Interleukin-17F increases the secretion of interleukin-8 and the expression of cyclooxygenase 2 in endometriosis

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1 Interleukin-17F increses the secretion of interleukin-8 nd the expression of cyclooxygense in endometriosis Tetsuy Hirt, M.D., Ph.D., Yutk Osug, M.D., Ph.D., Msshi Tkmur, M.D., Ako Sito, M.D., Ph.D., Akiko Hsegw, M.D., Ph.D., Kori Kog, M.D., Ph.D., Osmu Yoshino, M.D., Ph.D., Ysushi Hirot, M.D., Ph.D., Miyuki Hrd, M.D., Ph.D., nd Yuji Tketni, M.D., Ph.D. Deprtment of Ostetrics nd Gynecology, University of Tokyo, Tokyo, Jpn Ojective: To exmine the effects of interleukin (IL)-17F on the secretion of IL-8 nd the gene expression of cyclooxygense (COX) in endometriotic stroml cells. Design: In vitro experimentl study using humn smples. Setting: University hospitl. Ptient(s): Endometriotic tissues were otined from women with ovrin endometrioms undergoing lproscopic surgery. Intervention(s): Endometriotic stroml cells (ESCs) were cultured with IL-17F. Min Outcome Mesure(s): Concentrtions of IL-8 were mesured y specific ELISA, nd messenger RNA levels of IL-8 nd COX were mesured y rel-time reverse trnscription polymerse chin rection (PCR). Result(s): IL-17F incresed the secretion of IL-8 from ESCs, nd the effect ws inhiited y ntiodies for IL-17 receptor A nd IL-17 receptor C. Tumor necrosis fctor (TNF-) synergisticlly enhnced IL-17F-induced increse in IL-8 secretion from ESCs. The IL-17F incresed the gene expression of IL-8 nd COX in ESCs. Conclusion(s): These findings suggest tht IL-17F my stimulte the development of endometriosis y upregultion of IL-8 nd COX. (Fertil Steril Ò 11;9: Ó11 y Americn Society for Reproductive Medicine.) Key Words: Endometriosis, interleukin-17f, interleukin-8, cyclooxygense Endometriosis is n enigmtic disese, which is defined y the presence of endometriotic tissue outside the uterus. Although the precise etiology nd pthogenesis of endometriosis remins uncler, it is widely elieved tht endometril cells in retrogrde menstrution implnt nd grow in the pelvic cvity. Given this theory, puzzle emerges tht only frction of women develop endometriosis, wheres retrogrde menstrution is oserved in most women. Multiple lines of evidence suggest tht inflmmtion nd immune responses ply pivotl role in the pthogenesis of endometriosis (1 3). The Th17 cells re new nd distinct linege of CDþ helper T cells, which express interleukin (IL)-17A, ut not IL- nd interferon g (IFN-g). The Th17 cells ply crucil roles in vrious utoimmune diseses nd llergic diseses y promoting chronic inflmmtory responses (, 5). Previously, we demonstrted tht Th17 cells were present in endometriotic tissues nd tht IL-17A Received Novemer 7, 1; revised nd ccepted April 19, 11; pulished online My, 11. T.H. hs nothing to disclose. Y.O. hs nothing to disclose. M.T. hs nothing to disclose. A.S. hs nothing to disclose. A.H. hs nothing to disclose. K.K. hs nothing to disclose. O.Y. hs nothing to disclose. Y.H. hs nothing to disclose. M.H. hs nothing to disclose. Y.T. hs nothing to disclose. Supported in prt y Helth nd Lor Sciences Reserch Grnts from the Ministry of Helth, Lor nd Welfre of Jpn, nd Grnt-in-Aid for Scientific Reserch from the Ministry of Eduction, Culture, Sports, Science nd Technology. Reprint requests: Yutk Osug, M.D., Ph.D., Deprtment of Ostetrics nd Gynecology, Fculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, , Jpn (E-mil: yutkos-tky@ umin.c.jp). stimulted IL-8 secretion, cyclooxygensse- (COX) expression, nd cell prolifertion of endometriotic stroml cells (, 7). Other investigtors reported tht IL-17A concentrtions in peritonel fluid (PF) correlted with the severity of endometriosis nd infertility of this disorder (8). In ddition, recent study demonstrted tht high expression of IL-17A ws oserved in the fluid of endometriom positive for romtse (9). These findings suggest tht IL-17A nd Th17 re involved in the pthogenesis of endometriosis. Interleukin-17F is memer of the IL-17 fmily, which consists of six cytokines: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, nd IL- 17F (1). Interleukin-17F consists of 153 mino cids pproximtely 5% homologous with IL-17A. It is expressed in Th17 cells (11), sophils, mst cells (1), nd monocytes (13). Both IL-17A nd IL-17F ind to the heteromeric receptor complex comprised of IL-17 receptor C (IL-17RC) nd IL-17 receptor A (IL-17RA). However, ech lignd hs different ffinity for ech receptor. Interleukin-17F inds with n extremely low ffinity to IL-17RA, wheres it inds with higher ffinity to IL-17RC compred with IL-17A (1). Interleukin-17A nd IL-17F induce similr iologicl responses in mny iologicl events, ut lso elicit mrkedly different responses in severl inflmmtory situtions (15). It increses the production of vrious proinflmmtory cytokines nd chemokines in ronchil epithelil cells (1, 1), kertinocytes (17), colonic firolsts (18), nd endothelil cells (13). Recent studies suggest the involvement of IL-17F in the pthophysiology of llergic sthm (19), inflmmtory owel disese (), or psorisis (17). Although we hve demonstrted the presence of Th17 cells nd possile roles of IL-17A in endometriosis, the role of IL-17F in the disese hs not een ddressed. To study the possile roles of IL-17F in endometriosis, we exmined the effects of IL-17F on 15-8/$3. Fertility nd Sterility â Vol. 9, No. 1, July doi:1.11/j.fertnstert.11.. Copyright ª11 Americn Society for Reproductive Medicine, Pulished y Elsevier Inc.

2 the production of IL-8 nd on the expression of COX, possile key plyers in endometriosis, in endometriotic stroml cells (ESCs) (1 3). We lso studied coopertive effect of IL-17F nd tumor necrosis fctor (TNF-), typicl cytokine implicted in the disese (). MATERIALS AND METHODS Ptients nd Smples Endometriotic tissues were otined from ptients with ovrin endometrioms undergoing lproscopy. All ptients hd regulr menstrul cycles, nd none hd received hormonl tretment (HT) for t lest months efore surgery. The tissues, collected under sterile conditions, were processed for primry cell cultures. The experimentl procedures were pproved y the institutionl review ord of the University of Tokyo nd signed informed consent for use of the endometriotic tissues ws otined from ech ptient. Isoltion nd Culture of Mononucler Cells From Endometriotic Lesions Fresh endometriotic tissues collected in sterile medium were rinsed to remove red lood cells (RBC). The tissues were minced into smll pieces nd incuted in phenol red-free Dulecco s minimum essentil medium (DMEM)/F1 contining type I collgense (.5%; Wko Pure Chemicl Industries) nd deoxyrionuclese I (DNse; 15 IU/mL; Invitrogen) for 1 minutes t 37 C. The resulting dispersed cells were seprted y filtrtion through 1-mm nd 7-mm nylon cell striners (Becton Dickinson). The filtrte ws wshed twice with phosphte-uffered sline (PBS). This pellet ws resuspended in % Percoll (5 ml), lyered gently onto 7% Percoll, nd centrifuged t 1,8 rpm for minutes. The interfce ws recovered nd wshed in PBS, resuspended in RPMI 1 medium contining 1% chrcol-stripped fetl ovine serum (FBS; Hyclone), plted into 1-mm pltes (Iwki, Ashi Technology), nd llowed to dhere t 37 C overnight. Nondherent cells were collected nd used for the experimentl procedures s mononucler cells from endometriotic lesions (EMMCs). The EMMCs were resuspended in 1% FBS in RPMI 1 medium. The cells were stimulted with 5 ng/ml of phorol 1-myristte 13-cette (PMA; Sigm) nd 1 mg/ml of ionomycin (Sigm) for 8 hours. The conditioned medi ws hrvested for ssy. Isoltion nd Culture of ESCs Isoltion nd culture of humn ESCs were processed s descried previously (5, ). Fresh endometriotic tissues collected in sterile medium were rinsed to remove lood cells. The tissues were minced into smll pieces nd incuted in phenol-red free DMEM/F1 contining type I collgense (.5%) nd DNse I (15 IU/mL) for 1 minutes t 37 C. The resultnt dispersed endometriotic cells were seprted y filtrtion through 1-mm nylon cell striner nd 7-mm nylon cell striner. Stroml cells remining in the filtrte were collected y centrifugtion, resuspended in phenol-red free DMEM/F-1, nd plted 1-mm dishes nd llowed to dhere t 37 C for 1 hours. At the first pssge, the cells were plted into -, 1-, or 8-well culture pltes. The cells reched confluence in or 3 dys nd then were used for the experiments. The purity of ESCs ws more thn 95%, s judged y positive cellulr stining for vimentin nd negtive cellulr stining for cytokertin or CD5. Tretment of ESCs First, to exmine the effect of IL-17F on IL-8 production, ESCs were incuted in 5% FBS DMEM/F1 medium with vrious doses of IL-17F for hours. Second, for the time-course study of IL-8 nd COX gene expression, ESCs were incuted with 5% FBS medium with IL-17F (1 ng/ml) for different periods up to hours. Third, to exmine the effect of mouse nti-il- 17RA ntiody nd got nti-il-17rc ntiody (R&D Systems), ESCs were preincuted in 5% FBS DMEM/F-1 with ech ntiody or the control IgGs for 3 minutes nd then stimulted with 5 ng/ml IL-17F for hours. Finlly, to evlute the synergic effect of IL-17F nd TNF- on IL-8 secretion, the cells were stimulted with vrious doses of IL-17F (1 1 ng/ml) with or without TNF- (1 ng/ml). RNA Extrction, Reverse Trnscription, nd Rel-Time Quntittive Polymerse Chin Rection of IL-8 nd COX We extrcted totl RNA from ESCs cultured in 1-well plte using n RNesy minikit (QIAGEN). One microgrm of totl RNA ws reverse trnscried in -ml volume using reverse trnscription polymerse chin rection (RT-PCR) kit (TOYOBO). Rel-time quntittive PCR ws performed s reported previously (7, 8). To ssess IL-8 nd COX messenger RNA (mrna) expression, rel-time quntittive PCR nd dt nlysis were performed using Light Cycler (Roche Dignostics GmH). Expression of IL-8 nd COX mrna ws normlized to RNA loding for ech smple using glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna s n internl stndrd. The primers for IL-8 nd COX were the sme s those used previously (7). The PCR conditions were s follows: for IL-8, cycles t 95 C for 1 seconds, C for 1 seconds, 7 C for 11 seconds; for COX, 3 cycles t 95 C for 1 seconds, C for 1 seconds, 7 C for 13 seconds; for GAPDH, 3 cycles t 95 C for 1 seconds, C for 1 seconds, 7 C for 18 seconds. All PCR conditions were followed y melting curve nlysis. Immunocytochemistry The ESCs were cultured in 1-well chmer slides (Nunc) in humidified 5% CO 95% ir environment nd llowed to grow to out 5% confluence. The cells were fixed with cold methnol/cetone t C for minutes, wshed twice with PBS. Endogeneous peroxidse ws locked y incution for minutes with solution of.3% hydrogen peroxidse. Immunocytochemicl cell leling ws performed using the vidin-iotin peroxidse method. After locking with norml rit serum (Vector lortories), the cells were incuted with 1 mg/ml nti IL-17RC ntiody or got IgG for minutes t room temperture nd incuted with vidin-iotin peroxidse complex (Vectstin Elite, Vector Lortories), ccording to the mnufcturer s instructions. Stining ws detected with the diminoenzidine chromogen fter 3 minutes. All slides were counterstined with hemtoxylin nd evluted under light microscope. Mesurement of Cytokines Concentrtions of IL-17F or IL-8 in conditioned medi were mesured using specific ELISA kits (IL-17F, Pepro tech; IL-8, Genzyme/Techne). The intrssy nd interssy coefficients of vrition (CV) were less thn 5%. Sttisticl Anlysis Dt were evluted using nlysis of vrince (ANOVA) with Scheffe s post hoc nlysis for multiple comprisons nd Student s t-test for two groups. P<.5 ws ccepted s sttisticlly significnt. RESULTS Expression of IL-17F in EMMCs nd IL-RC in ESCs As shown in Figure 1A, EMMCs secreted IL-17F. The stimultion with PMA nd ionomycin significntly enhnced the secretion of IL-17F from EMMCs. The presence of immunorective IL-17RC ws demonstrted in ESCs (Fig. 1B). No stining ws seen when got IgG ws used s primry ntiody. Effect of IL-17F nd Anti-IL-17RA or Anti-IL-17RC Antiodies on IL-8 Secretion From ESCs As shown in Figure A, IL-17F t 1 ng/ml nd higher dose significntly incresed the secretion of IL-8 from ESCs. The increse of IL-8 secretion y IL-17F t 1 ng/ml ws.-fold of the control. Tretment with the neutrlizing ntiodies for IL-17RA or IL-17RC significntly diminished IL-17F-induced increse in IL-8 secretion, wheres the control IgGs hd no effect (Fig. B). 11 Hirt et l. IL-17F increses IL-8 in endometriosis Vol. 9, No. 1, July 11

3 FIGURE 1 (A) Interleukin (IL)-17F secretion from mononucler cells from endometriotic lesions (EMMCs). The EMMCs secreted IL-17F nd the stimultion with phorol ester (PMA; 5 ng/ml) nd ionomycin (1 mg/ml) enhnced the secretion of IL-17F from EMMCs. Concentrtion of IL-17F in the conditioned medium ws mesured using specific ELISA. Vlues re the mens SEM of pentplicte cultures. *P<.1 versus control. The result is representtive of three seprte experiments using smples from different ptients. (B) Immunocytochemistry of IL-17RC in endometriotic stroml cells. Cultured endometriotic stroml cells were immunostined with nti-il-17rc ntiody (i) or got IgG isotype mtched control (ii). Brs ¼ mm. FIGURE (A) Interleukin (IL)-17F stimultes IL-8 secretion from endometriotic stroml cells (ESCs). The ESCs were cultured in 5% fetl ovine serum with different doses of IL-17F for hours. (B) Effect of mouse nti-il-17ra ntiody or got nti-il-17rc ntiody on IL-17F-induced IL-8 secretion y ESCs. ESCs were preincuted in 5% fetl ovine serum medium with or without the ntiodies or the control ntiodies for 3 minutes nd then stimulted with IL-17F (5 ng/ml) for hours. (A, B) Concentrtion of IL-8 in the conditioned medium ws mesured using specific ELISA. Vlues re the mens SEM of pentplicte cultures. Different letters denote significnt differences etween groups (P<.5). The result is representtive of four seprte experiments using smples from different ptients. IL - 8 (ng/ml ) c control.1ng.1ng 1ng 1ng 1ng Synergistic Effect of IL-17F nd TNF- on IL-8 Secretion From ESCs The TNF-, together with IL-17F, triggered IL-8 secretion more thn the comined levels generted y ech stimulus lone (Fig. 3). This synergistic effect ws pprent when TNF- (1 ng/ ml) ws comined with 1 ng/ml IL-17F, nd mximl synergy ws otined t the highest dose of IL-17F tested (1 ng/ml). Effect of IL-17F on the Expression of IL-8 mrna nd COX mrna in ESCs We conducted time-course experiments to determine the effect of IL-17F on the expression of IL-8 mrna (Fig. A) nd COX mrna (Fig. B). Rel-time quntittive PCR nlysis demonstrted Fertility nd Sterility â 115

4 FIGURE 3 Effects of interleukin (IL)-17F on tumor necrosis fctor (TNF-)- medited IL-8 secretion from endometriotic stroml cells. Endometriotic stroml cells were treted with IL-17F or TNF- or in comintion for hours. The conditioned medium ws collected nd ssyed for IL-8 concentrtion using specific ELISA. All vlues re expressed s the men SEM of pentplicte cultures. *P<.1 versus ech control. The dt re representtive of four independent experiments. FIGURE Effect of interleukin (IL)-17F on the expression of IL-8 (A) nd cyclooxygense (COX) (B) messenger RNA in endometriotic stroml cells (ESCs). The ESCs were incuted with IL-17F (1 ng/ ml) for the indicted durtion. Expression of IL-8 nd COX messenger RNA in ESCs ws exmined y rel-time quntittive polymerse chin rection (PCR). The dt shown re the reltive rtio (A, IL-8 to glycerldehyde-3-phosphte dehydrogense (GAPDH); B, COX to GAPDH) mesured y rel-time quntittive PCR. Dt re the men SEM of five independent experiments using smples from five different ptients. Different letters denote significnt differences etween groups (P<.5). I L - 8 / G A P D H ( f o l d o f c o n t r o l ) h h h 8h 1h h tht IL-17F up-regulted IL-8 nd COX mrna. Mximl increses in IL-8 nd COX mrna were oserved t hours, followed y decrese with time up to hours. The mximl increse of IL-8 mrna ws.9-fold of the control nd tht of COX mrna ws 9.-fold of the control. DISCUSSION In the present study, we first demonstrted expression of IL-17F in EMMCs. We then showed tht IL-17F stimulted IL-8 secretion from ESCs. The IL-17F incresed IL-8 mrna nd COX mrna to the mximl levels t hours, followed y decrese with time up to hours. These response ptterns pper to reflect selfprotection of the cells from extreme inflmmtory rections tht might dmge the cells. We previously reported tht ESCs expressed IL-17RA (7). In the present study, we showed tht ESCs lso expressed IL-17RC nd tht ntiodies for IL-17RA nd IL-17RC inhiited IL-17F-induced IL-8 secretion. The TNF- synergisticlly enhnced IL-17F-induced IL-8 secretion. In ddition, IL-17F stimulted COX expression in ESCs. Pleiotropic functions of IL-8, such s chemottrction nd ctivtion of neutrophils, ngiogenesis, stimultion of prolifertion, nd survivl of endometril cells, re suggested to promote endometriosis (1, 1). The present study provided evidence tht IL-17F stimultes IL-8 secretion in ESCs, suggesting tht IL-17F prticiptes in the development of endometriosis. The present study lso showed tht IL-17F stimultes IL-8 secretion in ESCs through IL-17RA nd IL-17RC. Our previous report demonstrted tht IL-17A stimultes IL-8 secretion in ESCs through IL-17RA (7). It hs een shown tht IL-17RA nd IL-17RC re necessry for the ioctivity of IL-17A nd IL-17F (9). Furthermore, it hs een demonstrted tht solule form of IL-17RC could neutrlize the ctivity of IL-17A nd IL-17F (1). Accordingly, IL-17RA nd IL-17RC could e trget for novel therpy of endometriosis. C O X / G A P D H ( f o l d o f c o n t r o l ) h h h 8h 1h h The synergistic effect of IL-17F nd TNF- in stimulting secretion of IL-8 from ESCs ws remrkle. Similr findings were reported for humn ronchil epithelil cells (3) nd colonic myofirolsts (18). The TNF- is proinflmmtory cytokine tht plys multiple roles in the progression of endometriosis (). During the inflmmtory response, TNF- is secreted from vrious cell types, such s peritonel mcrophge, endometril epithelil, nd stroml cells. The IL-17F my ply role s n ccelertor of progression of endometriosis given tht chronic pelvic inflmmtion entils incresed TNF- levels in the endometriotic tissues. The COX, key enzyme in prostglndin iosynthesis, is upregulted in the lesions of endometriosis (31, 3). It plys n importnt role in the inflmmtory responses y producing prostglndins nd is involved in the development of endometriosis (33). The present finding tht IL-17F induced COX expression in ESCs provides further evidence for involvement of IL-17F in endometriosis. 11 Hirt et l. IL-17F increses IL-8 in endometriosis Vol. 9, No. 1, July 11

5 Interleukin-17A nd IL-17F induce similr inflmmtory responses, inducing the proinflmmtory cytokines nd chemokines in mny different cell types (3, 35). However, mice lcking either IL-17A or IL-17F exhiit distinct defects in experimentl model of sthm nd colitis (15). In ddition, IL-17A nd IL-17F re not lwys coexpressed or coregulted (3). Therefore, IL- 17A nd IL-17F re suggested to hve distinct roles, lthough overlpping in mny prts, in vrious physiologicl nd pthologicl events. The present study indicted similr roles of IL-17A nd IL-17F s proinflmmtory fctor in the development of endometriosis. An dditionl study my revel different roles, if ny, of these molecules in endometriosis. Our preliminry study showed tht mononucler cells from norml endometril tissues ppered to secrete lower levels of IL-17F in the sl sttus, wheres higher levels of IL-17F under PMA nd ionomycin stimultion compred with those in endometriotic tissues (dt not shown). 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