La Jolla, CA) labeled with [-³²P]ATP using an end-labeling kit (Megalabel, Takara, Shiga, Japan). Thereafter, the blots were washed with 6 x SSC (standard saline citrate) and SDS (0.1%, wt/vol) for 15 min at room temperature, twice with 2 x SSC and 0.1% SDS for 15 min at room temperature, and once with 2 x SSC and 0.1% SDS for 20 min at 65 C. Autoradiography of the membranes was performed at -80 C using Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY). The visualization of ethidium bromide-stained 28S ribosomal RNA subunits was used for neutralization. The autoradiographic bands were quantified using an NIH Image program.

Statistical analysis

The SD of the absorbance of MTT assay (percentage of control values) and thymidine incorporation were analyzed by one-way ANOVA, followed by Fisher’s protected least significant difference test. The data are presented as the mean ± SE. P < 0.05 was accepted as indicating statistical significance.

	Results

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Correlation between the concentrations of TNF and IL-8 in PF

We previously found that the levels of TNF and IL-8 in PF were significantly higher in patients with endometriosis than in patients without endometriosis (2, 3). For the present study we further analyzed the relation between TNF and IL-8 in the PF of patients with endometriosis. We found a significant positive correlation between the levels of TNF and IL-8 in the PF in endometriosis patients (r = 0.71; P < 0.001; Fig. 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Correlation between the levels of TNF and IL-8 in peritoneal fluid of women with endometriosis. A significant positive correlation was observed. y = -13.858 + 19.52x; r = 0.71; P < 0.001.

TNF-induced gene and protein production of IL-8 in endometriotic stromal cells

When endometriotic stromal cells were incubated in serum-free medium for 24 h, then treated with various concentrations of TNF, TNF induced the expression of IL-8 mRNA in endometriotic stromal cells (Fig. 2). The levels of IL-8 mRNA in endometriotic stromal cells increased depending upon the concentration of TNF (0–100 pg/mL; Fig. 2A). The increase in IL-8 mRNA was evident at 0.5 h, and the levels became highest at 24 h after the addition of 100 pg/mL TNF (Fig. 2B).

View larger version (42K): [in this window] [in a new window]   Figure 2. Induction of IL-8 mRNA in endometriotic stromal cells by TNF. Subconfluent endometriotic stromal cells were placed in serum-free medium for 24 h before incubation with culture medium containing TNF (0–100 pg/mL) for 24 h (A) or TNF (100 pg/mL) for 0–48 h (B). At the end of incubation period, total RNA was isolated from the cells. IL-8 mRNA was evaluated by Northern analysis of total RNA (20 mg/lane).

View larger version (24K): [in this window] [in a new window]   Figure 3. TNF stimulation of IL-8 production by endometriotic stromal cells in culture. IL-8 production was increased by the addition of TNF (0–100 pg/mL) in a dose-dependent manner for 24 h (A). IL-8 production was increased by the addition of TNF (100 pg/mL) in a time-dependent manner up to 48 h (B). Concentrations of IL-8 in the supernatants were measured by ELISA. The concentrations of IL-8 were expressed per 5 x 104 cells. *, P < 0.05.

Effect of TNF on endometriotic stromal cell proliferation

The MTT assay showed that the number of endometriotic stromal cells was increased in the presence of 20–100 pg/mL TNF (Fig. 4). The stimulatory effects were abolished not only by adding anti-TNF antibody, but also by adding anti-IL-8 antibody. The mouse IgG did not influence the effect of TNF. The addition of 20–100 pg/mL TNF increased DNA synthesis in endometriotic stromal cells (Fig. 5). These results suggest that the action of IL-8 mediates the stimulatory effects of TNF on stromal cell proliferation.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of TNF on the endometriotic stromal cell proliferation. Cell proliferation was determined spectrophotometrically by the incorporation of tetrazolium dye for 72 h (A) and 120 h (B). Results were expressed as a percentage of control values. Anti-TNF antibody (20 ng/mL), anti-IL-8 antibody (50 ng/mL), or mouse IgG (50ng/mL) was added to the culture medium containing TNF (100 pg/mL). Bars represent SEs. *, P < 0.05 vs. the control value. **, P < 0.05 vs. the addition of TNF (100 pg/mL).

View larger version (60K): [in this window] [in a new window]   Figure 5. Effect of TNF on DNA synthesis of endometriotic stromal cells. DNA synthesis was determined by tritiated thymidine incorporation after the addition of TNF for 48 h. Results were expressed as disintegrations per min. Anti-TNF antibody (20 ng/mL), anti-IL-8 antibody (50 ng/mL), or mouse IgG (50 ng/mL) was added to the culture medium containing TNF (100 pg/mL). Bars represent th SEs. *, P < 0.05 vs. the control value. **, P < 0.05 vs. the addition of TNF (100 pg/mL).

Expression of TNF receptor

RT-PCR illustrated the expression of TNFR-I and TNFR-II in cultured endometriotic stromal cells (Fig. 6).

View larger version (34K): [in this window] [in a new window]   Figure 6. RT-PCR revealed TNFR-I and -II mRNA expression in endometriotic stromal cells. The U937 cell lines (lymphoma) were used as a positive control for TNF receptors. Lane 1, U937 cells; lane 2, endometriotic stromal cells; lane 3, reverse transcriptase(-). Glycerol-3-phosphate dehydrogenase (G3PDH) was used as a positive control (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-8 is produced by many types of cells, including monocytes, lymphocytes, neutrophils, endothelial cells, and fibloblasts (12). Other peritoneal sources are macrophages and peritoneal mesothelial cells (12). Arici et al. reported that IL-8 is produced in the human endometrium in vivo, mainly in glandular cells (13), and that IL-8 induces the proliferation of endometrial stromal cells as a potential autocrine growth factor (9). Although they postulated that IL-8 may also play a role in the pathogenesis of endometriosis, no direct evidence concerning the growth-promoting effects of IL-8 have been obtained using endometriotic tissues. The results of our previous and present studies clearly demonstrated that IL-8 exerts its growth-promoting actions in normal endometrium as well as in endometriotic cells.

TNF is one of the pleiotropic cytokines, which exert cytotoxic as well as differentiation and growth modulatory activities on many different target cells. TNF activities are elicited by binding to two distinct receptors, type I (p55, TNFR-I) and type II (p75, TNFR-II), that are ubiquitously coexpressed on almost all cell types in various proportions (14, 15). Studies using TNFR-I- and TNFR-II-selective agonists showed that the majority of known TNF activities are mediated by TNFR-I. In contrast, TNFR-II mainly has an accessory function to TNFR-I signaling, either by enhancement and modulation of the TNFR-I in intracellular transduction pathways or by ligand passing (14). In mouse fibloblasts, distinct but overlapping roles of TNFR-I and TNFR-II are found, and a different regulation mechanism of signal transduction pathways under the control of both TNFRs is suggested (14). The coexpression of transcripts for TNFR-I and -II in the present study is consistent with a previous study that demonstrated that immunoreactive TNFR-I and -II are expressed in endometrium throughout the entire menstrual cycle (16).

The functional role of TNF in endometrial tissue is still unknown. However, TNF is thought to be an important contributor to cell turnover and normal tissue homeostasis in the endometrium. Supporting this, ovarian steroids, such as 17ß-estradiol and progesterone, regulate the expression of epithelial and stromal cell TNF in mouse and human endometrium (17, 18). In situ hybridization of TNF signals in both epithelial and stromal cells increase in intensity during the proliferative phase, whereas peak signal intensity is observed during the late secretary phase (17).

We also know that TNF has both growth inhibitory and growth stimulatory effects depending on its concentration and cell type (14, 19). Although a low dose of TNF reportedly induced angiogenesis, a high dose of TNF, in contrast, inhibited angiogenesis (20). Low doses of TNF (10–1000 pg) induced angiogenesis, which was maximum at 100 pg, whereas high doses (1 and 5 mg) inhibited it. The mitogenic or antiproliferative effects of TNF on neoplastic endometrial epithelial cells were shown to depend on the dose of this cytokine (21). A moderate (10–20%) growth stimulatory effect could be demonstrated in the picogram per mL concentration range, whereas a more dramatic (up to 45%) inhibitory effect was seen in the nanogram per mL concentration range (21). TNF, in a dose- and time-dependent fashion, reportedly inhibits proliferation and induces apoptosis in an endometrial epithelial cell line (16). The cell death induced by TNF in epithelial cells was associated with the characteristic morphological changes in apoptosis and fragmentation of DNA into oligonucleosome size fragments (16).

Peritoneal fluid levels of TNF were found to vary from 5–300 pg/mL in women with endometriosis (2, 22). Endometriotic tissues may be growth-stimulated in a low dose, picogram per mL concentration range, TNF environment. We also examined TNF effects on the eutopic endometrial stromal cells; the results of gene and protein expression of IL-8 were similar to those obtained using ectopic endometrial stromal cells (data not shown). Because tissue levels of this cytokine are currently unknown, TNF could potentially be involved in up- or down-regulatory endometrial cell proliferation.

It is well known that epithelial and stromal cells derived from the endometrium respond poorly to estrogen in vitro, whereas these endometrial cells are remarkably growth stimulated by estrogen in vivo (23), suggesting that a paracrine interaction between stromal and epithelial cells plays an important role in estrogen-induced growth of endometrial cells (24). Therefore, it is likely that the growth regulation in vivo of endometrial and endometriotic cells is controlled by a complex combination of cytokine and growth factors.

In conclusion, TNF action mediated by IL-8 may contribute to the pathogenesis of endometriosis by promoting the growth of endometriotic cells. TNF in the peritoneal fluid may be an essential factor in the pathogenesis of endometriosis.

Received February 26, 1999.

Revised September 22, 1999.

Accepted October 18, 1999.

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