Expression of TNFα Receptor Type-I Gene in Endometrial Cells from Women With and Without Endometriosis

Objectives: While retrograde menstruation occurs in most women, only some women develop endometriosis. Under normal conditions the phagocytic activity of peritoneal macrophages (PM) clears this body cavity of foreign cells while activation of PM causes secretion of biologically active substances that control inflammation. Women with endometriosis have elevated numbers of activated PM in their peritoneal fluid, yet the ectopic endometrial cells escape immune destruction. It is unclear why ectopic endometrial cells in women with endometriosis are significantly more resistant to PM-mediated phagocytosis or if these PM have a decreased capacity to mediate cytolysis of the misplaced endometrial cells. Endometriotic lesions produce haptoglobin (Hp), which in certain inflammatory states, has been shown to be immunomodulatory. Hence, this study evaluated the effects of Hp on PM attachment, the first step in phagocytosis, in women with and without endometriosis. Design: Peritoneal fluid was collected upon initiation of laparoscopic diagnosis for endometriosis or elective sterilization, with the approval of our Institutional Review Board. PM were isolated by density centrifugation, washed and plated at 100,000 cells in 96 well culture plates using RPMI media. The purity of PM preparations (.98%) was evaluated using antiCD68. Concentrations of 0, 0.1, 1.0 or 10 mg Hp were added, in duplicate, to replicate cultures. At 10, 20, 30 and 60 min, 10 mL aliquots of media containing non-adherent PM were removed after agitation and allowed to dry on glass slides. Cells on the slides were stained with anti-CD68 and quantified by a observer blinded to treatments. The adhesion index (AI) 5 1 2 (PM/mL supernatant) divided by (PM/mL original sample) 3 100 and reported as the mean6SEM. Three-way ANOVA (disease, treatment, time) and Tukey’s post hoc analyses were used to evaluate AI differences. Results: In the absence of Hp, PM from women with endometriosis exhibit a reduced AI as compared to women without the disease at 10 (94.2 6 0.4; 98.2 6 0.4), 20 (94.5 6 0.4; 98.0 6 0.4), 30 (95.5 6 0.4; 99.0 6 0.4) and 60 (96.6 6 0.4; 99.6 6 0.4) min (p,0.001). Hp further reduced peritoneal macrophage attachment. Further, the mean PM AI for women without endometriosis (98.760.2) was observed within 10 min whereas the mean PM AI for women with endometriosis (95.2 6 0.2) was not observed until after 30 min (p,0.001). And while neither time nor Hp had major effects on the PM AI in women without endometriosis, significant effects of time (P,0.001), Hp dose (P,0.001) and dose 3 time interactions (P,0.001) were observed in women with endometriosis. Conclusions: Localized expression of Hp by ectopic endometriotic cells and lesions in the peritoneal cavity suggests a Hp-mediated mechanism by which ectopic endometrial cells may alter peritoneal macrophage-mediated phagocytosis. (Supported by TAP Holdings, Inc.)

Monday, October 23, 2000 3:45 P.M. O-058 Epithelial Neutrophil-Activating Protein 78 (ENA-78) Is Elevated in the Peritoneal Fluid of Patients with Endometriosis. M. D. Mueller, D. I. Lebovic, R. N. Taylor. Center for Reproductive Sciences, University of California, San Francisco, CA. Objective: Immune cells are postulated to contribute to the pathophysiology of endometriosis. Chemokines of the a or CXC family have intrinsic angiogenic activity. Moreover, neutrophils themselves can secrete VEGF and may participate in endometrial vascularization (Mueller et al, in press). The current study tested the hypothesis that epithelial neutrophil-activating protein 78 (ENA-78), an a or CXC chemokine, might accumulate differentially in peritoneal fluid (PF) of women with and without endometriosis. Design: Case-control study. Setting: University hospital. Patients: Eighteen women with laparoscopic findings of endometriosis and eight women with no evidence of pelvic pathology. Main outcome measures: Peritoneal ENA-78 concentrations were measured blindly, in duplicate, by a specific quantitative sandwich enzyme immunoassay (sensitivity , 15 pg/mL). ENA-78 concentrations were compared among women with and without endometriosis (X2 and unpaired t-test). Results: Ten of the eighteen patients with endometriosis (56%) had elevated peritoneal ENA-78 values (Median: 65 pg/ml) whereas only one of eight subjects (13%) without pelvic pathology had detectable ENA-78

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values (X2: P,0.05). Mean 6 SD ENA-78 concentrations were higher in affected (44 6 40 pg/ml) than control subjects (2.2 6 3.8 pg/ml, unpaired t-test P,0.05). Conclusions: ENA-78 belongs to the ELR-CXC-chemokine class, known to have angiogenic activity. As angiogenesis occurs only when the balance of local factors promoting vascular growth exceeds those factors inhibiting angiogenesis, ENA-78 may contribute to the pathogenesis of endometriosis by promoting the neovascularization of ectopic endometrial implants. Immunohistochemical and in-situ hybridization studies are in progress to evaluate the cellular source(s) of ENA-78 production in endometriosis. (Supported by a grant of the SSMBS (MDM) and U54-HD37321 (DIL, RNT).

Monday, October 23, 2000 4:00 P.M. O-059 Altered Endometrial Expression of Endothelial Nitric Oxide Synthase (eNOS) in Women with Endometriosis. 1O. Khorram, 2B. A. Lessey. 1 Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI, 2University of North Carolina, Chapel Hill, NC. Objectives: Nitric oxide (NO) is a vasodilator synthesized from Larginine through the action of nitric oxide synthase (NOS). Recently we demonstrated that the endometrial expression of eNOS protein was higher during the secretory phase as compared to the proliferative phase, and exogenous steroids influenced eNOS expression. Based on these observations we postulated that NO may play a significant role in endometrial physiology through regulation of local blood flow. The purpose of this study was to determine if changes in the expression of endometrial eNOS in women with endometriosis could be a contributing factor to the pathogenesis of this disease. Design: Endometrial biopsies were obtained from women undergoing laparascopy for infertility 8 –10 days post LH surge. Subjects were assigned to a control (n59) or endometriosis group (n531) based on the surgical findings. In a separate study peritoneal fluid was obtained for NO measurement from women undergoing laparascopy for infertility. IRB approval was obtained for both studies. Materials and Methods: Tissue sections from the same luteal phase endometrial biopsies were immunostained for both eNOS and b3 integrin using human directed monoclonal antibodies. Intensity and distribution of staining was determined using the semiquantitative HSCORE. Peritoneal fluid levels of NO were measured by a NO analyzer. Results: A four fold (P,0.0001) and 1.5 fold higher (P,0.0002) eNOS staining intensity was found in the luminal and glandular epithelium respectively of women with endometriosis as compared to the control group. No differences in stromal eNOS staining was detected between the two groups. Peritoneal fluid levels of NO did not differ in women with and without endometriosis. A significant positive correlation (r5.37, P,0.05) between glandular eNOS protein and b3 was found in women with endometriosis but not in the control group. There were no other significant correlations found between eNOS and b3 expression in other uterine compartments in either group. Conclusions: A highly significant site-specific increase in endometrial eNOS expression was detected in women with endometriosis. Peritoneal fluid levels of NO, however, were not altered in women with endometriosis. A positive correlation between glandular eNOS and b3 was found only in women with endometriosis. These results suggest increased endometrial expression of eNOS in women with endometriosis may contribute to the pathogenesis of this disease, and point to an interaction between eNOS and b3 in glandular epithelium.

Monday, October 23, 2000 4:15 P.M. O-060 Expression of TNFa Receptor Type-I Gene in Endometrial Cells from Women With and Without Endometriosis. 1J. Ding, 2M. Gogacz, 1M. Shen, 1,3D. P. Braun, 1N. Rana, 1,3W. P. Dmowski. 1Institute for the Study and Treatment of Endometriosis, Chicago, Illinois, USA; 2Department of

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Surgical Gynecology, University School of Medicine, Lublin, Poland; 3 Rush Medical College, Chicago, IL. Objectives: We have previously demonstrated that in women with endometriosis spontaneous apoptosis in the uterine endometrium was significantly reduced and that tumor necrosis factor alpha (TNFa) stimulated proliferation of the endometrial cells in vitro. In healthy controls, endometrial cell proliferation was inhibited by TNFa. In the present study we investigated the expression of TNFa receptor type-I (TNFR-1) gene in the uterine endometrial cells and its modulation in response to TNFa treatment in vitro in women with and without endometriosis. Design: Expression of mRNA for TNFR-1 gene in the uterine endometrial cells and its response to TNFa treatment during in vitro culture were analyzed using RT-PCR in women with and without endometriosis. Materials and Methods: Eutopic endometria from women with (n516) and without (n519) endometriosis were treated with collagenase and DNase to obtain single cell suspensions of endometrial cells. One aliquot was then used immediately to extract mRNA (0 time point), while the others were cultured in the medium containing 0, 100 and 200 units/ml of recombinant human TNFa for 24 and 48 hours before extraction of RNA. RT-PCR for TNFR-1 mRNA was performed. The intensity of the PCR signal was scored from 1 (low) to 5 (high). Results: At 0 time point, TNFR-1 was expressed at a low level (scores #2, 8/9) in 9 of 18 control samples. At the equivalent time point in endometriosis, TNFR-1 was expressed in 13 of 16 samples and majority (7/13) had high (.2) scores. Statistically, the expression level (mean score 6 se) of TNFR-1 mRNA in the endometrial cells was significantly lower in controls than in women with endometriosis (0.5 6 0.17 vs 1.95 6 0.37, P,0.002). During in vitro culture, TNFR-1 mRNA expression was stimulated (P,0.05) by TNFa in the control group while it was inhibited (P,0.05) by TNFa in women with endometriosis (Table 1). Conclusion: These results suggest a difference in TNFR-1 expression in eutopic endometrial cells and a difference in their response to TNFa during in vitro culture in women with and without endometriosis. We conclude, based on these data and our previous findings, that in women with endometriosis spontaneous apoptosis in the endometrial cells is reduced because of the defective regulatory mechanism(s) in the TNFa/TNFR-1 signaling pathway which triggers the programmed cell death. Table 1. Effect of TNFa on expression of mRNA for TNFR-1 (mean score 6 SE) in endometrial cells from women with and without endometriosis. Hour of culture 24 48 0 100 200 0 100 200 TNFa dose (units/ml) Controls 2.160.4 2.360.5 2.960.4 2.160.3 2.260.3 2.760.4 Endometriosis 2.860.5 2.260.5 1.360.4 1.860.5 1.960.4 1.360.4

Monday, October 23, 2000 4:30 P.M. O-061 Interleukin-8 Increases Endometrial Stromal Cell Metalloproteinase Activity: Possible Role in Endometriosis. N. Mulayim, A. Savlu, A. Arici. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT. Objective: We have recently shown that interleukin-8(IL-8), a chemokine that is present at elevated levels in the peritoneal fluid of women with endometriosis, stimulates the adhesion of endometrial stromal cells to fibronectin. Recent evidence suggests that after the initial cell attachment, endometrial cells may invade the extracellular matrix of the peritoneum by way of an increased metalloproteinase activity. In this study we have evaluated the effect of IL-8 and different extracellular matrix proteins on the protease activity of endometrial stromal cells in culture. Materials and Methods: Endometrial tissue samples were obtained from human uteri after hysterectomy for benign disease. Endometrial stromal cells were prepared by standard enzyme digestion and filtration and were replicated to confluence in Ham’s F12:DME medium. Cells were trypsinized and plated on 24-well plates previously coated with poly-L-lysine (0.1 mg/ml), fibronectin (1 mg/cm2) or vitronectin (0.1 mg/cm2). Uncoated and poly-L-lysine-coated wells were compared with fibronectin and vitronectin-

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Abstracts

coated wells. In another set of experiments cells grown in uncoated, polyL-lysine-, fibronectin- or vitronectin-coated wells were treated with IL-8 (1ng/ml) or vehicle for 24 h. Metalloproteinase activity in endometrial stromal cell conditioned media was measured using the thiopeptolide substrate in a spectrophotometric assay. Same samples were also analyzed with substrate gel zymography. Results: Endometrial stromal cells that were grown in fibronectin and vitronectin-coated dishes demonstrated 1.4- and 1.8-fold increase, respectively, in metalloproteinase activity as compared to cells grown in uncoated or poly-L-lysine coated dishes. In uncoated and poly-L-lysine-coated dishes IL-8 increased the protease activity by 1.7-fold. In fibronectin- and vitronectin-coated dishes protease activity was increased with IL-8 treatment 2-fold and 2.4-fold, respectively. Results obtained in spectrophotometry were confirmed in zymograms. 92kD bands in zymograms showed that MMP-9 is the major metalloproteinase regulated by IL-8. Conclusion: IL-8 may play a role in the pathogenesis of endometriosis by increasing metalloproteinase activity in endometrial cells. In addition it may also potentiate the integrin-dependent protease activity of these cells.

Monday, October 23, 2000 4:45 P.M. O-062 Eutopic and Ectopic Human Endometrium Expresses Cannabinoid Receptors and Produces their Endogenous Ligand. 1E. Zupi, 2M. Sbracia, 3A. Rossi, 3M. Maccarrone, 2F. Scarpellini, 1D. Marconi. 1Department of Obstetrics and Gynecology, 2CEMR, 3Department of Sperimental Medicine “Tor Vergata” University, Rome, Italy. Objectives: It has been reported that the rat endometrium expresses cannabinoid receptors and their putative endogenous ligand, the anandamyde. We previously showed that human endometrium expresses both cannabinoid-receptors, CB1-R and CB2-R. Furthermore, our unpublished data showed the presence of anandamyde production in human endometrium, the putative endogenous cannabinoid ligand. In this study we investigated if eutopic and ectopic human endometrium of women affected by endometriosis produces anandamide and expresses the receptors for this substance. Design: Expression of CB1-R and CB2-R in eutopic and ectopic endometrium of women with endometriosis were studied by immunohistochemestry and RT-PCR, and production of anandamyde in eutopic and ectopic endometrium was studied by the assay for the activity of the enzyme synthesizing anandamide. Materials and Methods: We evaluated tissue specimens of eutopic and ectopic endometrium obtained from 10 patients with endometriosis underwent to operative laparoscopy for class III and class IV endometriosis. The specimens of endometriosis lesions, ovarian cysts and peritoneal adhesions or implants, were obtained during operative laparoscopy. The indirect avidin-biotin complex (Vectastin ABC-peroxidase kit) immunoperoxidase assay was performed on dewaxed and rehydrated 4 mm thick sections of 10% formalin fixed, paraffin embedded tissue. Anti-CB1-R and anti-CB2-R monoclonal primary mouse antibodies with a 1:20 dilution phosphatebuffered solution were used. Part of the endometrial biopsy were used to establish culture of epithelial and stromal cells. Briefly, after first digestion by 0.5% collagenase for 2 hours at 37°C in a shaking water bath in DMEM/F-12 medium, the cells clumps were separated by filtration through a nylon mesh filter (40mm); the epithelial cells were retained by the filters and dissociated by another enzimatic digestion, with trypsin for an hour. Epithelial and stromal cells were cultured in DMEM/F-12 medium supplemented with FCS 10%. The total RNA extracted from the cells cultured was reverse-transcribed and then amplified using reagents from Ez r Tth RNA Kit. For RT-PCR total RNA was extracted from each sample by TRIzol. RT-PCR was performed to reveal the presence of the mRNA of CB1-R and CB2-R using the published primers. Part of the endometrial biopsy and peritoneal endometrial implants were analysed to assess the activity of anandamide hydrolase, the enzyme synthesizing this substance, following the published procedures. Results: The immunohistochemistry for the receptors CB1-R and CB2-R showed their presence in eutopic and ectopic endometrium. In both cellular component of endometrium, epithelial and stromal cells, the RT-PCR evidenced the presence of specific cDNA for CB1-R and CB2-R. The produc-

Vol. 74, No. 3, Suppl. 1, September 2000