help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0749
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirata, T.
Right arrow Articles by Taketani, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirata, T.
Right arrow Articles by Taketani, Y.
Endocrinology Vol. 149, No. 3 1260-1267
Copyright © 2008 by The Endocrine Society

Interleukin (IL)-17A Stimulates IL-8 Secretion, Cyclooxygensase-2 Expression, and Cell Proliferation of Endometriotic Stromal Cells

Tetsuya Hirata, Yutaka Osuga, Kahori Hamasaki, Osamu Yoshino, Mika Ito, Akiko Hasegawa, Yuri Takemura, Yasushi Hirota, Emi Nose, Chieko Morimoto, Miyuki Harada, Kaori Koga, Toshiki Tajima, Shigeru Saito, Tetsu Yano and Yuji Taketani

Department of Obstetrics and Gynecology (T.H., Y.O., K.H., O.Y., A.H., Y.Takem., Y.H., E.N., C.M., M.H., K.K., T.T., T.Y., Y.Taket.), Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan; and Department of Obstetrics and Gynecology (M.I., S.S.), University of Toyama, Toyama 930-0194, Japan

Address all correspondence and requests for reprints to: Yutaka Osuga, M.D., Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: yutakaos-tky{at}umin.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-17A is secreted from Th17 cells, a discovery leading to revision of the mechanism underlying the role of Th1/Th2 in the immune response. Strong evidence suggests that immune responses associated with inflammation are involved in the pathogenesis of endometriosis. In the present study, we first demonstrated that the presence of Th17 cells in peritoneal fluid of endometriotic women by flow cytometric analysis and IL-17A-positive cells in endometriotic tissues by immunohistochemistry. To investigate the role of IL-17A in the development of endometriosis, we then studied the effect of IL-17A on IL-8 production, cyclooxygensase-2 expression, and cell proliferation of cultured endometriotic stromal cells (ESCs). IL-17A enhanced IL-8 secretion from ESCs in a dose-dependent manner. The IL-17A-induced secretion of IL-8 from ESCs was suppressed by anti-IL-17 receptor A antibodies or inhibitors of p38 MAPK, p42/44 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase. Addition of TNF{alpha} synergistically increased IL-17A-induced IL-8 secretion from ESCs. IL-17A also enhanced the expression of cyclooxygensase-2 mRNA and proliferation of ESCs. IL-17A may play a role in the development of endometriosis by stimulating inflammatory responses and proliferation of ESCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIOSIS, DEFINED BY the presence of viable endometriotic tissue outside the uterus, is an enigmatic disease. Implantation and growth of endometrial cells from the overflow of menstrual blood into the peritoneal cavity is a widely accepted hypothesis for the pathogenesis of endometriosis. Although retrograde menstruation is observed in most women, only a fraction develop endometriosis. Multiple lines of evidence suggest that inflammation and immune responses play a pivotal role in the pathogenesis of endometriosis (1, 2).

Recent expeditious understanding of Th17 cells substantially revised the conventional Th1/Th2 hypothesis of T cell immunology (3, 4, 5). Th17 cells, along with Th1 and Th2 cells, differentiate from naïve T cells. Interferon-{gamma} and IL-4 are specific cytokines secreted from Th1 and Th2 cells, respectively. IL-17A is a representative cytokine secreted from Th17 cells. IL-17A, a disulfide-linked homodimeric glycoprotein consisting of 155 amino acids, has been described in various immune responses and inflammation (6).

Elevated levels of inflammatory substances and cells in the peritoneal fluid (PF) of women with endometriosis is highly indicative of pelvic cavity inflammation (7). A recent study demonstrated that increases in the level of IL-17A in PF correlate with the severity of endometriosis and infertility associated with this disorder (8).

In view of the emerging significance of IL-17A in a novel paradigm in immunology, we investigated the role of IL-17A in endometriosis. In the present study, we first examined presence of IL-17A immunoreactive cells in endometriotic tissues. In particular, we demonstrated Th17 cells in peritoneal fluid mononuclear cells (PFMCs). Thereafter, we studied effects of IL-17A on endometriotic stromal cells (ESCs). To this end, we mainly measured production of IL-8 in ESCs because IL-8 is a possible key player in endometriosis. It has been reported that IL-8 concentrations are increased in PF of endometriotic women, and IL-8 stimulates proliferation, matrix metalloproteinase activity, invasive capability, Fas ligand protein expression, and adhesion capability of endometrial stromal cells (9, 10, 11). We also examined cooperative effect of IL-17A to TNF{alpha}, another proinflammatory cytokine important for the disease (12). Finally, we studied direct proliferative effect of IL-17A on ESCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and materials
Human recombinant IL-17A, TNF{alpha}, goat antihuman IL-17A, goat IgG control, mouse antihuman IL-17 receptor (IL-17RA) and mouse IgG1 isotype control were purchased from R&D systems (Minneapolis, MN). Antibodies of human CD3, CD4, and mouse IgG1 isotype control and Goldistop were purchased from BD Bioscience (San Jose, CA). Antihuman IL-17A antibody and isotype control IgG1 were purchased from eBioscience (San Diego, CA). Antibodies of human p38 MAPK, phospho p38 MAPK, p42/44 MAPK, phospho p42/44 MAPK, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and phospho SAPK/JNK were purchased from Cell Signaling Technology (Beverly, MA). MAPK inhibitors SB202190, PD98059, and SP600125 were from Calbiochem (La Jolla, CA). Collagenase was obtained from WAKO (Osaka, Japan). The antibiotic mixture of penicillin, streptomycin, amphotericin B phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma (St. Louis, MO). Charcoal-stripped fetal bovine serum (FBS) was from Hyclone (Logan, UT). DMEM/Ham’s F12 (DMEM/F12) and deoxyribonuclease I were from Invitrogen (Rockville, MD).

Patients and samples
Endometriotic tissues and PF were obtained from patients with ovarian endometriomas undergoing laparoscopy. The severity of the disease was determined according to the revised American Society for Reproductive Medicine classification. The final diagnosis was confirmed by histopathological examination. Laparoscopic excision of ovarian endometrioma was performed as follows. After inspection of the pelvis, the ovary was freed from any adhesions. The cyst wall of endometrioma was stripped away from the normal ovarian tissue gently and completely. Endometriotic tissue were obtained from the excised cyst wall of ovarian endometrioma and transported to the laboratory in DMEM/F12 on ice under sterile condition. The PF was obtained from the patients, who were diagnosed as stage III or IV. All patients had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before surgery. The tissues collected under sterile conditions were processed for the generation of primary cell cultures. The peritoneal fluid was collected under sterile conditions before any manipulative procedure. PFMCs were collected as previously described (13, 14). Briefly, the collected PF was centrifuged at 200 x g for 5 min, and the supernatants were removed. The cell pellet was resuspended in PBS, layered onto Ficoll-Paque (Amersham Biosciences, Piscataway, NJ), and centrifuged at 150 x g for 30 min. PMFCs were recovered from the interface.

The experimental procedures were approved by the Institutional Review Board of the University of Tokyo and signed informed consent for use of the endometriotic tissue was obtained from each patient.

Immunohistochemistry
Paraffin-embedded specimens were sliced at a 5-µm thickness. These slide sections were deparaffinized and rehydrated. Antigens were retrieved by buffer at 98 C. Endogenous peroxidase was blocked by incubation for 20 min with a solution of 1% hydrogen peroxidase. Immunohistochemical tissue labeling was performed using the avidin-biotin peroxidase methods. After blocking with normal rabbit serum (Vector Laboratories, Burlingame, CA), the sections were incubated with 1 µg/ml anti IL-17A antibody or goat IgG for 60 min at room temperature and incubated with avidin-biotin peroxidase complex (Vectastain Elite; Vector Laboratories), according to the manufacture’s instructions. The pattern of immunoreactivity was visualized using Vector VIP (Vector Laboratories) as substrate. All sections were counterstained with hematoxylin and evaluated under a light microscope.

Flow cytometric analysis
PFMCs were resuspended in 10% FBS RPMI 1640 medium. The cells were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) for 5 h in the presence of Goldistop. Cells were firstly stained extracellularly with anti-CD3 and anti-CD4 antibodies, then fixed and permeabilized with Perm/Fix solution (eBioscience), and finally stained intracellularly with anti-IL-17A antibody. Samples were analyzed using FACSCalibur (BD Bioscience) and Cell Quest Pro (BD Bioscience).

Isolation and culture of ESCs
The isolation and culture of human ESCs were performed as described previously (15, 16). Fresh endometriotic tissue collected in sterile medium was rinsed to remove blood cells. The tissue was minced into small pieces and incubated in phenol-red free DMEM/F12 containing type I collagenase (0.25%) and deoxynuclease I (15 IU/ml) for 120 min at 37 C. The resultant dispersed endometriotic cells were separated by filtration through a 100-µm nylon cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ) and 70 µm nylon cell strainer. Stromal cells remaining in the filtrate were collected by centrifugation, resuspended in phenol-red free DMEM/F-12, plated onto 100-mm dishes (Iwaki, Asahi technology Co., Tokyo, Japan), and allowed to adhere at 37 C for 12 h. At the first passage, the cells were plated into six-well plates at 4 x 105 cells/well, 12-well plates at 2 x 105 cells/well, or 48-well plates at 1 x 105 cells/well. Once the cells reached confluence, in 2 or 3 d, they were used for experiments. The purity of ESCs was greater than 95%, according to positive cellular staining for vimentin and negative cellular staining for cytokeratin or CD45, CD68, and von Willebrand factor.

Treatment of the cells
First, to examine the effect of IL-17A on IL-8 production, the cells were incubated for 24 h in 5% FBS DMEM/F12 medium with varying doses of IL-17A. Second, to examine the effect of the anti-IL-17RA antibody, ESCs were preincubated in 5% FBS DMEM/F12 with the antibody for 30 min and then stimulated with 10 ng/ml IL-17A for 24 h. Third, to evaluate the effect of IL-17A on MAPK phosphorylation in ESCs, the cells were incubated with 5% FBS media with IL-17A (10 ng/ml) for different time periods. Fourth, to evaluate the effect of MAPK inhibitors, the cells were preincubated with each MAPK inhibitor for 1 h before the addition of IL-17A and then incubated for 24 h. Fifth, to evaluate the synergic effect of IL-17A and TNF{alpha} on IL-8 secretion, the cells were stimulated with varying doses of IL-17A (1–100 ng/ml) with or without TNF{alpha} (1 ng/ml). Finally, for time-course experiments examining the expression of IL-8 and cyclooxygenase (COX)-2 mRNA, ESCs were incubated with 5% FBS medium with IL-17A (10 ng/ml) for different time periods up to 24 h.

RNA extraction, reverse transcription, and PCR of IL-17A, IL-17RA, IL-8, and COX2
We extracted total RNA from endometriotic tissues and PFMCs by the acid guanidinium-phenol-chloroform method using ISOGEN (Nippongene, Toyama, Japan). Using an RNeasy minikit (QIAGEN, Hilden, Germany), we extracted total RNA from ESCs cultured in a 12-well plate. One microgram of total RNA was reverse transcribed in a 20-µl vol using an RT-PCR kit (TOYOBO, Osaka, Japan). Standard PCR was performed using Rever Tra Dash (TOYOBO) according to the manufacturer’s instructions. Human glyceraldehyde dehydrogenase (GAPDH) primers (TOYOBO) were used as a positive control for RNA levels. Primer pairs for IL-17A, IL-17RA, IL-8, and COX2 used in PCR are shown in Table 1Go. PCR conditions for amplification were 30 cycles (for IL-17RA, IL-8, COX-2, and GAPDH) or 35 cycles (for IL-17A) at 98 C for 10 sec, 60 C for 2 sec, and 74 C for 14 sec. Each PCR product was purified with a QIAEX II gel extraction kit (QIAGEN), and the identity of PCR products was confirmed using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Primer pairs used for PCR analysis

 
Real-time quantitative PCR
Real-time quantitative PCR was performed as reported previously (17). To assess IL-8 and COX2 mRNA expression, real-time quantitative PCR and data analysis were performed using Light Cycler (Roche Diagnostics GmbH, Mannheim, Germany). Expression of IL-8 and COX2 mRNA was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. The primers for IL-8 and COX2 were the same as those used for standard PCR. PCR conditions were as follows: for IL-8, 40 cycles at 95 C for 10 sec, 66 C for 10 sec, 72 C for 11 sec; for COX2, 30 cycles at 95 C for 10 sec, 66 C for 10 sec, 72 C for 13 sec; for GAPDH, 30 cycles at 95 for 10 sec, 64 C for 10 sec, 72 C for 18 sec. All PCR conditions were followed by melting curve analysis.

Counting cell numbers
Cell counting was performed using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), according to the manufacturer’s instruction.

5-Bromo-2'-deoxyuridine (BrdU) incorporation assay
BrdU incorporation assay was performed as reported previously (15, 18, 19). The effects of IL-17A and IL-8 on the proliferation of ESCs was examined by measuring BrdU incorporation into DNA using the Biotrak cell proliferation ELISA system (Amersham Biosciences) according to the manufacture’s instructions. Briefly, ESCs were seeded into a 96-multiwell plate (Becton Dickinson) at a density of 5 x 104 cells/well in 100 µl of the culture medium. After 24 h, cells were stimulated with IL-17A or IL-8 for 48 h. Then 10 µl BrdU solution were added and incubated at 37 C for an additional 2 h. After removing the culture medium, the cells were fixed and the DNA denatured by the fixative. The peroxidase-labeled anti-BrdU bound to the BrdU incorporated in the newly synthesized, cellular DNA. The immune complexes were detected by the subsequent substrate reaction, and the resultant color was read at 450 nm in the DigiScan microscope reader (ASYS Hitech GmbH, Eugendorf, Austria).

Immunocytochemistry
ESCs were cultured in 16-well chamber slides (Nunc, Naperville, IL) in a humidified 5% CO2-95% air environment and allowed to grow to approximately 50% confluence. The cells were fixed with cold methanol/acetone at –20 C for 20 min, washed twice with PBS, blocked for 20 min with 5% bovine serum in PBS, and incubated with an anti-IL-17RA antibody (10 µg/ml in 1.5% BSA in PBS) or IgG2b mouse IgG isotype control for 40 min at room temperature. After three washes with PBS, the slides were incubated with peroxidase-conjugated secondary antibody (goat antimouse Envision plus; Dako, Glostrup, Denmark) for 30 min at room temperature. Staining was detected with the diaminobenzidine chromogen after 3 min. All slides were counterstained with hematoxylin and evaluated under a light microscope.

Western blotting
Cultured cells in 6-well plates were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue. The lysates were further diluted with lysis buffer to give a final concentration of 1 mg total protein per milliliter. Samples were resolved using 10% SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane and incubated with rabbit antibodies to p38 MAPK (1:1000), phospho-specific p38 MAPK (1:1000), p42/44 MAPK (1:1000), phospho-specific p42/44 MAPK, SAPK/JNK (1:1000), phospho-specific SAPK/JNK (1:1000) as primary antibodies, and antirabbit horseradish peroxidase antibody (1:1000) as a secondary antibody. Immune complexes were visualized by use of the ECL Western blotting system (Amersham Biosciences).

Measurement of IL-8
The concentration of IL-8 in conditioned media was measured using a specific ELISA kit (Genzyme/Techne, Minneapolis, MN). The sensitivity of the assay was 15.6 pg/ml. The intraassay and interassay coefficients of variation were less than 5%.

Statistical analysis
Data were evaluated using ANOVA with Scheffé’s post hoc analysis for multiple comparisons and Student’s t test for two groups. P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-17A and IL-17RA mRNA in endometriotic tissue, PFMCs, and ESCs
The expression of IL-17A mRNA was detected in endometriotic tissues and peritoneal cells, but not in ESCs, by standard RT-PCR analysis. The expression of IL-17RA mRNA was detected in endometriotic tissues, PFMCs, and ESCs (Fig. 1AGo).


Figure 1
View larger version (66K):
[in this window]
[in a new window]

  		FIG. 1. A, Expression of IL-17A and IL-17RA mRNA in endometriotic tissues (lane 1), PFMCs (lane 2), and ESCs (lane 3). Endometriotic tissues, PFMCs, and ESCs without stimulation were analyzed by RT-PCR. B, Expression of IL-17A in the cyst wall of endometrioma (i, ii, iii, and iv) and ovary (v). Sections were immunostained with antihuman IL-17A antibody (i, ii, iv, and v) or goat IgG (iii). IL-17A-positive cells were detected in the stroma immediately beneath epithelium (i, white rectangular area). Arrowheads indicate IL-17A-positive cells in the stroma immediately beneath epithelium (ii). IL-17A-positive cells (arrowheads) were detected at the site of hemosiderin deposits (iv, white circle). No IL-17A-positive cells were detected in ovarian surface (v). Magnification, x100 (i), x400 (ii and iii), and x200 (iv and v). Scale bars, 200 µm (i, iv, and v); 100 µm (ii and iii). C, Th17 cells in PFMCs of patients with endometriosis. PFMCs were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) and labeled with an antibody specific to lymphocytes (CD3 and CD4) and IL-17A. Th17 cells were CD4 positive and IL-17A positive. The data shown are representative of four separate experiments. D, Immunocytochemistry of IL-17RA in ESCs. Cultured ESCs were immunostained with anti-IL-17RA antibody (upper photo). Lower photo shows the control with mouse IgG1 isotype. Magnification, x100.

 
In vivo expression of IL-17A in the endometriotic lesion
As shown in Fig. 1BGo, the presence of immunoreactive IL-17A was detected in the cyst wall of endometrioma. Intense IL-17A immunoreactive cells were localized in the stroma immediately beneath epithelium and at the site of hemosiderin deposits. No IL-17A immunoreactivity was visualized in endometriotic epithelial cells. Few IL-17A-positive cells were observed in the endometrium of proliferative and secretary phases (data not shown). No IL-17A-positive cells were detected in ovarian surface. No staining was observed when normal goat IgG was used as a primary antibody.

Th17 cells in PFMCs of patients with endometriosis
The presence of IL-17A-producing cells was evaluated by flow cytometry on T cells (CD3+ cells) from PFMCs after stimulation with PMA and ionomycin. As shown in Fig. 1CGo, IL-17A-producing T cells were detected in PFMCs of patients with endometriosis. We also found IL-17A-producing T cells were predominantly CD4+ T cells.

Expression of IL-17RA protein in ESCs
The presence of immunoreactive IL-17RA was demonstrated in ESCs (Fig. 1DGo). No staining was observed when mouse IgG1 was used as a primary antibody.

Effects of IL-17A on IL-8 secretion by ESCs
As shown in Fig. 2Go, IL-17A at 1 ng/ml and higher significantly enhanced the secretion of IL-8 from ESCs. The maximum effect was observed with IL-17A at 10 ng/ml. The magnitude of increase with 10 ng/ml IL-17A varied between patients from 1.8- and 5.3-fold, with a median increase of 3.5-fold (n = 9).


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 2. IL-17A stimulates IL-8 secretion in ESCs. ESCs were cultured in 5% FBS with different doses of IL-17A for 24 h. Concentration of IL-8 in the conditioned medium was measured using a specific ELISA. Values are the mean ± SEM of pentaplicate cultures. Different letters denote significant differences between groups (P < 0.05). The result is representative of nine separate experiments using samples from different patients.

 
Effect of anti-IL-17RA antibody on IL-17A-induced IL-8 secretion in ESCs
Treatment with the neutralizing antibodies for IL-17RA significantly diminished the IL-17A-induced increase in IL-8 secretion in a dose-dependent manner, whereas the control IgG had no effect (Fig. 3Go).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 3. Effect of anti-IL-17RA antibody (aIL-17R) on IL-17A-induced IL-8 secretion by ESCs. ESCs were preincubated in 5% FBS medium with or without mouse IgG1 and aIL-17R for 30 min and then stimulated with or without IL-17A (10 ng/ml) for 24 h. Concentrations of IL-8 in the conditioned media were measured using a specific ELISA. All value are expressed as the mean ± SEM of pentaplicate cultures. Different letters denote significant differences between groups (P < 0.05). The result is representative of four separate experiments using samples from different patients. Cont, Control.

 
Effect of IL-17A on MAPK phosphorylation in ESCs
The phosphorylation of three MAPKs (p42/44 MAPK, p38 MAPK, and SAPK/JNK) by IL-17A was determined in cultured ESCs (Fig. 4AGo). An increase in MAPK phosphorylation was apparent after 5–15 min. Phosphorylation levels reached a maximum after 5 min for P42/44 MAPK and after 15 min for p38 MAPK and SAPK/JNK, respectively.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 4. Involvement of MAPKs in IL-17A induced IL-8 secretion from ESCs. A, phosphorylation of p38 MAPK, p42/44 MAPK, and SAPK/JNK induced by IL-17A in ESCs. ESCs were incubated with IL-17A (10 ng/ml) for the indicated time (0–120 min). Cell lysates were assayed for phosphorylated p38 MAPK (phospho-p38), total p38 MAPK (total-p38), phosphorylated p42/44 MAPK (phospho-p42/44), total p42/44 (total-p42/44), phosphorylated SAPK/JNK (phospho-SAPK/JNK), and total SAPK/JNK (total-SAPK/JNK) by Western blotting. The data are representative of four independent experiments. B, Effects of MAPK inhibitors on IL-17A-induced IL-8 secretion in ESCs. ESCs were pretreated with or without inhibitors of p38 MAPK (SB202190), p42/44 MAPK (PD98059), and SAPK/JNK (SP600125) for 1 h and stimulated with IL-17A (10 ng/ml) for 24 h. The conditioned medium was collected and assayed for IL-8 concentration using a specific ELISA. All values are expressed as the mean ± SEM of pentaplicate cultures. Different letters denote significant differences between groups (P < 0.05). The data are representative of four independent experiments. DMSO, Dimethylsulfoxide.

 
Effect of MAPK inhibitors on IL-17A-induced IL-8 secretion
The intracellular mechanism of IL-17A-induced secretion of IL-8 by ESCs was investigated by examining the effect of MAPK inhibitors. As shown in Fig. 4BGo, the addition of inhibitors for p38MAPK, p42/44 MAPK, and SAPK/JNK significantly diminished IL-17A-induced IL-8 secretion.

Synergic effects IL-17A and TNF{alpha} on IL-8 secretion in ESCs
We chose TNF{alpha} as a representative proinflammatory cytokine known to induce IL-8 secretion from ESCs (12). TNF{alpha} together with IL-17A triggered IL-8 secretion above the combined levels generated by each stimulus alone (Fig. 5Go). This synergistic effect was apparent when TNF{alpha} (1 ng/ml) was combined with 1 ng/ml IL-17A, and maximal synergy was obtained at the highest dose of IL-17A tested (100 ng/ml).


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 5. Effect of IL-17A on TNF{alpha}-mediated IL-8 secretion from ESCs. ESCs were treated with IL-17A or TNF{alpha} or in combination for 24 h. The conditioned medium was collected and assayed for IL-8 concentration using a specific ELISA. All values are expressed as the mean ± SEM of pentaplicate cultures. *, P < 0.0001 vs. each control. The data are representative of four independent experiments.

 
Effect of IL-17A on the expression of IL-8 mRNA and COX2 mRNA in ESCs
Time-course experiments were conducted to examine the effect of IL-17A on the expression of IL-8 mRNA and COX2 mRNA in ESCs (Fig. 6AGo). Real-time quantitative PCR analysis demonstrated that IL-17A up-regulated IL-8 and COX2 mRNA. Maximal increases in IL-8 and COX2 mRNA were observed at 4 h, followed by a decrease with time up to 24 h (Fig. 6Go, B and C). The maximal increase of IL-8 mRNA was 6.2-fold of the control, and that of COX2 mRNA was 13.6-fold of the control.


Figure 6
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 6. Effect of IL-17A on the expression of IL-8 and COX2 mRNA in ESCs. ESCs were incubated with IL-17A (10 ng/ml) for the indicated duration. A, Expression of IL-8 and COX2 mRNA in ESCs was examined by RT-PCR. The data shown are representative of four separate experiments using samples from different women. B, Expression of IL-8 mRNA in ESCs was examined by real-time quantitative PCR. The data shown are the relative ratio (IL-8 to GAPDH) measures by real-time quantitative PCR. Data are the mean ± SEM of five independent experiments using samples from five different women. Different letters denote significant differences between groups (P < 0.05). C, Expression of COX2 mRNA in ESCs was examined by real-time quantitative PCR. The data shown are the relative ratios (COX2 to GAPDH) measured by real-time quantitative PCR. Data are the mean ± SEM of five independent experiments using samples from 5 different women. Different letters denote significant differences between groups (P < 0.05).

 
Effect of IL-17A on cell proliferation of ESCs
The effect of IL-17A on cell proliferation was determined in ESCs (Fig. 7AGo). IL-17A at 10 and 100 ng/ml significantly increased cell number by 106 and 111%, respectively, after exposure for 48 h. As shown in Fig. 7BGo, IL-17A at 1–100 ng/ml significantly increased BrdU incorporation into DNA in ESCs. The maximal effect (3.98-fold of control) was observed at 10 ng/ml.


Figure 7
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 7. Effect of IL-17A on proliferation of ESCs. A, The effect of IL-17A on proliferation of ESCs was examined by measuring cell number with cell-counting kit. ESCs were cultured in 1% FBS with different doses of IL-17A for 48 h. Values are the mean ± SEM of pentaplicate cultures. *, P < 0.05; **, P < 0.01 vs. control. The result is representative of four separate experiments using samples from different patients. B, The effect of IL-17A on the proliferation of ESCs was examined by measuring BrdU incorporation into DNA by using a cell proliferation ELISA. ESCs were treated with IL-17A at different concentrations for 48 h. Values are the mean ± SEM of the pentaplicate cultures. Different letters denote significant differences between groups (P < 0.05). The data shown are representative of four independent experiments using samples from four different patients.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we first demonstrated that presence of IL-17A-positive cells in the endometriotic tissue. In addition, the presence of Th17 cells in PFMCs was clearly shown by flow cytometric analysis. These findings instigated us to examine possible roles of IL-17A in endometriosis. We then showed that IL-17A stimulated the secretion of IL-8 from ESCs. ESCs expressed IL-17RA, and the anti-IL-17RA antibody inhibited IL-17A-induced IL-8 secretion. IL-17A stimulated the activation of p38 MAPK, p42/44 MAPK, and SAPK/JNK, and inhibitors of these kinases suppressed IL-17A-induced IL-8 secretion. TNF{alpha} synergistically enhanced IL-17A-induced IL-8 secretion. IL-17A also stimulated ESC proliferation and COX2 expression in ESCs.

A recent study on the Th1/ Th2 concept of T cell immunology revealed that endometriosis is an inflammatory disease with a Th2 immune response component (20, 21). The emerging concept of the Th17 pathway has challenged the conventional paradigm of Th1/Th2 hypothesis (4, 5). Together with the recent discovery of Treg, our understanding of the mechanisms underlying T cell immunology has advanced into a new era. In this context, the presence of Th17 cells in PFMCs demonstrated in our study might lead the concept of immune response in endometriosis to a novel direction. In particular, abundant IL-17A-positive cells in the endometriotic tissue imply possible Th17 immune response therein. The present study has demonstrated multiple functions of IL-17A in ESCs. Given that IL-17A is a key effector molecule of Th17 cells, our findings form the foundation for understanding the etiology of endometriosis under the novel concept of T cell differentiation and regulation.

Substantial evidence points to IL-8 as a pivotal factor involved in the progression of endometriosis. IL-8 exerts pleiotropic functions, such as chemoattraction and activation of neutrophils, angiogenesis, stimulation of proliferation, and survival of endometrial cells (1, 9). Our previous studies suggest that expression of IL-8 in endometriotic cells is regulated by various cytokines and enzymes (15, 16, 22). Notably, these IL-8-inducing molecules are derived from macrophages, neutrophils, and mast cells, which are suggested to play important roles in the development of the disease. The present study provides evidence that IL-17A is an additional stimulant of IL-8 secretion from ESCs and strongly suggests that Th17 cells participate in the development of endometriosis.

The synergistic effect of IL-17A and TNF{alpha} in stimulating secretion of IL-8 from ESCs was remarkable. TNF{alpha} is a proinflammatory cytokine that plays multiple roles in the progression of endometriosis. The importance of TNF{alpha} in endometriosis is underpinned by recent findings that TNF{alpha}-targeted suppression by specific drugs inhibits the development of endometriosis in baboons (23, 24). During the inflammatory response TNF{alpha} is secreted from various cells type, such as peritoneal macrophage, endometrial epithelial, and stromal cells. IL-17A may be an accelerator of endometriosis during chronic pelvic inflammation accompanied by increased TNF{alpha} production.

IL-17A-induced phosphorylation of p42/44MAPK, p38MAPK, and SAPK/JNK in ESCs, and the IL-17A-induced increase in IL-8 secretion from ESCs was suppressed by inhibitors of these kinases. This finding suggests that these enzymes are involved in the pathway of IL-17A-induced IL-8 secretion. A similar finding was reported for human airway smooth muscle cells (25). Interestingly, our previous study showed that IL-1β stimulated phosphorylation of these MAPKs, and IL-1β-induced IL-8 secretion from ESCs was inhibited by the same MAPK inhibitors (22). IL-1β is a well-known central player of the inflammatory condition of endometriosis, and thus, it can be speculated that cytokines promoting endometriosis, such as IL-1β and IL-17A, share similar pathways to induce IL-8 secretion from ESCs.

COX2, a key enzyme in prostaglandin biosynthesis, is up-regulated in endometriotic stromal cells (26, 27, 28). COX2 plays an important role in the inflammatory response associated with endometriosis and appears to function in the pathogenesis of endometriosis (29, 30). The present finding of IL-17A-induced COX2 expression in ESCs provides further evidence for a key role of IL-17A in endometriosis.

In the present study, IL-17A stimulated ESC proliferation. Human tumors show increased expression of IL-17A (31), and IL-17A promotes angiogenesis and tumor growth (32). IL-17A also enhances the proliferation of normal airway epithelial cells (33). Because IL-17A appears to exert a mitogenic effect on various cell types, IL-17A may stimulate progression of endometriosis as a result of this mitogenic effect, possibly in addition to other functions. Interestingly, IL-8 has been shown to stimulate the proliferation of endometrial stromal cells (34). Therefore, the proliferative effect of IL-17A on ESCs may be partially attributable to the increased production of IL-8 induced by IL-17A.

The proinflammatory and mitogenic action of IL-17A in ESCs demonstrated in the present study revealed possible roles of the molecule in the development of endometriosis. The prevailing medical treatment for endometriosis is suppression of ovarian hormones by GnRH analogs. The adverse effects of this treatment induced by the hypoestrogenic status often lead to a lack of compliance. IL-17A is well placed as a candidate target molecule for novel treatment strategies of endometriosis. Further investigation would benefit the development of an improved treatment option involving IL-17A.

In summary, the present study demonstrated that IL-17A stimulates inflammatory responses and proliferation of ESCs, suggesting a role for IL-17A in the pathogenesis of endometriosis.


   Footnotes
 
First Published Online December 13, 2007

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; COX, cyclooxygenase; ESC, endometriotic stromal cell; FBS, fetal bovine serum; GAPDH, glyceraldehyde dehydrogenase; IL-17RA, antihuman IL-17 receptor; JNK, c-Jun N-terminal kinase; PF, peritoneal fluid; PFMC, PF mononuclear cell; PMA, phorbol 12-myristate 13-acetate; SAPK, stress-activated protein kinase.

This work was partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology and the Ministry of Health, Labor, and Welfare.

Disclosure Statement: The authors of this manuscript have nothing to declare.

Received June 6, 2007.

Accepted for publication December 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Harada T, Iwabe T, Terakawa N 2001 Role of cytokines in endometriosis. Fertil Steril 76:1–10[CrossRef][Medline]
  2. Lebovic DI, Mueller MD, Taylor RN 2001 Immunobiology of endometriosis. Fertil Steril 75:1–10[CrossRef][Medline]
  3. Iwakura Y, Ishigame H 2006 The IL-23/IL-17 axis in inflammation. J Clin Invest 116:1218–1222[CrossRef][Medline]
  4. Reiner SL 2007 Development in motion: helper T cells at work. Cell 129:33–36[CrossRef][Medline]
  5. Steinman L 2007 A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med 13:139–145[CrossRef][Medline]
  6. Kolls JK, Linden A 2004 Interleukin-17 family members and inflammation. Immunity 21:467–476[CrossRef][Medline]
  7. Osuga Y, Koga K, Tsutsumi O, Yano T, Maruyama M, Kugu K, Momoeda M, Taketani Y 2002 Role of laparoscopy in the treatment of endometriosis-associated infertility. Gynecol Obstet Invest 53(Suppl 1):33–39
  8. Zhang X, Xu H, Lin J, Qian Y, Deng L 2005 Peritoneal fluid concentrations of interleukin-17 correlate with the severity of endometriosis and infertility of this disorder. BJOG 112:1153–1155[Medline]
  9. Arici A 2002 Local cytokines in endometrial tissue: the role of interleukin-8 in the pathogenesis of endometriosis. Ann NY Acad Sci 955:101–109[Medline]
  10. Mulayim N, Savlu A, Guzeloglu-Kayisli O, Kayisli UA, Arici A 2004 Regulation of endometrial stromal cell matrix metalloproteinase activity and invasiveness by interleukin-8. Fertil Steril 81(Suppl 1):904–911
  11. Selam B, Kayisli UA, Garcia-Velasco JA, Akbas GE, Arici A 2002 Regulation of fas ligand expression by IL-8 in human endometrium. J Clin Endocrinol Metab 87:3921–3927[Abstract/Free Full Text]
  12. Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M, Terakawa N 2000 Tumor necrosis factor-{alpha} promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 85:824–829[Abstract/Free Full Text]
  13. Hirota Y, Osuga Y, Koga K, Yoshino O, Hirata T, Harada M, Morimoto C, Yano T, Tsutsumi O, Sakuma S, Muramatsu T, Taketani Y 2005 Possible implication of midkine in the development of endometriosis. Hum Reprod 20:1084–1089[Abstract/Free Full Text]
  14. Yoshino O, Osuga Y, Koga K, Hirota Y, Tsutsumi O, Yano T, Morita Y, Momoeda M, Fujiwara T, Kugu K, Taketani Y 2003 Concentrations of interferon-{gamma}-induced protein-10 (IP-10), an antiangiogenic substance, are decreased in peritoneal fluid of women with advanced endometriosis. Am J Reprod Immunol 50:60–65[Medline]
  15. Hirota Y, Osuga Y, Hirata T, Harada M, Morimoto C, Yoshino O, Koga K, Yano T, Tsutsumi O, Taketani Y 2005 Activation of protease-activated receptor 2 stimulates proliferation and interleukin (IL)-6 and IL-8 secretion of endometriotic stromal cells. Hum Reprod 20:3547–3553[Abstract/Free Full Text]
  16. Hirota Y, Osuga Y, Hirata T, Yoshino O, Koga K, Harada M, Morimoto C, Nose E, Yano T, Tsutsumi O, Taketani Y 2005 Possible involvement of thrombin/protease-activated receptor 1 system in the pathogenesis of endometriosis. J Clin Endocrinol Metab 90:3673–3679[Abstract/Free Full Text]
  17. Hirata T, Osuga Y, Hirota Y, Koga K, Yoshino O, Harada M, Morimoto C, Yano T, Nishii O, Tsutsumi O, Taketani Y 2005 Evidence for the presence of toll-like receptor 4 system in the human endometrium. J Clin Endocrinol Metab 90:548–556[Abstract/Free Full Text]
  18. Morimoto C, Osuga Y, Yano T, Takemura Y, Harada M, Hirata T, Hirota Y, Yoshino O, Koga K, Kugu K, Taketani Y 2005 GnRH II as a possible cytostatic regulator in the development of endometriosis. Hum Reprod 20:3212–3218[Abstract/Free Full Text]
  19. Takemura Y, Osuga Y, Yoshino O, Hasegawa A, Hirata T, Hirota Y, Nose E, Morimoto C, Harada M, Koga K, Tajima T, Yano T, Taketani Y 2007 Metformin suppresses interleukin (IL)-1β-induced IL-8 production, aromatase activation, and proliferation of endometriotic stromal cells. J Clin Endocrinol Metab 92:3213–3218[Abstract/Free Full Text]
  20. Antsiferova YS, Sotnikova NY, Posiseeva LV, Shor AL 2005 Changes in the T-helper cytokine profile and in lymphocyte activation at the systemic and local levels in women with endometriosis. Fertil Steril 84:1705–1711[CrossRef][Medline]
  21. Podgaec S, Abrao MS, Dias Jr JA, Rizzo LV, de Oliveira RM, Baracat EC 2007 Endometriosis: an inflammatory disease with a Th2 immune response component. Hum Reprod 22:1373–1379[Abstract/Free Full Text]
  22. Yoshino O, Osuga Y, Hirota Y, Koga K, Hirata T, Harada M, Morimoto C, Yano T, Nishii O, Tsutsumi O, Taketani Y 2004 Possible pathophysiological roles of mitogen-activated protein kinases (MAPKs) in endometriosis. Am J Reprod Immunol 52:306–311[Medline]
  23. D’Hooghe TM, Nugent NP, Cuneo S, Chai DC, Deer F, Debrock S, Kyama CM, Mihalyi A, Mwenda JM 2006 Recombinant human TNFRSF1A (r-hTBP1) inhibits the development of endometriosis in baboons: a prospective, randomized, placebo- and drug-controlled study. Biol Reprod 74:131–136[Abstract/Free Full Text]
  24. Falconer H, Mwenda JM, Chai DC, Wagner C, Song XY, Mihalyi A, Simsa P, Kyama C, Cornillie FJ, Bergqvist A, Fried G, D’Hooghe TM 2006 Treatment with anti-TNF monoclonal antibody (c5N) reduces the extent of induced endometriosis in the baboon. Hum Reprod 21:1856–1862[Abstract/Free Full Text]
  25. Wuyts WA, Vanaudenaerde BM, Dupont LJ, Van Raemdonck DE, Demedts MG, Verleden GM 2005 Interleukin-17-induced interleukin-8 release in human airway smooth muscle cells: role for mitogen-activated kinases and nuclear factor-{kappa}B. J Heart Lung Transplant 24:875–881[CrossRef][Medline]
  26. Chishima F, Hayakawa S, Sugita K, Kinukawa N, Aleemuzzaman S, Nemoto N, Yamamoto T, Honda M 2002 Increased expression of cyclooxygenase-2 in local lesions of endometriosis patients. Am J Reprod Immunol 48:50–56[Medline]
  27. Fagotti A, Ferrandina G, Fanfani F, Legge F, Lauriola L, Gessi M, Castelli P, Barbieri F, Minelli L, Scambia G 2004 Analysis of cyclooxygenase-2 (COX-2) expression in different sites of endometriosis and correlation with clinico-pathological parameters. Hum Reprod 19:393–397[Abstract/Free Full Text]
  28. Ota H, Igarashi S, Sasaki M, Tanaka T 2001 Distribution of cyclooxygenase-2 in eutopic and ectopic endometrium in endometriosis and adenomyosis. Hum Reprod 16:561–566[Abstract/Free Full Text]
  29. Attar E, Bulun SE 2006 Aromatase and other steroidogenic genes in endometriosis: translational aspects. Hum Reprod Update 12:49–56[Abstract/Free Full Text]
  30. Matsuzaki S, Canis M, Darcha C, Dallel R, Okamura K, Mage G 2004 Cyclooxygenase-2 selective inhibitor prevents implantation of eutopic endometrium to ectopic sites in rats. Fertil Steril 82:1609–1615[CrossRef][Medline]
  31. Langowski JL, Zhang X, Wu L, Mattson JD, Chen T, Smith K, Basham B, McClanahan T, Kastelein RA, Oft M 2006 IL-23 promotes tumour incidence and growth. Nature 442:461–465[CrossRef][Medline]
  32. Numasaki M, Fukushi J, Ono M, Narula SK, Zavodny PJ, Kudo T, Robbins PD, Tahara H, Lotze MT 2003 Interleukin-17 promotes angiogenesis and tumor growth. Blood 101:2620–2627[Abstract/Free Full Text]
  33. Inoue D, Numasaki M, Watanabe M, Kubo H, Sasaki T, Yasuda H, Yamaya M, Sasaki H 2006 IL-17A promotes the growth of airway epithelial cells through ERK-dependent signaling pathway. Biochem Biophys Res Commun 347:852–858[CrossRef][Medline]
  34. Arici A, Seli E, Zeyneloglu HB, Senturk LM, Oral E, Olive DL 1998 Interleukin-8 induces proliferation of endometrial stromal cells: a potential autocrine growth factor. J Clin Endocrinol Metab 83:1201–1205[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum ReprodHome page
A. Hasegawa, Y. Osuga, Y. Hirota, K. Hamasaki, A. Kodama, M. Harada, T. Tajima, Y. Takemura, T. Hirata, O. Yoshino, et al.
Tunicamycin enhances the apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand in endometriotic stromal cells
Hum. Reprod., February 1, 2009; 24(2): 408 - 414.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirata, T.
Right arrow Articles by Taketani, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirata, T.
Right arrow Articles by Taketani, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals