Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. Email: yutakaos-tky{at}umin.ac.jp
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Abstract |
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Introduction |
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The regurgitated endometrial debris bathes in peritoneal fluid (PF), and this is a significant constituent that influences the destiny of the refluxed endometrial cells. Mounting evidence that concentrations of various substances such as prostaglandins, growth factors, cytokines and chemokines in PF are aberrant in women with endometriosis (Osuga et al., 1999, 2000
, 2002
; Koga et al., 2000
; Yoshino et al., 2003
) suggests that these molecules are involved in the pathogenesis of the disease. In addition, immune cells in PF in endometriosis have been shown to be defective in numbers or activities, which may indicate the association of the immune system with the pathogenesis (Berkkanoglu and Arici, 2003
). Given that anti-apoptosis of refluxed endometrial cells is an etiological event in endometriosis, it would be reasonable to speculate that certain molecules in PF modulate apoptosis of the endometrial cells and influence the development of endometriosis.
Osteoprotegerin (OPG) has recently been shown to be a survival factor in several cell types (Malyankar et al., 2000; Holen et al., 2002
; Shipman and Croucher, 2003
;). The survival effect is mainly exerted by binding to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily (Emery et al., 1998
). TRAIL induces the apoptosis of susceptible cells upon binding to its receptors, TRAIL receptor 1 (TRAIL-R1, DR4) (Pan et al., 1997
) and TRAIL-R2 (DR5) (Walczak et al., 1997
), while OPG inhibits TRAIL-induced apoptosis by antagonizing TRAIL. Despite the wide-ranging expression of OPG in human tissues and its distinctive anti-apoptotic property, the relevance of OPG in endometriosis has not yet been studied.
We hypothesized that the aberrant OPG-related cytokine system in the peritoneal cavity is related to the development of endometriosis. To address this in the present study, we measured concentrations of OPG and TRAIL in PF of women with or without endometriosis.
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Materials and methods |
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PF was collected via a laparoscopic cannula introduced into the cul-de-sac before any manipulative procedure. The fluid was centrifuged at 400 g for 10 min, and the supernatants were frozen and stored at 80°C until assay.
Peritoneum and endometriotic tissues were also collected from the women with endometriosis. Peritoneal bone marrow-derived cells (PBMC) from these women were obtained according to the method we previously described (Yoshino et al., 2003).
TRAIL and OPG concentrations in the PF were measured in duplicate in a blind fashion, using a specific enzyme-linked immunosorbent assay (ELISA) for TRAIL (R&D Systems Inc., Minneapolis, MN) and for OPG (Biomedica, Vienna, Austria). The minimum detectable concentration was 2.86 pg/ml for TRAIL and 0.14 pmol/l for OPG. The intra and interassay CVs were <10% for each.
Total RNA was extracted from PBMC, peritoneum and endometriotic tissues using an RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was reverse-transcribed in a 20 µl volume using a TOYOBO RTPCR kit (TOYOBO, Osaka, Japan). Standard PCR was performed using TOYOBO Rever Tra Dash (TOYOBO) according to the manufacturer's instructions. The PCR primers for OPG, DR4 and DR5 were as follows: OPG sense primer, 5'-ATAAAGGCATGCAAACCCAG-3'; OPG antisense primer, 5'-GCCTCAAGTGCCTGAGAAAC-3' (Simonet et al., 1997); DR4 sense primer, 5'-ACAGCAATGGGAACATAGCC-3'; DR4 antisense primer, 5'-GTCACTCCAGGGCGTACAAT-3' (Pan et al., 1997
); DR5 sense primer, 5'-TGCAGCCGTAGTCTTGATTG-3'; DR5 antisense primer, 5'-GCACCAAGTCTGCAAAGTCA-3' (Walczak et al., 1997
). The PCR amplification protocols for OPG, DR4 and DR5 were all as follows: (denaturing 98°C for 10 s, annealing 60°C for 4 s, extension 74°C for 20 s) x30 cycles.
The data were described as median and interquartile range (IQR). MannWhitney test was used for the statistical analysis. Statistical significance was defined as P<0.05.
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Results |
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The concentrations of TRAIL and OPG for all PF samples were above the lower limits of the assay. The OPG concentrations in the PF of the women with endometriosis (median, 6.82 pmol/l; IQR, 5.0711.32) were significantly higher than those of the women without endometriosis (5.12 pmol/l, 3.726.78; P=0.006). The women with endometriosis, then, were subdivided into those with stage I/II and those with stage III/V, for further analysis. Next, we compared the concentrations of OPG and TRAIL in the proliferative phase and those in the secretory phase. As shown in Table I, no remarkable difference in the concentrations of OPG or in those of TRAIL was observed between the phases in each group. Accordingly, we dealt with the data from both phases as one homogenous group.
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When we compared the concentrations of OPG and TRAIL, and TRAIL/OPG ratios between fertile and infertile women in the non-endometriosis group, to address whether infertility has any relevance to these concentrations, the values were similar between the groups (data not shown).
Figure 2 shows the expression of OPG in the peritoneum, the endometriotic tissues and the PBMC in the PF. OPG mRNA expression was detected in all the tissues and cells examined.
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Discussion |
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OPG mRNA was found to be expressed in a number of tissues, including lung, heart, kidney, liver, stomach, intestine, brain, spinal cord, thyroid gland and bone (Simonet et al., 1997; Yasuda et al., 1998
). In addition, the present study showed the expression of OPG mRNA in PBMC, the peritoneum and the endometriotic tissue, which could be a possible source of OPG in PF.
The anti-apoptotic effect of OPG is exerted by antagonizing TRAIL, which induces apoptosis by binding to its receptors, DR4 and DR5. The expression of DR5 in endometriotic tissues, shown in the present study, implies that the TRAIL/OPG system regulates the susceptibility to apoptosis in endometriotic cells. Therefore, our findings of an increase in OPG concentrations in PF with a decrease in TRAIL/OPG concentration ratios in stage III/IV endometriosis, as compared to those in non-endometriosis and in stage I/II endometriosis, imply that the peritoneal environment is less apoptotic for endometriotic cells and is permissive for the progress of endometriosis.
Similar to TRAIL receptors, TNF receptor (TNFR) has an intracellular death domain that mediates the signal for apoptosis. TNF activates TNFR and induces apoptosis, while soluble TNFR (sTNFR), which is derived from the extracellular portion of TNFR, antagonizes the apoptosis-inducing effect of TNF
. Interestingly, concentrations of sTNFR in PF are elevated in women with endometriosis (Koga et al., 2000
). As TNFR are expressed in endometriotic tissues, the increase in sTNFR concentrations in PF was suggested to promote the development of endometriosis by sparing the cells from apoptosis. Combined with the present study, these findings may underscore the idea that anti-apoptotic molecules play important roles in the pathophysiology of endometriosis.
Apart from its ability to provoke apoptosis, TRAIL exhibits immunomodulatory capacities. In animal models of autoimmunity, systemic TRAIL blockade led to exacerbation of the disease without affecting the degree of apoptosis in inflammatory cells (Song et al., 2000; Hilliard et al., 2001
). In addition, TRAIL has been shown to inhibit T cell activation and proliferation without inducing T cell death (Lunemann et al., 2002
). Given that immune dysfunction is involved in the pathophysiology of endometriosis (Nothnick, 2001
; Berkkanoglu and Arici, 2003
), it is feasible that aberration of the TRAIL/OPG system causes the derangement of immune homeostasis in the peritoneal cavity in women with endometriosis.
The limitation of the current study was the inability to determine whether the findings are causes or results of endometriosis. In this sense, the increase in TRAIL concentrations in stage I/II endometriosis is interesting, in that it might be due to the physiological response of the body to inhibit the progress of the disease beyond these early stages.
In summary, the present study demonstrated an increase in OPG concentrations in the PF of women with endometriosis, which may contribute to the pathophysiology of endometriosis through anti-apoptotic and immunomodulatory mechanisms.
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References |
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Berkkanoglu M and Arici A (2003) Immunology and endometriosis. Am J Reprod Immunol 50, 4859.[CrossRef][ISI][Medline]
Dmowski WP, Gebel H and Braun DP (1998) Decreased apoptosis and sensitivity to macrophage mediated cytolysis of endometrial cells in endometriosis. Hum Reprod Update 4, 696701.
Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, Dul E, Appelbaum ER, Eichman C, DiPrinzio R et al. (1998) Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 273, 1436314367.
Garcia-Velasco JA and Arici A (2003) Apoptosis and the pathogenesis of endometriosis. Semin Reprod Med 21, 165172.[CrossRef][ISI][Medline]
Gebel HM, Braun DP, Tambur A, Frame D, Rana N and Dmowski WP (1998) Spontaneous apoptosis of endometrial tissue is impaired in women with endometriosis. Fertil Steril 69, 10421047.[CrossRef][ISI][Medline]
Hilliard B, Wilmen A, Seidel C, Liu TS, Goke R and Chen Y (2001) Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis. J Immunol 166, 13141319.
Holen I, Croucher PI, Hamdy FC and Eaton CL (2002) Osteoprotegerin (OPG) is a survival factor for human prostate cancer cells. Cancer Res 62, 16191623.
Koga K, Osuga Y, Tsutsumi O, Okagaki R, Momoeda M, Yano T, Fujiwara T, Takai Y, Kugu K, Morita Y et al. (2000) Increased concentrations of soluble tumour necrosis factor receptor (sTNFR) I and II in peritoneal fluid from women with endometriosis. Mol Hum Reprod 6, 929933.
Lebovic DI, Mueller MD and Taylor RN (2001) Immunobiology of endometriosis. Fertil Steril 75, 110.[CrossRef][ISI][Medline]
Lunemann JD, Waiczies S, Ehrlich S, Wendling U, Seeger B, Kamradt T and Zipp F (2002) Death ligand TRAIL induces no apoptosis but inhibits activation of human (auto)antigen-specific T cells. J Immunol 168, 48814888.
Malyankar UM, Scatena M, Suchland KL, Yun TJ, Clark EA and Giachelli CM (2000) Osteoprotegerin is an alpha vbeta 3-induced, NF-kappa B-dependent survival factor for endothelial cells. J Biol Chem 275, 2095920962.
Nothnick WB (2001) Treating endometriosis as an autoimmune disease. Fertil Steril 76, 223231.[CrossRef][ISI][Medline]
Osuga Y, Koga K, Tsutsumi O, Igarashi T, Okagaki R, Takai Y, Matsumi H, Hiroi H, Fujiwara T, Momoeda M et al. (2000) Stem cell factor (SCF) concentrations in peritoneal fluid of women with or without endometriosis. Am J Reprod Immunol 44, 231235.[CrossRef][ISI][Medline]
Osuga Y, Koga K, Tsutsumi O, Yano T, Maruyama M, Kugu K, Momoeda M and Taketani Y (2002) Role of laparoscopy in the treatment of endometriosis-associated infertility. Gynecol Obstet Invest 53 (Suppl 1), 3339.[CrossRef][ISI][Medline]
Osuga Y, Tsutsumi O, Okagaki R, Takai Y, Fujimoto A, Suenaga A, Maruyama M, Momoeda M, Yano T and Taketani Y (1999) Hepatocyte growth factor concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod 14, 16111613.
Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J and Dixit VM (1997) The receptor for the cytotoxic ligand TRAIL. Science 276, 111113.
Shipman CM and Croucher PI (2003) Osteoprotegerin is a soluble decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand and can function as a paracrine survival factor for human myeloma cells. Cancer Res 63, 912916.
Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T et al. (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89, 309319.[ISI][Medline]
Song K, Chen Y, Goke R, Wilmen A, Seidel C, Goke A, Hilliard B and Chen Y (2000) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J Exp Med 191, 10951104.
Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, Timour MS, Gerhart MJ, Schooley KA, Smith CA et al. (1997) TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J 16, 53865397.
Yasuda H, Shima N, Nakagawa N, Mochizuki SI, Yano K, Fujise N, Sato Y, Goto M, Yamaguchi K, Kuriyama M et al. (1998) Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 139, 13291337.
Yoshino O, Osuga Y, Koga K, Hirota Y, Tsutsumi O, Yano T, Morita Y, Momoeda M, Fujiwara T, Kugu K et al. (2003) Concentrations of interferon-gamma-induced protein-10 (IP-10), an antiangiogenic substance, are decreased in peritoneal fluid of women with advanced endometriosis. Am J Reprod Immunol 50, 6065.[CrossRef][ISI][Medline]
Submitted on February 24, 2004; accepted on June 7, 2004.
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