possible mechanism for the altered immunological functions in

J. Clin. Endocrinol. Metab. 2004 89: 5094-5100, doi: 10.1210/jc.2004-0354

Masakazu Nishida, Kaei Nasu, Junichiro Fukuda, Yasushi Kawano, Hisashi Narahara and Isao Miyakawa

Possible Mechanism for the Altered Immunological Functions in EndometriosisDysregulated Expression of CXC Chemokines in Endometriotic Stromal Cells: A

Down-Regulation of Interleukin-1 Receptor Type 1 Expression Causes the

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Down-Regulation of Interleukin-1 Receptor Type 1Expression Causes the Dysregulated Expression of CXCChemokines in Endometriotic Stromal Cells: A PossibleMechanism for the Altered Immunological Functionsin Endometriosis

MASAKAZU NISHIDA, KAEI NASU, JUNICHIRO FUKUDA, YASUSHI KAWANO,HISASHI NARAHARA, AND ISAO MIYAKAWA

Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Oita 879-5593, Japan

To evaluate the involvement of chemokines in the pathogen-esis of endometriosis, we investigated the expression of CXCchemokines in cultured ovarian endometriotic cyst stromalcells (ECSC), endometrial stromal cells with endometriosis(ESCwE), and normal endometrial stromal cells (NESC). Us-ing ELISA, TNF-� significantly enhanced the production ofIL-8, growth-related oncogene �, and epithelial neutrophil-activating peptide-78 in all cases of ECSC (n � 10), ESCwE (n �6), and, NESC (n � 10). IL-1� did not affect the production ofthese chemokines in eight of 10 cases of ECSC. In contrast,IL-1� significantly enhanced the expression of these chemo-kines in all cases of ESCwE (n � 6) and NESC (n � 10). Westernblot analysis revealed down-regulation of expression of IL-1receptor type 1 (IL-1-R1) in all cases of ECSC with low re-

sponse to IL-1� (n � 8). In contrast, significant IL-1-R1 ex-pression was detected in all cases of NESC. Although IL-1-R1expression was detected in all cases of ESCwE (n � 6), itsexpression in ESCwE tended to decrease compared with thatin NESC. Moreover, phosphorylation of inhibitor �B-� wasdetected in all cases of ESCwE and NESC after stimulationwith IL-1�, but not in ECSC with low response to IL-1� (n � 8).In contrast, significant IL-1-R2 expression was detected in allcases of ECSC, ESCwE, and NESC. The present findings sug-gest that the dysregulation of IL-1/IL-1-R system relates toimmunological dysfunction in endometriosis. The alterationof the CXC chemokines expression may be important for elu-cidation of the pathogenesis of endometriosis. (J Clin Endo-crinol Metab 89: 5094–5100, 2004)

ENDOMETRIOSIS IS A common chronic inflammatorydisease affecting 3–10% of reproductive women; it is

associated with chronic pelvic pain, dysmenorrhea, and in-fertility (1). Infertility is often determined during examina-tion of the patients with endometriosis and is a critical prob-lem for these patients. In addition to the obstruction offallopian tubes due to adhesion, endometriosis negativelyaffects embryo implantation and development in the earlystage and sperm motility, which suggests the involvement ofimmunological changes associated with this disease (1–5).However, the underlying fundamental mechanisms of al-tered immunological status are still unknown.

The most widely accepted theory about the pathogenesisof endometriosis is that it is a consequence of implantationof viable endometrial tissues in the pelvis via retrogrademenstruation (6, 7). It has been suggested that the ip envi-ronment is important in the formation of endometriotic le-sions (1, 7, 8). Estrogen encourages the development of en-

dometriotic lesions, and progesterone inhibits diseaseprogression (9). Besides these ovarian steroid hormones, re-cent studies have reported that a variety of cytokines, suchas IL-1 (10), IL-6 (11), IL-8 (12), interferon-� (IFN-�) (13), andTNF-� (14), form complex networks and play important rolesin the growth and expansion of endometriotic lesions.

Chemokines are a large superfamily of structurally andfunctionally related molecules with chemotactic activity tar-geted at specific leukocyte populations. They are 70–90amino acids in length and are divided into four major sub-families based on the relative positions of their cysteine res-idues (CC, CXC, C, and CX3C) (15–17). The CXC chemokinesubfamily includes IL-8, growth-related oncogene � (GRO�),GRO�, GRO�, epithelial neutrophil-activating peptide 78(ENA-78), granulocyte chemotactic protein-2, and neutro-phil-activating protein-2, many of which have been shown tochemoattract and activate neutrophils (15–18). Normal en-dometrial stromal cells (NESC) have been reported to pro-duce and secrete various CXC chemokines, including IL-8(19), GRO� (20), and ENA-78 (21), especially after stimula-tion with proinflammatory cytokines, such as IL-1 andTNF-�. The expression of these chemokines has been sug-gested to be important in menstruation and implantation andin the maintenance of early pregnancy (22). These chemo-kines are also reported to be expressed in endometriotictissues (23, 24).

Many studies have reported the immunological disorders

Abbreviations: ECSC, Endometriotic cyst stromal cell; ENA-78, epi-thelial neutrophil-activating peptide-78; ESCwE, endometrial stromalcell with endometriosis; FBS, fetal bovine serum; GAPDH, glyceralde-hyde-3-phosphate-dehydrogenase; GRO, growth-related oncogene; I�B-�,inhibitor �B-�; IFN-�, interferon-�; IL-1-R1, IL-1 receptor type 1; NESC,normal endometrial stromal cell; TNF-R1, TNF receptor type 1.JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endo-crine community.

0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(10):5094–5100Printed in U.S.A. Copyright © 2004 by The Endocrine Society

doi: 10.1210/jc.2004-0354

5094

in the pelvic cavity of endometriosis. A variety of cytokines,such as IL-1 (10), IL-6 (8), IL-8 (12), and ENA-78 (25), havebeen reported to be increased in the peritoneal fluid of en-dometriotic patients. These chemokines are considered veryimportant in the pathogenesis of endometriosis, but therehave been few reports on the expression of chemokines ineutopic and ectopic endometrial tissue of endometrioticpatients.

In this study we investigated the production of CXC che-mokines (IL-8, GRO�, and ENA-78) in cultured endometri-otic cyst stromal cells (ECSC), eutopic endometrial stromalcells with endometriosis (ESCwE), and NESC and the ex-pression of IL-1 receptor type 1 (IL-1-R1), IL-1-R2, TNF re-ceptor type 1 (TNF-R1), and TNF-R2 in these cells, and wediscuss the involvement of altered immunological functionin the pathogenesis of endometriosis.

Materials and MethodsECSC, ESCwE, and NESC isolation procedures and cellculture conditions

NESC (n � 10) were obtained from premenopausal patients who hadundergone hysterectomies for leiomyoma. ESCwE (n � 6) were obtainedfrom premenopausal patients who had undergone hysterectomies forendometriosis. ECSC were obtained from premenopausal patients whohad undergone salpingo-oophorectomy or evisceration for ovarian en-dometriotic cysts (n � 10). All patients had been free of any hormonaltreatments before the operation. All specimens were diagnosed as beingin the mid to late secretory phase using a standard histological exam-ination of endometrial tissues. This study was approved by the insti-tutional review board of the Faculty of Medicine, Oita University, andwritten informed consent was obtained from all patients.

ECSC were isolated from endometriotic tissues according to themethod described by Osteen et al. (26). Briefly, the tissues were mincedin Hanks’ balanced salt solution and digested with 0.5% collagenase(Invitrogen Life Technologies, Gaithersburg, MD) in DMEM (InvitrogenLife Technologies) at 37 C for 40 min. The dispersed cells were filteredthrough a 70-�m pore size nylon mesh to remove the undigested tissuepieces. The filtrated fraction was separated from epithelial cell clumpsby differential sedimentation at unit gravity as follows. The cells wereresuspended in 2 ml culture medium and layered slowly over 10 ml ofthe medium in a centrifuge tube. Sealed tubes were placed in an uprightposition at 37 C in 5% CO2 in air for 30 min. After sedimentation, the top8 ml medium were collected. Finally, the medium containing stromalcells was filtered through a 40-�m pore size nylon mesh. Final purifi-cation was achieved by allowing stromal cells (which attach rapidly toplates) to adhere selectively to culture dishes for 30 min at 37 C, followedby the removal of nonadhering epithelial cells. ESCwE and NESC werealso isolated by digesting the endometrial tissues fragments with 0.5%collagenase, as previously described (21). Isolated ECSC, ECSCwE, andNESC were cultured in DMEM supplemented with 100 IU/ml penicillin(Invitrogen Life Technologies), 50 mg/ml streptomycin (Invitrogen LifeTechnologies), 2.5 �g/ml amphotericin B (Sigma-Aldrich Corp., St.Louis, MO), and 10% heat-inactivated fetal bovine serum (FBS; Invitro-gen Life Technologies) at 37 C in 5% CO2 in air.

ECSC, ESCwE, and NESC in monolayer culture after the third pas-sage were more than 99% pure, as analyzed by immunocytochemicalstaining with antibodies to vimentin (V9; Dako, Copenhagen, Denmark),cytokeratin (Dako), factor VIII (Dako), and leukocyte common antigen(2B11�PD7/26, Dako) and were used for the following experiments.

Detection by ELISA of IL-8, GRO�, and ENA-78 in culturemedia of ECSC, ESCwE, and NESC

To study the production of IL-8, GRO�, and ENA-78 by ECSC,ESCwE, and NESC, we plated 5 � 106 cells on six-well culture plates(Corning Glass, Corning, NY) and cultured the cells until they were fullyconfluent. The supernatant was then replaced with 1 ml fresh culturemedium containing various amounts of recombinant human IL-1�

(0.001–1 ng/ml; R&D Systems, Minneapolis, MN) and recombinant hu-man TNF-� (0.1–100 ng/ml; R&D Systems). Under these conditions, thesupernatant was collected 24 h after stimulation and stored at �70 Cuntil assay. Isolated cells from individual patients were used in eachexperiment, and each experiment was performed in triplicate. All ex-periments were performed in the presence of 10% heat-inactivated FBS.The concentrations of IL-8, GRO�, and ENA-78 were determined in eachsupernatant with commercially available ELISA kits (R&D Systems).The sensitivities of the assay were 10 pg/ml for IL-8, 10 pg/ml for GRO�,and 15 pg/ml for ENA-78.

Western blotting analysis

ECSC, ESCwE, and NESC were plated in 100-mm dishes and culturedto confluence. Then TNF-� (100 ng/ml) and IL-1� (1 ng/ml) were addedto the dishes, which were cultured for 5 min. The time for stimulationwas determined by a time-course study performed as a backgroundexperiment. After stimulation, the cells were washed with PBS, andwhole cell extracts were prepared by lysing the cells in lysis buffer (50mM Tris-HCl, 125 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA, 50 mmNaF, and 0.1% phenylmethylsulfonylfluoride). The suspension was cen-trifuged at 15,000 rpm for 15 min at 4 C, and the supernatant wascollected. The total protein concentration was quantified using the Coo-massie protein assay regent (Pierce Chemical Co., Rockford, IL). Thewhole cell protein extract was resolved with SDS-PAGE using a 10%polyacrylamide gel under reduced conditions. After transfer to Immo-bilon-P transfer membrane (Millipore Corp., Bedford, MA), the proteinwas stained with Ponceau S (Sigma-Aldrich Corp.) to verify uniformloading and transfer. Membranes were blocked with 5% skim milk (BDBiosciences, Sunnyvale, CA) in Tris-buffered saline with Tween 20 (50mm Tris-HCl, 150 mm NaCl, and 0.1% Tween 20, pH 7.4; TBS-T) over-night and subsequently incubated with primary antibodies [IL-1-R1,IL-1-R2, TNF-R1, and TNF-R2 (Rockland, Gilbertsville, PA); nonphos-phoserine inhibitor �B-� (I�B-�; non-pI�B-�) and phosphoserine(pI�B-�; Cell Signaling, Beverly, MA); and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Ambion, Inc., Austin, TX)] with appropriatedilution for 1 h at room temperature. The membrane was washed threetimes with TBS-T and incubated with the appropriate horseradish per-oxidase-conjugated secondary antibody for 1 h at room temperature.Subsequently, the membrane was washed three times with TBS-T andanalyzed by enhanced chemiluminescence (Amersham Biosciences, Ar-lington Heights, IL).

Preparation of IL-1-R1 cDNA fragment by RT-PCR

To evaluate the expression of IL-1-R1 mRNA in ECSC, ESCwE, andNESC by Northern blot analysis, we amplified the IL-1-R1 transcript bymeans of the RT-PCR method using an RNA PCR kit with AMV reversetranscriptase (Takara, Tokyo, Japan) as previously described (27). TotalRNA was isolated from NESC using TRIzol reagent (Invitrogen LifeTechnologies) according to the manufacturer’s instructions and wasreverse transcribed into cDNA. To perform the PCR, primer sets forIL-1-R1 (sense primer, 5�-AAGGTGGAGGATTCAGGACAT-3�; anti-sense primer, 5�-AGCCTATCTTTGACTCCACTA-3�) (28) were synthe-sized by the phosphoramide method on a DNA synthesizer (model 8700;Biosearch, San Rafael, CA) and purified on Sephadex G-50 columns(Pharmacia Biotech, Piscataway, NJ) and by HPLC. The predicted sizeof the PCR product was 284 bp. The cDNA transcribed from 1 �g totalRNA was amplified using a thermal cycler (model PJ2000; PerkinElmer,Norwalk, CT). The PCR with primer pairs for IL-1-R1 was performed for35 cycles, with each cycle consisting of a denaturation step of 94 C for45 sec, an annealing step of 55 C for 45 sec, and an extension step of 72C for 60 sec. The PCR products were separated by 1.5% agarose gel(Takara) electrophoresis and visualized by ethidium bromide (Takara)staining. The PCR products were cloned using a TA cloning kit (In-vitrogen Life Technologies, Leek, The Netherlands) and used as theprobe for Northern blot analysis. Sequence analysis of the PCR productswas also performed to confirm that the amplified cDNA was IL-1-R1.

Northern blot analysis for IL-1-R1 mRNA expression inECSC, ESCwE, and NESC

To study the expression of IL-1-R1 mRNA in ECSC, ESCwE, andNESC, 5 � 106 cells of ECSC with low responses to IL-� (n � 4), ESCwE

Nishida et al. • Involvement of Chemokines in Pathogenesis of Endometriosis J Clin Endocrinol Metab, October 2004, 89(10):5094–5100 5095

(n � 4), and NESC (n � 4) were plated on 75-cm2 culture flasks (CorningGlass) in 15 ml culture medium with 10% heat-inactivated FBS andcultured until fully confluent. Total RNA was isolated from ECSC,ESCwE, and NESC using TRIzol reagent according to the manufacturer’sinstructions. Northern blotting was performed as previously described(27). The expression of �-actin mRNA was also examined as an internalcontrol. The relative expression of IL-1-R1 and �-actin mRNA imageswas analyzed using the public domain Image program 1.61 developedat National Institutes of Health (Bethesda, MD).

Statistical analysis

Data are presented as the mean � sd and were appropriately ana-lyzed using the Mann-Whitney U test and the Bonferroni/Dunn testwith StatView 4.5 (Abacus Concepts, Inc., Berkeley, CA). P � 0.05 wasaccepted as statistically significant.

Results

Levels of IL-8, GRO�, and ENA-78 in the culture mediumwithout cells were below the levels of detection. Low levelsof IL-8, GRO�, and ENA-78 protein were detected in theculture medium of nonstimulated ECSC, ESCwE, and NESCincubated for 24 h. As shown in Fig. 1, the concentrations ofthese three chemokines stimulated by TNF-� (10 ng/ml)were higher (�2.5-fold) than those without stimulation in allspecimens of ECSC (n � 10), ESCwE (n � 6), and NESC (n �

10). With IL-1� stimulation (0.1 ng/ml), the chemokine con-centrations were increased more than 2.5-fold comparedwith those under nonstimulated conditions in all ESCwE(n � 6) and NESC (n � 10) specimens, whereas only two of10 ECSC specimens showed more than a 2.5-fold increase inthe chemokine concentration.

As shown in Fig. 2, levels of IL-8, GRO�, and ENA-78 wereincreased in parallel with the addition of TNF-� in all casesof ECSC (n � 10). In contrast, IL-1� did not affect the pro-duction of IL-8, GRO�, or ENA-78 in eight of 10 specimensof ECSC (low response group). The results for the remainingtwo specimens (high response group) were similar to thoseof ESCwE and NESC (date not shown). Levels of IL-8, GRO�,and ENA-78 increased with the addition of increasingamounts of TNF-� and IL-1� in all cases of NESC (n � 10)and ESCwE (n � 6).

To analyze the underlying mechanisms of the above find-ings, we evaluated the signal pathways of IL-1�- and TNF-�-stimulated CXC chemokine production in ECSC, ESCwE,and NESC. As shown in Fig. 3, significant expression ofIL-1-R1 was detected in all cases of NESC (n � 10), but onlya faint expression of IL-1-R1 was observed in all cases ofECSC with low response to IL-1� (n � 8). IL-1-R1 expression

FIG. 1. Comparison of IL-8, GRO�, and ENA-78 production among ECSC, ESCwE, and NESC after stimulation by TNF-� (A–C; 10 ng/ml) andIL-1� (D–F; 0.1 mg/ml). The data are presented as relative percentages for nonstimulated controls. The concentrations of three chemokinesstimulated by TNF-� showed more than a 2.5-fold increase compared with nonstimulated controls in all specimens of ECSC (n � 10), ESCwE(n � 6), and NESC (n � 10). Whereas IL-1� stimulation induced more than a 2.5-fold increase in the levels of the three chemokines in all samplesof NESC (n � 10) and ESCwE (n � 6), only two of 10 samples in ECSC showed an elevation greater than 2.5-fold. *, P � 0.0001 vs. NESC (byMann-Whitney U test).

5096 J Clin Endocrinol Metab, October 2004, 89(10):5094–5100 Nishida et al. • Involvement of Chemokines in Pathogenesis of Endometriosis

in ECSC with high response to IL-1� (n � 2) and ESCwE (n �6) was also detected; however, its expression in these cellstended to decrease compared with that in NESC. Significantexpression of IL-1-R2, TNF-R1, and TNF-R2 was detected in

all cases of ECSC (n � 10), ESCwE (n � 6), and NESC (n �10). The expression of these four receptors after 24-h stim-ulation by TNF-� and IL-1� was unchanged compared withthat under unstimulated conditions (date not shown). As

FIG. 2. Concentrations of IL-8, GRO�, and ENA-78 in the culture media of ECSC, ESCwE, and NESC after 24-h stimulation with TNF-� (A–C)and IL-1� (D–F). �, ECSC; o, ESCwE; f, NESC. *, P � 0.005; **, P � 0.0001 (vs. unstimulated controls, by Bonferroni/Dunn test). The dataare expressed as the mean � SD of triplicate samples of a representative experiment.

FIG. 3. Expression of IL-1-R1, IL-1-R2, TNF-R1, TNF-R2, and GAPDH protein in nonstimulated ECSC (n � 10), ESCwE (n � 6), and NESC(n � 10).


shown in Fig. 4, nonstimulated ECSC, ESCwE, and NESCrevealed significant non-pI�B-� and faint pI�B-� protein ex-pression. pI�B-� protein was not detected after 5-min stim-ulation by IL-1� (1 ng/ml) in ECSC with a low response toIL-1� (n � 8), whereas a significant increase in pI�B proteinwas observed in ECSC with a high response to IL-1� (n � 2),ESCwE (n � 6), and NESC (n � 10). In contrast, after stim-ulation by TNF-� (100 ng/ml), a significant amount of pI�B-�protein was detected in all cases of ECSC (n � 10), ESCwE(n � 6), and NESC (n � 10). GAPDH was detected in allsamples at almost equal quantities.

As shown in Fig. 5, significant expression of IL-1R1 mRNAin ESCwE (n � 4) and NESC (n � 4) was observed. However,the expression of IL-1-R1 mRNA was significantly decreasedin ECSC with a low response to IL-1� (n � 4). IL-1-R1 mRNAexpression tended to decrease in ESCwE compared with thatin NESC. �-Actin mRNA was detected in all samples atalmost equal quantities.

Discussion

CXC chemokines are believed to be involved in the patho-genesis of endometriosis (1–5). However, only IL-8 expres-sion has been evaluated in endometriotic tissues (23). In thepresent study we demonstrated that the other CXC chemo-kines, GRO� and ENA-78, are also expressed in culturedECSC. Furthermore, the expression of IL-8, GRO�, andENA-78 was up-regulated in ECSC, ESCwE, and NESC afterstimulation by TNF-�. Phosphorylation of pI�B-� was de-tected after stimulation with TNF-� in ECSC, ESCwE, andNESC. The effects of TNF-� on ECSC and ESCwE weresimilar to those on NESC. IL-1� also enhanced the expressionof these three chemokines by ESCwE and NESC, but IL-1�stimulation did not affect the expression of these three che-mokines in most cases of ECSC. These results are supportedby the observation that the expression of IL-1-R1 mRNA and

protein was detected in NESC, but significantly decreased inECSC. Furthermore, phosphorylation of I�B-� after IL-1-�stimulation, an important intracellular signaling procedurein CXC chemokine production (29), was detected in ESCwEand NESC, but not in ECSC.

These results suggest that the regulatory mechanisms oflocal chemokine expression are partially altered in endome-triosis. Previous studies have demonstrated that the levels ofvarious substances in the pelvic cavity that are important inthe pathogenesis of endometriosis are altered when the dis-ease is present (1, 7, 8). Sakamoto et al. (30) reported that IL-8was produced in endometriotic tissues, especially afterTNF-� stimulation. Their findings are consistent with ourpresent results. Lebovic et al. (31, 32) reported that the pro-duction of regulated upon activation, normal T cell-expressed and secreted (RANTES), vascular endothelialgrowth factor, and IL-6 was enhanced by IL-1� stimulation.Akoum et al. (33) reported that macrophage inflammatoryprotein-1� expression was increased by estradiol and IL-1�-stimulation. However, relatively high doses of IL-1� wereused in these studies. In the present study we demonstratedthat the expression of IL-1-R1 protein and mRNA was down-regulated in most cases of ECSC, and IL-1� did not affect theproduction of CXC chemokines in these cases of ECSC. Fur-thermore, we indicated that the expression of IL-1-R1 proteinand mRNA in ESCwE tended to decrease compared with thatin NESC. It is suggested that the alteration of this receptorexpression has already begun at a eutopic location before thedevelopment of endometriotic lesions. Furthermore, thismay mean that the down-regulation of the IL-1/IL-1-R sys-tem in endometriotic lesions induces changes in various cel-lular functions in affected cells in addition to the alteredchemokine production, causes abnormality of the immunesystem in the pelvic cavity, and leads to infertility, especiallybecause of implantation failure of blastocysts.

FIG. 4. Effects of TNF-� (A; 100 ng/ml) and IL-1� (B; 1 ng/ml) stimulation on the levels of non-pI�B-�, pI�B-�, and GAPDH protein in ECSC,ESCwE, and NESC. The figure shows a representative result.


Iwabe et al. (23) demonstrated that IL-8 exerts its growth-promoting actions in endometriotic cells as well as in normalendometrial cells. Like IL-8, ENA-78 and GRO� belong to theCXC chemokine family, so it is expected that these chemo-kines may play similar roles in the development ofendometriosis.

There is much evidence suggesting that cytokines such asIL-1 play a critical role in the process of blastocyst implan-tation (34–37). The expression of IL-1-R1 mRNA occurs dur-ing the entire menstrual cycle in normal human endometrialstromal and epithelial cells, but it is significantly increased inboth the early and late secretory phases (34, 38). Severalstudies have reported that blocking of IL-1-R1 prevents em-bryo implantation. Lindhard et al. (36) reported that micetreated with an IL-1-R antagonist express a lower level ofintegrins that are active during the adhesion process duringendometrial receptivity, and that the murine embryostreated with an IL-1-R antagonist had the capacity to implantand develop normally when transferred to normal mice.Given this finding, IL-1-R seems to be an important cytokinewhen pregnancy, especially implantation, occurs.

Various cytokines, such as IL-1�, TNF-�, IL-8, IL-13, IL-15,and IFN-�, have been reported to be differently expressedamong eutopic and ectopic endometrial cells with endome-triosis and NESC (24, 39). Sotnikova et al. (24) reported the

differential expression of cytokines, including TNF-�, IL-1�,IFN-�, and IL-8 in eutopic and ectopic endometrial cells withendometriosis and NESC. They reported that TNF-� expres-sion in eutopic and ectopic endometrial cells with endome-triosis was significantly enhanced compared with that inNESC. In contrast, IL-1� expression in ectopic endometrialcells with endometriosis was reduced compared with that ineutopic endometrial cells with or without endometriosis.These results are consistent with our present results, whichindicated the decrease in IL-1-R1 mRNA levels in ECSC.However, the underlying mechanism of this phenomenon isstill unclear. Additional research on genomic rearrangementis required. Akoum et al. (40) reported that the expression ofIL-1-R2, which is described as an IL-1 decoy receptor andplays an important physiological role in IL-1 regulation as acompetitor of IL-1-R1; in endometriotic cells, it decreasescompared with the level in NESC. However, in our presentstudy there were no differences in IL-1-R2 expression amongECSC, ESCwE, and NESC.

In summary, we demonstrated for the first time that IL-1-R1 protein and mRNA expressions are down-regulated inmost cases of ECSC, leading to dysregulated expression ofCXC chemokines. A poorly regulated immunological statusdue to the lack of IL-1-R1 in ECSC may lead to reproductivefailure. At present, the significance of our findings is not fullyunderstood, but we hope that these discoveries will be usefulin elucidating the pathogenesis of endometriosis.

Acknowledgments

Received February 23, 2004. Accepted July 20, 2004.Address all correspondence and requests for reprints to: Dr.

Masakazu Nishida, Department of Obstetrics and Gynecology, Facultyof Medicine, Oita University, Hasama-machi, Oita 879-5593, Japan. E-mail: [email protected].

This work was supported in part by Grant-in-Aid 16591672 (to K.N.)for Scientific Research from the Japan Society for the Promotion ofScience and a grant-in-aid from the Yamaguchi Endocrine ResearchAssociation (to K.N.).

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