ENDOMETRIOSIS, DEFINED AS the presence of endometrial

Transcription

1 X/05/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 90(3): Printed in U.S.A. Copyright 2005 by The Endocrine Society doi: /jc Regulation of Interleukin-8 Expression in Human Endometrial Endothelial Cells: A Potential Mechanism for the Pathogenesis of Endometriosis Janelle Luk, Yasemin Seval, Umit A. Kayisli, Murat Ulukus, Cagnur E. Ulukus, and Aydin Arici Departments of Obstetrics and Gynecology (J.L., Y.S., U.A.K., M.U., A.A.) and Pathology (C.E.U.), Yale University School of Medicine, New Haven, Connecticut 06520; Department of Histology and Embryology, Akdeniz University School of Medicine (Y.S., U.A.K.), Antalya 07070, Turkey; Department of Obstetrics and Gynecology, Ege University School of Medicine (M.U.), Izmir 35100, Turkey; and Department of Pathology, Dokuz Eylul University School of Medicine (C.E.U.), Izmir 35340, Turkey The elevation of the proinflammatory chemoattractant cytokine levels in ectopic and eutopic endometrium of endometriosis implies an inflammatory basis for this disease. The relationship between endothelial cells and leukocytes is likely to be important in the regulation of inflammatory mediators of endometriosis. The aim of this study was to describe the temporal and spatial expression of IL-8 in human endometrial endothelial cells (HEEC) in vivo and to compare the in vitro regulation of IL-8 expression by sex steroids in HEEC from women with or without endometriosis. Eutopic endometrial tissues and endometriosis implants were grouped according to menstrual cycle phase and examined by immunohistochemistry for IL-8 expression. Endothelial cells of endometriotic implants expressed higher IL-8 immunoreactivity compared with endothelial cells of eutopic endometrium from women with or without endometriosis (P < 0.02). For in vitro studies, First Published Online December 21, 2004 Abbreviations: C/EBP, CCAAT/enhancer-binding protein; ER, estrogen receptor; FBS, fetal bovine serum; HEEC, human endometrial endothelial cells; HSCORE, histological score; NF- B, nuclear factor- B. JCEM is published monthly by The Endocrine Society ( endo-society.org), the foremost professional society serving the endocrine community HEEC were isolated from women with or without endometriosis and grown to preconfluence. The purity of cultured HEEC (90 95%) was confirmed by immunocytochemistry using endothelium-specific markers, CD31 and CD146. The effects of estradiol ( M), progesterone (10 7 M), or both on IL-8 mrna and protein levels were analyzed by RT-PCR and ELISA, respectively. Sex steroids reduced the expression of IL-8 mrna and protein in HEEC from women without endometriosis. In contrast, both estradiol and progesterone stimulated IL-8 mrna and protein expression in HEEC from women with endometriosis. We postulate that the stimulation of chemokine expression by sex steroids in HEEC of women with endometriosis may play a role in the inflammatory aspect of this disease. (J Clin Endocrinol Metab 90: , 2005) ENDOMETRIOSIS, DEFINED AS the presence of endometrial tissue outside the uterine cavity, is a gynecological disease that causes pelvic pain and infertility in about 10% of women in reproductive age. Although our current understanding of the pathogenesis and pathophysiology of the disease is poor, the most widely accepted theory is that viable endometrial cells are shed into the peritoneal cavity by retrograde menstruation, with subsequent implantation and growth (1). Retrograde menstruation is nearly a universal phenomenon among cycling women (2, 3), but it is not clear why endometrial tissue will implant and grow in the peritoneal cavity of only a subgroup of women. Several studies have shown that there is an elevation of proinflammatory chemoattractant cytokines, such as IL-8, in the peritoneal fluid from patients with endometriosis (4 6). IL-8, a cytokine with neutrophil chemotactic/activating activity and T cell chemotactic activity (4, 7), plays a major role in the recruitment of leukocytes to endometrium. Some of the known functions of IL-8 include increased expression of surface adhesion molecules on neutrophils, angiogenesis, and mitogenesis of epidermal and vascular smooth muscle cells (8). Moreover, it induces the proliferation of endometrial stromal cells, acting as an autocrine growth factor to the endometrium (9). In the human endometrium, IL-8 shows a cyclical pattern throughout the menstrual cycle at the protein and mrna levels, with peak levels during the late secretory and early to midproliferative phases (4). Moreover, Milne et al. (10) demonstrated that the endometrium obtained from women who received progesterone for 4 d revealed increased expression of IL-8 mrna 48 h after the withdrawal of progesterone, which simulated luteal regression. In women with endometriosis, the peritoneal fluid levels of IL-8 are elevated and correlate with the stage of the disease (5, 11). Potential sources of IL-8 are macrophages, fibroblasts, mesothelial cells, and ovarian cells. However, chemokine production by endometrial endothelial cells has never been explored. Endothelial cells form a monolayer that lines the vascular system. Their structure and function are fundamental to the maintenance of blood vessel wall homeostasis as well as leukocyte migration. However, during each menstrual cycle human endometrial endothelial cells (HEEC) undergo cyclic remodeling under the influence of hormonal changes. In this sense, HEEC differ from endothelial cells at other parts of the body. We have developed an efficient method in isolating HEEC (12). Moreover, we have carried out proliferation as-

2 1806 J Clin Endocrinol Metab, March 2005, 90(3): Luk et al. Regulation of IL-8 Expression in HEEC says and angiogenic tubular assays in which the HEEC have been shown to be responsive to steroid hormones (12). Through immunohistochemistry, we have previously shown that IL-8 is expressed in endometrial endothelial cells, glandular cells, and perivascular stroma (4). However, the effect of ovarian steroids on IL-8 expression in HEEC is still unknown, and the role of this effect in the pathogenesis of endometriosis has not been investigated. Because HEEC are the principle regulators of leukocyte extravasation into the endometrium, we analyzed the influence of steroid hormones on IL-8 expression in HEEC from women with or without endometriosis to study the steroid-mediated regulation of HEEC function in eutopic endometrium and endometriotic tissues. Materials and Methods Tissue collection Endometrial tissues were obtained from human uteri after hysterectomy or from endometrial biopsies performed at the time of laparoscopy from women who did not receive any hormonal medications in the preceding 3 months. The presence of endometriosis was confirmed by histological examination of the specimens. Tissue samples were grouped according to the menstrual cycle phase based on the last menstrual period and were confirmed by histological examination of the endometrium. The presence of endometriosis was confirmed by histological examination of the biopsies. We evaluated seven (three proliferative and four secretory) samples of endometrium from women without endometriosis confirmed by laparoscopy and eight eutopic (four proliferative and four secretory) and seven homologous ectopic endometrial samples (five ovarian endometriomas and two peritoneal implants) from women with endometriosis confirmed by laparoscopy or hysterectomy for immunohistochemical analysis. Informed consent in writing was obtained from each patient before surgery. Consent forms and protocols were approved by the human investigation committee of Yale University. The mean age of patients without endometriosis was 38.3 yr (range, yr). The mean age of patients with endometriosis was 27.4 yr (range, yr). For the cell cultures, the tissues were placed in Hanks balanced salt solution and transported to the laboratory for endometrial endothelial cell isolation and long-term culture. Each experimental set-up was repeated on at least three occasions using cells obtained from different patients. Immunohistochemistry Formalin-fixed slides were embedded in paraffin and cut into 5- m sections. Slides were deparaffinized in xylene and rehydrated in a graded series of ethanol, then boiled in citrate buffer (10 mm; ph 6.0) for 30 min for antigen retrieval. Then sections were immersed in 1% H 2 O 2 in PBS for 15 min to block endogenous peroxidase. Slides were incubated with blocking horse serum (Vector Laboratories, Inc., Burlingame, CA) for 10 min at room temperature in a humidified chamber. Excess serum was drained, and primary antibody [goat antihuman IL-8 (affinitypurified goat IgG), 1:50 dilution; R&D Systems, Inc., Minneapolis, MN] was added to the sections for 1 h. For the negative controls, nonspecific goat IgG isotypes were used at the same concentrations. The sections were rinsed, then biotinylated horse antigoat antibody (1.5 mg/ml; Vector Laboratories, Inc.) was added at a 1:250 dilution for 45 min at room temperature. The antigen-antibody complex was detected using an avidin-biotin-peroxidase kit (LabVision, Fremont, CA). 3,3-Diaminobenzidine tetrahydrochloride dihydrate was used as the chromogen, and the sections were counterstained with hematoxylin and mounted with Permount (Fisher Chemicals, Springfield, NJ). Immunohistochemical staining of endothelial cells for IL-8 was evaluated in a semiquantitative fashion [i.e. 0 (absent) to 3 (most intense)]. For each slide, an HSCORE (histological score) value was derived by summing the percentages of cells staining at each intensity multiplied by the weighted intensity of staining, that is, HSCORE P i (i 1), where i is the intensity score, and is the corresponding percentage of the cells. In each slide, five different areas were evaluated under a light microscope ( 40 magnification), and the percentage of the cells for each intensity within these areas was determined at different times by two investigators blinded to the type and source of the tissues. The inter- and intraindividual coefficient of variations were 15% and 7%, respectively, for the HSCORE evaluation. The average score of 2 was used. Isolation and culture of human endometrial stromal cells Endometrial stromal and glandular cells were separated and maintained in monolayer culture as described previously (13). Briefly, endometrial tissue was minced with a sterile stainless surgical blade and digested by incubation of tissue minces in Hanks balanced salt solution (Sigma-Aldrich Corp., St. Louis, MO) that contained HEPES (25 mmol), penicillin (200 U/ml), streptomycin (200 mg/ml), collagenase H (1 mg/ ml; 15 U/mg; Roche, Mannheim, Germany), and deoxyribonuclease (0.1 mg/ml; 1500 U/mg; Roche) for min at 37 C with agitation every 5 min using a 20-ml syringe. The dispersed endometrial cells were separated by filtration through a wire sieve (73- m diameter pore; Sigma-Aldrich Corp.). The endometrial glands (largely undispersed) were retained by the sieve, whereas the dispersed stromal and endothelial cells passed through the sieve into the filtrate. The stromal cells were plated in Ham s F-12/DMEM (1:1, vol/vol; Sigma-Aldrich Corp.) and fetal bovine serum (FBS; 10% vol/vol; Invitrogen Life Technologies, Gaithersburg, MD). Cells were plated in plastic flasks (75 cm 2 ; Falcon, Franklin Lakes, NJ), maintained at 37 C in a humidified atmosphere (5% CO 2 in air), and allowed to attach to the flask. On the second day of the culture, the medium was changed to remove unattached cells, dead cells, and erythrocytes. Preparation of microbead-conjugated anti-cd105-coated petri dishes CD105 (endoglin) is a glycoprotein and its expression is highly restricted to endothelium in all tissues except bone marrow (14). Microbead-conjugated anti-cd105 (20 l/ml; Miltenyi Biotec, Auburn, CA) solution was prepared in 50 mm Tris-Cl, ph 9.5. After adding the anti-cd105 microbead solution to petri dishes, the outside bottom surface of petri dishes was enforced with a magnet to enhance and stabilize antibody binding to dishes. Petri dishes (60 mm diameter; Falcon) were then incubated with the microbead-conjugated, anti-cd105 solution (3 ml for each petri dish) for 2 h at 37 C. Thereafter, the solution was removed, and each dish was rinsed three times with 0.15 m NaCl. Dishes were then incubated with BSA [0.1% (w/vol) in PBS] for 30 min at room temperature. The BSA solution was discarded before adding the cells. Isolation of HEEC On the second day of the endometrial stromal/endothelial cell culture ( 60 80% confluence), cells were washed with PBS, harvested by standard methods of trypsinization, and centrifuged at 1800 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in 2 ml/10 7 cells cell dilution buffer (PBS, ph 7.2, supplemented with 0.5% BSA and 2 mm EDTA). Thereafter, cells were filtered through 30- m pore size nylon mesh (Miltenyi Biotec) to prevent cells from clumping. After applying the filtered cell suspension onto the dishes coated with the CD105 antibody, the dishes were placed on a shaker, and CD105- positive cells were allowed to attach to antibody-covered beads with gentle agitation for 7 10 min at 8 10 C. Afterward, unattached cells were discarded, and dishes were rinsed several times with cell dilution buffer. Finally, remaining attached cells were cultured with EGM MV-Microvascular endothelial cell medium supplemented with SingleQuots containing growth factors, cytokines, and endothelial growth supplements (Bulletkits, Cambrex-Clonetics, Baltimore, MD). The medium was replaced each day. The HEEC were grown to 80 90% confluence in 37 C and 95% air with 5% CO 2 incubator. Cells were then split 1:4 for passaging to 60-mm culture dishes (Falcon) or to four-well chamber slides (Falcon). Immunocytochemistry of endothelial cells, glandular cells, and leukocyte-specific markers were carried out on second passage for cellular characterization. Second passage HEEC were also cultured in 12- well culture plates, and their appearance was recorded microscopically every 24 h until confluence. When cells reached 70 80% confluence, culture medium was switched for 24 h to a medium that contained 2%

3 Luk et al. Regulation of IL-8 Expression in HEEC J Clin Endocrinol Metab, March 2005, 90(3): charcoal-stripped, steroid-depleted FBS. All experiments were then carried out in a phenol red-free medium containing 2% charcoal-stripped, steroid-depleted FBS. Experimental set-up The passage of endothelial cells was performed by standard methods of trypsinization, plated in 12-well culture plates (3.8-mm diameter), and six-well culture plates (9.6-mm diameter), as appropriate for the experimental design, and allowed to replicate to confluence before commencement of each experiment. Experiments were performed using three replicate wells for each condition in a 12- or six-well culture plate. Supernatant from each 12-well plate was tested in a single ELISA, and the cells from the six-well plate were used for RNA isolation and mrna analysis. Treatments were estradiol ( m), progesterone (10 7 m), and estradiol ( m) plus progesterone (10 7 m). The concentrations and durations used in this study were determined based on our previous studies in which we have observed the HEEC are most responsive (12). Semiquantitative RT-PCR analysis Total RNA from cell cultures of women with (n 3) or without (n 3) endometriosis was extracted using TRIzol (Invitrogen Life Technologies, Inc., Carlsbad, CA) according to the instructions provided by the manufacturer. An aliquot of 0.5 g total RNA was reverse transcribed to cdna using AMV reverse transcriptase (Roche, Indianapolis, IN) for 1 h at 42 and 95 C for 5 min. The cdna was amplified using Taq polymerase (Promega Corp., Madison, WI) and primers for IL-8 (sense, 5 -GACAAGAGCCAGGAAGAAAC-3 ; antisense, 5 -CTACAACA- GACCCACACAATAC-3 ). PCR amplification of a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) was also performed to normalize the IL-8 PCR product. PCR was performed for 30 cycles under the following conditions: denaturation for 5 min at 95 C and extension FIG. 1. The expression of IL-8 in eutopic and ectopic endometrial samples. A, Proliferative endometrium of a woman without endometriosis (magnification, 40). B, Secretory endometrium of woman without endometriosis (magnification, 40). C, Proliferative eutopic endometrium of a woman with endometriosis (magnification, 60). D, Secretory eutopic endometrium of a woman with endometriosis (magnification, 20). E and F, Ectopic endometrial implant on the ovary (magnification, 20 and 60, respectively). Arrows indicate endothelial IL-8 immunostaining. for 1 min at 72 C in a thermal Master Cycler Gradient PCR System (Eppendorf, Westbury, NY). The annealing temperatures for IL-8 and glyceraldehyde-3-phosphate dehydrogenase were 54 and 55 C, respectively. Ten microliters of each RT-PCR product were electrophoresed in 2% agarose gel containing ethidium bromide and visualized under UV light. The bands were analyzed by a Kodak (version 3.6.1) software program (Eastman Kodak Co., Rochester, NY). IL-8 immunoassay Immunoreactive IL-8 in culture supernatant was quantified using an ELISA from R&D Systems (Minneapolis, MN). According to the manufacturer, there is no measurable cross-reactivity with other known cytokines in this assay. The sensitivity for IL-8 was 0.47 pg/100 l sample. Each experiment was performed using three replicate wells for each condition, and supernatant from each well was tested in a single ELISA assay. Each experimental setup was repeated on at least three occasions using endothelial cells obtained from three different patients. The intra- and interassay coefficients of variation were 7.95% and 10.2%, respectively. Statistical analysis Differences in HSCORE values among the proliferative and secretory phases of normal and eutopic endometrium of women with endometriosis were analyzed using t test. One-way ANOVA and Student-Newman-Keuls methods were used to determine the difference in endothelial IL-8 expression among normal, eutopic, and ectopic endometrial samples. IL-8 mrna levels were evaluated by ANOVA with the nonparametric Mann-Whitney U test for multiple comparisons. All statistical analyses were performed using SigmaStat for Windows, version 2.0 (Jandel Scientific Corp., San Rafael, CA). Data are presented as the mean sem. Differences were considered significant at P 0.05.

4 1808 J Clin Endocrinol Metab, March 2005, 90(3): Luk et al. Regulation of IL-8 Expression in HEEC Results Immunohistochemical localization of IL-8 in endometrial endothelial cells from women with and without endometriosis We first compared the endothelial IL-8 HSCORE values among the normal, eutopic, and ectopic endometrial samples. The IL-8 HSCORE values were normally distributed as determined by Kolmogorov-Smirnov test. Representative micrographs of endothelial IL-8 expression in eutopic endometrium from women with and without endometriosis and in ectopic endometriosis are shown in Fig. 1. We did not observe a significant difference in the HSCORE between the eutopic endometrium of women without endometriosis compared with the eutopic endometrium of women with endometriosis (Fig. 2A). In ectopic endometrium, endothelial IL-8 HSCORE values were significantly higher than those in normal eutopic endometrium (P 0.027). In contrast, when we compared the IL-8 HSCORE values according to the menstrual cycle phase, we did not find a significant difference between the eutopic endometrium of women with and without endometriosis (Fig. 2B). We did not observe a menstrual cycle phase-dependent variation in endothelial cell IL-8 expression in eutopic endometrium of women with and without endometriosis (data not shown). Regulation of IL-8 mrna expression in HEEC by sex steroids Confluent HEEC from women with or without endometriosis were treated for 4 and 6 h with medium alone or with medium containing estradiol ( m) and progesterone (10 7 m), alone or in combination. Total RNA was extracted, and IL-8 mrna levels were semiquantitatively assessed by RT-PCR. The amount of IL-8 mrna was not normally distributed, as determined by Kolmogorov-Smirnov test. In the HEEC from women without endometriosis, estradiol treatment decreased IL-8 mrna levels by 35% and 45% below control levels at 4 and 6 h, respectively (P 0.005; Fig. 3). In contrast, in HEEC from women with endometriosis, estradiol treatment induced 29% and 32% increases in IL-8 mrna levels at 4 and 6 h, respectively (P 0.05; Fig. 4). In HEEC from women without endometriosis, progesterone treatment induced a 28% inhibition of the IL-8 mrna level at6h(p 0.01; Fig. 3). Treatment with progesterone in HEEC from women with endometriosis induced an 11% increase in the IL-8 mrna level at 4h(P 0.05; Fig. 4). Finally, estradiol plus progesterone decreased the IL-8 mrna level in HEEC from women without endometriosis by 45% at 6 h below the control levels (P 0.008; Fig. 3). In contrast, estradiol plus progesterone increased the IL-8 mrna level by 34% in HEEC from women with endometriosis at 6 h (Fig. 4). FIG. 2. A, The distribution of endometrial endothelial (proliferative and secretory combined) IL-8 immunostaining intensity (HSCORE) in eutopic and ectopic endometrial samples of women with and without endometriosis. Bars represent the mean SEM. NE, Endometrium from women without endometriosis; EEE, eutopic endometrium from women with endometriosis; ECE, ectopic endometrium from women with endometriosis. *, P 0.05 between normal and ectopic endometrium in pairwise comparison. B, The distribution of endometrial endothelial IL-8 immunostaining intensity (HSCORE) in normal and eutopic endometrium of women with endometriosis according to the menstrual cycle phase. Bars represent the mean SEM. Regulation of IL-8 protein production in HEEC by sex steroids Confluent HEEC were treated for 24 h with medium alone or with estradiol ( m) or progesterone (10 7 m), alone or in combination. Supernatants were collected, and IL-8 protein levels were measured using a specific ELISA. Estradiol inhibited IL-8 protein levels by 30% below the control levels in HEEC from women without endometriosis (P 0.005; Fig. 5), but induced an increase of 80% over control values in HEEC from women with endometriosis (P 0.005; Fig. 6). Progesterone treatment caused a 23% decrease in IL-8 protein levels in HEEC from women without endometriosis (P 0.001; Fig. 5). This contrasts with a 2-fold increase in IL-8 protein production over the control value in HEEC from women with endometriosis (P 0.002; Fig. 6). The combination of estradiol ( m) plus progesterone (10 7 m) caused a 22% decrease in IL-8 protein production in HEEC from women without endometriosis compared with controls (P 0.05; Fig. 5). Strikingly, the same combination treatment caused a 140% increase in IL-8 protein levels in HEEC from

5 Luk et al. Regulation of IL-8 Expression in HEEC J Clin Endocrinol Metab, March 2005, 90(3): FIG. 3. The regulation of IL-8 mrna expression in HEEC of women without endometriosis by estradiol and progesterone. HEEC in culture were treated with medium alone (C; control) or with medium containing progesterone (P; 10 7 M), 17 -estradiol (E; M), or estradiol ( M) plus progesterone (10 7 M) for 4 and 6 h. IL-8 mrna was evaluated by semiquantitative PCR analysis. *, Significantly different compared with control (P 0.05). women with endometriosis compared with controls (P 0.001; Fig. 6). Finally, we also found that the stimulation of IL-8 protein production by estradiol in HEEC from women with endometriosis is concentration dependent, with an increase in protein production at concentrations of 10 8 and 10 7 m (Fig. 7). FIG. 4. The regulation of IL-8 mrna expression in HEEC of women without endometriosis by estradiol and progesterone. HEEC in culture were treated with medium alone (C; control) or with medium containing progesterone (P; 10 7 M), 17 -estradiol (E; M), or estradiol ( M) plus progesterone (10 7 M) for 4 and 6 h. IL-8 mrna was evaluated by semiquantitative PCR analysis. *, Significantly different compared with control (P 0.05).

6 1810 J Clin Endocrinol Metab, March 2005, 90(3): Luk et al. Regulation of IL-8 Expression in HEEC FIG. 5. Regulation of IL-8 protein expression in HEEC of women without endometriosis in culture by estradiol and progesterone. HEEC in culture were treated with medium alone (C; control) or with medium containing progesterone (P; 10 7 M), estradiol (E; M), and estradiol ( M) plus progesterone (10 7 M) for 24 h. IL-8 was quantified by ELISA and normalized to total protein. *, Significantly different compared with control (P 0.05). Data are the mean SEM for three replicates. Discussion Although the precise role of cytokines in endometriosis is currently unknown, a compelling body of evidence suggests an immunological basis for the pathogenesis of endometriosis. Endometriosis mainly affects women of reproductive age, and the expression of cytokines such as IL-8 in the endometrium fluctuates in a predictable manner throughout the menstrual cycle (4). Endocrine factors are involved in modulation of the tissue-specific changes in endometrium and the expression of chemotactic factors that are involved in the recruitment of the leukocytes during the menstrual cycle. The present results suggest that sex steroids regulate chemokine expression differently in HEEC in women with or without endometriosis. Endothelial cells play a role in leukocyte extravasation into the endometrium and in the regulation of the tissue-specific changes associated with the leukocyte migration. In other words, the endothelial cell FIG. 6. Regulation of IL-8 protein expression in HEEC of women with endometriosis in culture by estradiol and progesterone. HEEC in culture were treated with medium alone (C; control) or with medium containing progesterone (P; 10 7 M), estradiol (E; M), and estradiol ( M) plus progesterone (10 7 M) for 24 h. IL-8 was quantified by ELISA and normalized to total protein. *, Significantly different compared with control (P 0.05). Data are the mean SEM for three replicates. FIG. 7. Regulation of IL-8 production by HEEC of women with endometriosis in culture by estradiol. HEEC were cultured in phenol red-free medium containing 2% charcoal-stripped calf serum for 24 h before incubation with culture medium alone (control) or medium containing various concentrations of estradiol ( M) for 24 h. IL-8 was quantified by ELISA and normalized to total protein. Data are the mean SEM for three replicates. layer acts as the gatekeeper of the endometrium for immune cells. Using immunohistochemistry, we first confirmed that IL-8 is expressed in HEEC. The antibody used in this study revealed a significant increase in cytoplasmic IL-8 staining in endothelial cells of ectopic samples compared with eutopic endometrium of women with or without endometriosis. We did not observe a significant increase in the IL-8 immunostaining of the endothelial cells of eutopic endometrium of women with endometriosis compared with eutopic endometrium of women without endometriosis (P 0.1). We observed that IL-8 mrna and protein production increase markedly in the cultured HEEC of women with endometriosis in response to sex steroids, but we did not observe a significant increase in in vivo IL-8 immunoreactivity in endothelial cells from the same group of patients. One possible explanation is that even though IL-8 protein production is markedly increased in women with endometriosis, IL-8 being a secretory protein may result in the nonsignificant increase in cellular IL-8 immunostaining intensity in HEEC, because IL-8 would not be retained in the cells. Our speculation is also confirmed by the peritoneal fluid levels of IL-8 that are markedly elevated in women with endometriosis (5, 11). We propose that an increase in IL-8 expression in HEEC of women in the reproductive age may contribute to the pathophysiology of the development of endometriosis. As a result, the increase in IL-8 expression may potentiate leukocyte extravasation and migration into the endometrial tissue under the effects of steroids, especially estradiol. A previous study reported that progesterone inhibits IL-8- and IL-1-induced IL-8 mrna expression in rat uterine fibroblasts (15). However, it is still unknown how estradiol or progesterone suppresses IL-8 transcription. It is likely that estradiol and progesterone exert their effects by similar mechanisms, because their combination was not synergistic. One possible mechanism is that each hormone-bound receptor may inhibit the transcriptional activity and/or DNA binding of transcriptional activators on the IL-8 promoter. How does estradiol induce IL-8 in women with endometri-

7 Luk et al. Regulation of IL-8 Expression in HEEC J Clin Endocrinol Metab, March 2005, 90(3): osis while suppressing IL-8 in healthy women? It is known that the effect of estradiol on IL-8 protein expression is dependent on the presence of specific ligand-activated receptors in target tissues. The presence of coactivators and/or cosuppressors in the cell is another possible reason for the difference. Each hormone-bound receptor may inhibit the transcriptional activity and/or DNA binding of transcriptional activators on the IL-8 promoter, resulting in IL-8 suppression in HEEC in women without endometriosis. In contrast, for HEEC from women with endometriosis, there may be an as yet undefined coactivator that may promote the transcription and/or translation of IL-8 under the influence of sex steroids, especially estradiol. We and others reported that there is a higher proportion of estrogen receptor (ER) than ER present in the human endothelial cells (12, 16). As a result, estradiol exerts its effects via intracellular ER, which may up- or down-regulate the transcription of various genes by binding to the estrogen response element of target genes (17) or by interacting with other transcription factors. It is known that several transcriptional activators cooperate with one another through protein-protein interaction to activate the IL-8 promoter (18). The sequences from 98 to 63 bp appeared to direct the constitutive IL-8 transcription in fibroblasts and may be the main target for hormone-induced transcriptional repression. This region includes adjacent elements for CCAAT/enhancer-binding protein (C/EBP) ( 94 to 81) and nuclear factor- B (NF- B; 80 to 70). It is reported that NF- B subunit p65 and C/EBP form a ternary complex with this region of the IL-8 promoter, which results in synergistic transcriptional activation (19). It is thus suggested that each sex hormone/receptor complex may interact with NF- B and/or C/EBP family proteins and alter their conformation, which may repress their DNA-binding and/or the cooperative transcriptional activity on the IL-8 promoter. It is possible that the HEEC employ the same mechanism in steroidregulated expression of IL-8. We have reported for the first time that sex steroids are able to regulate IL-8 secretion and transcription in HEEC in vitro. Moreover, the substantial increase in IL-8 secretion in HEEC of women with endometriosis leads us to propose that endometrial endothelial cells not only serve as a regulator of the immune response in the endometrium, but may also play a major role in the leukocyte extravasation into the endometrium in women with endometriosis. Our results suggest a new role for the HEEC as one of the main players in the pathophysiology of endometriosis. HEEC could be a crucial regulator of the immune response in the human endometrium as a result of the many physiological as well as pathological events in the endometrium. Acknowledgments Received September 13, Accepted December 9, Address all correspondence and requests for reprints to: Dr. Aydin Arici, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut aydin.arici@yale.edu. References 1. Sampson J 1927 Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 14: Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM 1984 Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 64: Kruitwagen RF, Poels LG, Willemsen WN, de Ronde IJ, Jap PH, Rolland R 1991 Endometrial epithelial cells in peritoneal fluid during the early follicular phase. Fertil Steril 55: Arici A, Seli E, Senturk LM, Gutierrez LS, Oral E, Taylor HS 1998 Interleukin-8 in the human endometrium. J Clin Endocrinol Metab 83: Ryan IP, Tseng JF, Schriock ED, Khorram O, Landers DV, Taylor RN 1995 Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil Steril 63: Gazvani MR, Christmas S, Quenby S, Kirwan J, Johnson PM, Kingsland CR 1998 Peritoneal fluid concentrations of interleukin-8 in women with endometriosis: relationship to stage of disease. Hum Reprod 13: Larsen CG, Anderson AO, Appella E, Oppenheim JJ, Matsushima K 1989 The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes. Science 243: Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM 1992 Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 258: 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: Milne SA, Critchley HO, Drudy TA, Kelly RW, Baird DT 1999 Perivascular interleukin-8 messenger ribonucleic acid expression in human endometrium varies across the menstrual cycle and in early pregnancy decidua. J Clin Endocrinol Metab 84: Arici A, MacDonald PC, Casey ML 1996 Progestin regulation of interleukin-8 mrna levels and protein synthesis in human endometrial stromal cells. J Steroid Biochem Mol Biol 58: Kayisli UA, Luk J, Guzeloglu-Kayisli O, Seval Y, Demir R, Arici A 2004 Regulation of angiogenic activity of human endometrial endothelial cells in culture by ovarian steroids. J Clin Endocrinol Metab 89: Arici A, Head JR, MacDonald PC, Casey ML 1993 Regulation of interleukin-8 gene expression in human endometrial cells in culture. Mol Cell Endocrinol 94: St Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, Lal A, Riggins GJ, Lengauer C, Vogelstein B, Kinzler KW 2000 Genes expressed in human tumor endothelium. Science 289: Ito A, Imada K, Sato T, Kubo T, Matsushima K, Mori Y 1994 Suppression of interleukin 8 production by progesterone in rabbit uterine cervix. Biochem J 301: Critchley HO, Brenner RM, Henderson TA, Williams K, Nayak NR, Slayden OD, Millar MR, Saunders PT 2001 Estrogen receptor, but not estrogen receptor, is present in the vascular endothelium of the human and nonhuman primate endometrium. J Clin Endocrinol Metab 86: Beato M 1989 Gene regulation by steroid hormones. Cell 56: Roebuck KA 1999 Regulation of interleukin-8 gene expression. J Interferon Cytokine Res 19: Kunsch C, Lang RK, Rosen CA, Shannon MF 1994 Synergistic transcriptional activation of the IL-8 gene by NF- B p65 (RelA) and NF-IL-6. J Immunol 153: JCEM is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community.