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Decreased peritoneal concentrations of interleukin-15 in women with advanced stage endometriosis
European Journal of Obstetrics & Gynecology and Reproductive Biology 129 (2006) 169–173 www.elsevier.com/locate/ejogrb
Decreased peritoneal concentrations of interleukin-15 in women with advanced stage endometriosis Jun Lin *, Xinmei Zhang, Deng Lin, Qingxian Fang, Yuli Qian Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310006, China Received 6 March 2005; received in revised form 29 October 2005; accepted 2 January 2006
Abstract Objective: To assess the concentrations of interleukin-15 (IL-15) in peritoneal fluid from women with endometriosis and fertile disease-free controls. Study design: Peritoneal fluid samples were obtained from 50 women with endometriosis and 29 fertile women having tubal ligation. Concentrations of IL-15 were measured. Results: The mean (S.D.s) concentration of IL-15 in peritoneal fluid was 11.17 pg/mL (3.89) for women with endometriosis, and 12.59 pg/ mL (4.11) for fertile disease-free controls. The difference of peritoneal IL-15 concentrations between endometriosis and control women was not statistically significant. However, peritoneal IL-15 concentrations were significantly lower in women with moderate/severe endometriosis when compared with those in women with minimal/mild endometriosis, and in controls (P < 0.05). In addition, peritoneal IL-15 concentrations did not correlate with the phase of menstrual cycle in endometriosis or control women. Conclusions: Our results suggest that the decreased peritoneal IL-15 concentrations in women with moderate/severe endometriosis imply a role of IL-15 in the pathogenesis of advanced endometriosis as compared to those with minimal/mild endometriosis and fertile disease-free controls. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Endometriosis; Interleukin-15; Cytokine; Peritoneal fluid
1. Introduction There is increasing evidence that peritoneal cytokines play a critical role in the pathogenesis of endometriosis [1]. Recent studies have shown that the level of many cytokines including interleukin (IL)-6, IL-8, IL-10, leptin, tumour necrosis factor (TNF)-a and vascular endothelial growth factor (VEGF) is increased in peritoneal fluid for women with endometriosis [2–6]. These cytokines may be involved in the development and progression of this disorder by mediating the local inflammatory and cellular immune response to retrograded endometrial cells within the pelvic cavity or increasing the neo-angiogenesis of pelvic peritoneum. * Corresponding author. Tel.: +86 571 88854260 (home)/+7061501 2112 (office); fax: +86 571 87061878. E-mail address: [email protected] (J. Lin).
The aetiology of endometriosis may be multifactorial; yet, abnormal immune response has been considered a key factor in the pathophysiology of this disease. In women with endometriosis, there is an impaired activation of peritoneal T cells predominant by Th1 (T cell helper type 1) inflammatory cells and a decreased natural killer (NK) cell activity in peritoneal fluid. These could lead to the implantation and growth of regurgitated endometrial cells, and the formation of endometriotic lesions [7–10]. Interestingly, the activity of peritoneal T and NK cells in women with endometriosis can be restored by the treatment of the standard gonadotropin-releasing hormone agonists (GnRH-a) [8] or Danazol [9]. IL-15 has been recently described as a cytokine that activates the beta and gamma chains of IL-2 receptor, and also a key regulator of local innate tissue inflammatory and adaptive immunity [11]. Although IL-15 shares similar biological activity and receptor component with IL-2, it
0301-2115/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2006.01.005
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J. Lin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 129 (2006) 169–173
facilitates the generation and persistence of NK and NK-T cells, favors persistence of memory phenotype CD8+ T cells in vivo, and assists maintenance of a long-lasting immune response to pathogens and vaccines [12]. As a T cell growth factor, IL-15 has also been shown to promote NK cell and lymphocyte proliferation, to induce the production of interferon (INF)-g, and to enhance lymphocyte cytotoxic killing ability [13]. It has been reported that IL-15 is produced by a wide variety of cells and tissues including epithelial cell lines, monocytes, macrophages, decidual tissue, eutopic and ectopic endometrial tissues [14–17]. In normal human endometrial cells, the production of IL-15 is regulated by ovarian steroid hormones, suggesting an important role that IL-15 plays in human reproductive physiology [18]. Two recent studies have demonstrated a trend toward higher peritoneal IL-15 concentrations in women with endometriosis as compared to healthy controls [19,20]. However, in one study, the concentrations of IL-15 in peritoneal fluid were not significantly different [19]. In the other study, peritoneal IL-15 concentrations were not related to the presence of ovarian endometriotic cyst although peritoneal IL-15 concentrations were associated with the presence and severity of endometriosis [20]. In this study, the concentrations of IL-15 in peritoneal fluid of women with endometriosis were measured, and the role of IL-15 in the pathogenesis of endometriosis was evaluated.
of Women’s Hospital, Zhejiang University School of Medicine, China approved the study protocol. Peritoneal fluid was aspirated from the cul-de-sac within the pelvic cavity at the beginning of the operation, right after the abdominal cavity was entered. The fluid was collected into a sterile syringe and centrifuged at 400 g for 10 min under 4 8C to remove cells. The samples were aliquoted, and frozen at 70 8C until assayed. Peritoneal IL-15 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using a human IL-15 ELISA kit (Human IL15 D1500, Bender Medsystems, CA, USA) designed specifically to measure IL-15 in body fluids according to the manufacturer’s instructions. The sensitivity of the assay was less than 2 pg/mL, and the intra- and interassay coefficients of variation are both less than 5%. In order to avoid subjective bias, an experienced technician blind to the status of subject at laboratory conducted the detection of peritoneal IL-15 concentrations. For each sample, duplicate peritoneal fluids were assayed. For statistical analysis, nonparametric data were described as medians (ranges) and parametric data as means (S.D.s). Unpaired t-test was used for the comparison of means. Mann–Whitney U-test was used to compare medians. Confidence intervals were calculated at the 95% level. IL-15 concentrations in peritoneal fluid were correlated with clinical and operative findings as well as the phase of the menstrual cycle and previous abortion.
3. Results 2. Materials and methods Seventy-nine consecutive patients undergoing laparoscopic surgery from July 2002 through July 2003 at Women’s Hospital were recruited for this study. Gynecologic indications for laparoscopy included evaluation of infertility, abdominal pain, pelvic mass or elective tubal sterilization. The control group consisted of 29 patients who were healthy, fertile women with normal pelvic anatomy undergoing tubal sterilization. The case group consisted of 50 patients diagnosed with different stages of endometriosis by laparoscopic and histological examinations. The day of the menstrual cycle was established from the women’s menstrual history and verified by histological examination of the endometrium in the study and control group. None of the patients had been on any medication for at least 3 months before the laparoscopy, and none was on any long-acting drugs. Besides drug use, clinical data, menstrual history and obstetric history were also collected in all study participants. Furthermore, operative findings such as the presence, location, volume, and degree of endometriosis and the condition of the pelvis, tubes, ovaries, Pouch of Douglas, and bowels were also recorded. Endometriosis was graded according to the Revised American Society for Reproductive Medicine (ASRM) (1997). Informed consent was obtained from each participant before surgery, and the ethic committee
Peritoneal fluid was obtained from all women undergoing laparoscopic assessment. The study group (n = 50) consisted of patients with evidence of endometriosis, and the control group consisted of patients who were free of endometriosis (n = 29). The primary indication for surgery was infertility (n = 21), abdominal pain (n = 13) and pelvic mass (n = 16) for women with endometriosis. All patients were parous women in the control group and had no abdominal pain. Of these 50 patients with endometriosis, 17 had early stage (stage I + II) and 33 had advanced stage (stage III + IV). In the vast majority of cases, the primary location of endometriosis was the ovaries (n = 36), followed by the Pouch of Douglas (n = 10) and the lateral pelvic wall (n = 4). Fifty-three women were in the follicular phase (of whom 36 had evidence of endometriosis and 17 were controls), and 26 women were in the luteal phase (of whom 14 had evidence of endometriosis and 12 were controls). There was no statistically significant difference between the study and control groups with respect to their age, previous abortion, and the phase of menstrual cycle. The mean age was 33.0 years (S.D. = 7.2) in the endometriosis group and 34.8 years (S.D. = 8.4) in the control group. The mean concentrations of IL-15 in peritoneal fluid were 11.17 pg/mL (S.D. = 3.89) in women with endometriosis, and 12.59 pg/ mL (S.D. = 4.11) in women without endometriosis. There was
J. Lin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 129 (2006) 169–173 Table 1 Study subjects’ demographics and peritoneal fluid concentrations of IL-15 Parameter
No endometriosis (n = 29)
Endometriosis (n = 50)
Agea, years (S.D.) Abortionb (range) Concentration of IL-15a, pg/mL (S.D.) Follicular phase concentration of IL-15a, pg/Ml (S.D.) Luteal phase concentration of IL-15a, pg/mL (S.D.)
34.8 (8.4) 1 (0–4) 12.59 (4.11)
33.0 (7.2) 1 (0–4) 11.17 (3.89)
12.38 (4.64)
11.28 (4.02)
11.89 (4.09)
10.87 (3.63)
a b
Mean. Median.
a trend toward lower peritoneal IL-15 concentration in the endometriosis group as compared to the control group, but the difference of peritoneal IL-15 concentrations between two groups was not statistically significant. Median abortion was 1 (range 0–4) in both groups (Table 1). On staging of endometriosis, the mean concentrations of IL-15 in peritoneal fluid were 13.09 4.53 pg/mL in minimal/mild stage, and 10.18 3.14 pg/mL in moderate/ severe stage of the disease. There was a significantly lower peritoneal concentration of IL-15 in the patients with moderate/severe stage endometriosis than those with minimal/mild stage endometriosis and those without endometriosis (Fig. 1).
Fig. 1. Peritoneal concentration of IL-15 in relation to the severity of endometriosis. Non-EM: no endometriosis (controls), EM: endometriosis, early: minimal/mild stage, advanced: moderate/severe stage. (*)Advanced vs. controls: P = 0.011; advanced vs. early: P = 0.01.
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The concentrations of IL-15 in peritoneal fluid did not show differences according to the phase of the cycle in either group. In the patients with endometriosis, the mean concentrations of IL-15 in peritoneal fluid were 11.28 4.02 pg/mL in the follicular phase, and 10.87 3.63 pg/mL in the luteal phase. In women without endometriosis, concentrations were 12.38 4.64 pg/mL in the follicular phase and 11.89 4.09 pg/mL in the luteal phase. In women with endometriosis, there was no significant difference in peritoneal IL-15 concentrations in terms of infertility. The mean concentrations of IL-15 in peritoneal fluid were 11.26 4.06 pg/ml in the patients with endometriosis-associated infertility and 11.80 3.84 pg/ml in the endometriosis patients without infertility. In addition, peritoneal IL-15 concentrations did not correlate with the complaints of abdominal pain and the history of previous abortion in the patients with endometriosis.
4. Comment In the present study we demonstrated that peritoneal IL15 concentrations did not show significant difference between the patients with and without endometriosis. This result is in agreement with the previous report by Chegini et al. [19]. Although a higher peritoneal IL-15 concentration in the patients with endometriosis, which was associated with the severity of this disorder, was reported in the study by Arici et al. [20], only 23.6% (13/55) of the patients with endometriosis had ovarian endometriomas. Peritoneal IL-15 concentrations did not reach statistical significance in the patients with ovarian endometriomas. In our study, there were 36 (72%) patients with ovarian endometriomas. There was a significantly lower concentration of IL-15 in peritoneal fluid from patients with moderate/severe stage endometriosis. These indicated that peritoneal IL-15 might play an important role in the pathogenesis of endometriosis. IL-15, a T cell growth factor, has been shown to induce the proliferation of NK cells from premature to mature, and to enhance and sustain lymphocyte cytotoxicity when human body is invaded by pathogens [21,22]. It is indicated that reduced activity or level of IL-15 may impair the ability of NK cells to fight against pathogens due to decreased immune function. Decreased cellular immune response in women with endometriosis has been considered as a crucial role in the pathogenesis of the disease [7–10]. Collectively, it is intriguing to speculate that a trend toward lower rather than higher peritoneal IL-15 concentration in the patients with endometriosis may suggest IL-15 playing in the pathogenesis of this disorder. It has been suggested that decreased cellular immune response including an impaired activity of NK cells mainly occurs in the advanced stage of endometriosis [23,24]. IL-15 is the physiologic NK cell hematopoietic factor, and also sustains NK cell survival [25,26]. Furthermore, exogenous administration of IL-15 could rapidly expand the production
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of NK cells, and could also activate the cytotoxicity and cytokine-producing function of NK cells [27]. Although IL15 shares similar biological activity and receptor component with IL-2 [12], cytokine therapy with IL-15 could be better or more efficient than IL-2 in expanding the NK cell compartment in immunotherapy of tumors [12,28,29]. Thus, reduced peritoneal IL-15 concentrations in the patients with moderate/severe endometriosis may imply a key role of IL15 in the pathogenesis of advanced disease stage. It has been shown that an impaired activation of peritoneal T cells predominant by Th1 inflammatory cells and a decreased NK cell activity in peritoneal fluid of women with endometriosis may facilitate the implantation and growth of regurgitated endometrial cells, and the formation of endometriotic lesions [7–10]. In this study we found that women with moderate/severe stage endometriosis had a significant lower concentration of IL-15 in peritoneal fluid as compared to women with minimal/mild stage endometriosis or healthy fertile controls. IL-15, a Th1 proinflammatory cytokine [30], can enhance the immune response of Th1 predominant proinflammatory cytokines [31], the proliferation of NK cells and lymphocyte cytotoxicity [32]. It is suggested that reduced concentrations of peritoneal IL-15 in women with moderate/severe stage endometriosis may be insufficient to activate NK and T cells and the immune response of Th1 predominant proinflammatory cytokines, thus leading to the development and progression of advanced endometriosis. Much evidence has suggested that the higher concentrations of IL-8, TNF-a, IL-6, IL-1 and monocyte chemoattractant protein (MCP)-1 in peritoneal fluid from women with endometriosis play an important role in the pathogenesis of this disease [2–4,33,34]. IL-15 can strongly inhibit the production of IL-8 and MCP-1 in colonic epithelial cells [35], and act as a potent autocrine regulator of macrophage proinflammatory cytokine production TNF-a, IL-6 and IL-1 [36]. Consequently, reduced or abnormal concentrations of peritoneal IL-15 in women with moderate/severe endometriosis may play a role in the pathogenesis of advanced stage endometriosis by inversely increasing the production of IL-8 and MCP-1 or regulating the production of TNF-a, IL-6 and IL-1. In summary, our results suggest that decreased peritoneal IL-15 concentrations in women with moderate/severe stage endometriosis may imply IL-15 playing an important role in the pathogenesis of advanced disease stage. However, due to differences between the previous reports and our study, it is necessary to validate these findings in a larger study. Further work is needed to explore the use of this potent molecule in animal and in vitro models of endometriosis.
Reference ¨ ski P, Marianowski L. Role of cytokines in pathogen[1] Barcz E, KamiA esis of endometriosis. Med Sci Monit 2000;6:1042–6.
[2] Mahnke JL, Dawood MY, Huang JC. Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril 2000;73:166–70. [3] Barcz E, Rozewska ES, Kaminski P, Demkow U, Bobrowska K, Marianowski L. Angiogenic activity and IL-8 concentrations in peritoneal fluid and sera in endometriosis. Int J Gynaecol Obstet 2002;79:229–35. [4] Punnonen J, Teisala K, Ranta H, Bennett B, Punnonen R. Increased levels of interleukin-6 and interleukin-10 in the peritoneal fluid of patients with endometriosis. Am J Obstet Gynecol 1996;174:1522–6. [5] Matarese G, Alviggi C, Sanna V, et al. Increased leptin levels in serum and peritoneal fluid of patients with pelvic endometriosis. J Clin Endocrinol Metab 2000;85:2483–7. [6] Bedaiwy MA, Falcone T, Sharma RK, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod 2002;17:426–31. [7] Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75:1–10. [8] Umesaki N, Tanaka T, Miyama M, Mizuno K, Kawamura N, Ogita S. Increased natural killer cell activities in patients treated with gonadotropin releasing hormone agonist. Gynecol Obstet Invest 1999;48:66– 8. [9] Matalliotakis I, Neonaki M, Zolindaki A, Hassan E, Georgoulias V, Koumantakis E. Changes in immunologic variables (TNF-a, sCD8 and sCD4) during danazol treatment in patients with endometriosis. Int J Fertil Womens Med 1997;42:211–4. [10] Ho HN, Wu MY, Yang YS. Peritoneal cellular immunity and endometriosis. Am J Reprod Immunol 1997;38:400–2. [11] Perera LP. Interleukin 15: its role in inflammation and immunity. Arch Immunol Ther Exp (Warsz) 2000;48:457–64. [12] Waldmann TA. IL-15 in the life and death of lymphocytes: immunotherapeutic implications. Trends Mol Med 2003;9:517–21. [13] Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood 2001;97:14–32. [14] Ohteki T. Critical role for IL-15 in innate immunity. Curr Mol Med 2002;2:371–80. [15] Okada S, Okada H, Sanezumi M, Nakajima T, Yasuda K, Kanzaki H. Expression of interleukin-15 in human endometrium and decidua. Mol Hum Reprod 2000;6:75–80. [16] Kitaya K, Yasuda J, Yagi I, Tada Y, Fushiki S, Honjo H. IL-15 expression at human endometrium and decidua. Biol Reprod 2000;63:683–7. [17] Dunn CL, Critchley HO, Kelly RW. IL-15 regulation in human endometrial stromal cells. J Clin Endocrinol Metab 2002;87:1898– 901. [18] Okada H, Nakajima T, Sanezumi M, Ikuta A, Yasuda K, Kanzaki H. Progesterone enhances interleukin-15 production in human endometrial stromal cells in vitro. J Clin Endocrinol Metab 2000;85:4765–70. [19] Chegini N, Roberts M, Ripps B. Differential expression of interleukins (IL)-13 and IL-15 in ectopic and eutopic endometrium of women with endometriosis and normal fertile women. Am J Reprod Immunol 2003;49:75–83. [20] Arici A, Matalliotakis I, Goumenou A, et al. Increased levels of interleukin-15 in the peritoneal fluid of women with endometriosis: inverse correlation with stage and depth of invasion. Hum Reprod 2003;18:429–32. [21] Yoshikai Y, Nishimura H. The role of interleukin 15 in mounting an immune response against microbial infections. Microbes Infect 2000;2:381–9. [22] Lu J, Giuntoli 2nd RL, Omiya R, Kobayashi H, Kennedy R, Celis E. Interleukin 15 promotes antigen-independent in vitro expansion and long-term survival of antitumor cytotoxic T lymphocytes. Clin Cancer Res 2002;8:3877–84. [23] Garzetti GG, Ciavattini A, Provinciali M, Muzzioli M, Di Stefano G, Fabris N. Natural killer activity in stage III and IV endometriosis: impaired cytotoxicity and retained lymphokine responsiveness of natural killer cells. Gynecol Endocrinol 1995;9:125–30.
J. Lin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 129 (2006) 169–173 [24] Garzetti GG, Ciavattini A, Provinciali M, Amati M, Muzzioli M, Governa M. Decrease in peripheral blood polymorphonuclear leukocyte chemotactic index in endometriosis: role of prostaglandin E2 release. Obstet Gynecol 1998;91:25–9. [25] Prlic M, Blazar BR, Farrar MA, Jameson SC. In vivo survival and homeostatic proliferation of natural killer cells. J Exp Med 2003; 197:967–76. [26] Cooper MA, Bush JE, Fehniger TA, et al. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood 2002;100:3633–8. [27] Naora H, Gougeon ML. Enhanced survival and potent expansion of the natural killer cell population of HIV-infected individuals by exogenous interleukin-15. Immunol Lett 1999;68:359–67. [28] Sun R, Fan J, Wei H, Zhang C, Tian Z. Use of interleukin-15 for preparation of adherent NK cells from human peripheral blood: comparison with interleukin-2. J Immunol Meth 2003;279:79–90. [29] Kobayashi H, Carrasquillo JA, Paik CH, Waldmann TA, Tagaya Y. Differences of biodistribution, pharmacokinetics, and tumor targeting between interleukins 2 and 15. Cancer Res 2000;60:3577–83. [30] Mizuno Y, Takada H, Nomura A, et al. Th1 and Th1-inducing cytokines in Salmonella infection. Clin Exp Immunol 2003;131:111–7.
173
[31] Strengell M, Sareneva T, Foster D, Julkunen I, Matikainen S. IL-21 upregulates the expression of genes associated with innate immunity and Th1 response. J Immunol 2002;169:3600–5. [32] Fehniger TA, Cooper MA, Caligiuri MA. Interleukin-2 and interleukin-15: immunotherapy for cancer. Cytokine Growth Factor Rev 2002;13:169–83. [33] Wu MY, Ho HN. The role of cytokines in endometriosis. Am J Reprod Immunol 2003;49:285–96. [34] Pizzo A, Salmeri FM, Ardita FV, Sofo V, Tripepi M, Marsico S. Behaviour of cytokine levels in serum and peritoneal fluid of women with endometriosis. Gynecol Obstet Invest 2002;54:82–7. [35] Lugering N, Kucharzik T, Maaser C, Kraft M, Domschke W. Interleukin15 strongly inhibits interleukin-8 and monocyte chemoattractant protein-1 production in human colonic epithelial cells. Immunology 1999;98:504–9. [36] Alleva DG, Kaser SB, Monroy MA, Fenton MJ, Beller DI. IL-15 functions as a potent autocrine regulator of macrophage proinflammatory cytokine production: evidence for differential receptor subunit utilization associated with stimulation or inhibition. J Immunol 1997; 159:2941–51.
Decreased peritoneal concentrations of interleukin-15 in women with advanced stage endometriosis Jun Lin *, Xinmei Zhang, Deng Lin, Qingxian Fang, Yuli Qian Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310006, China Received 6 March 2005; received in revised form 29 October 2005; accepted 2 January 2006
Abstract Objective: To assess the concentrations of interleukin-15 (IL-15) in peritoneal fluid from women with endometriosis and fertile disease-free controls. Study design: Peritoneal fluid samples were obtained from 50 women with endometriosis and 29 fertile women having tubal ligation. Concentrations of IL-15 were measured. Results: The mean (S.D.s) concentration of IL-15 in peritoneal fluid was 11.17 pg/mL (3.89) for women with endometriosis, and 12.59 pg/ mL (4.11) for fertile disease-free controls. The difference of peritoneal IL-15 concentrations between endometriosis and control women was not statistically significant. However, peritoneal IL-15 concentrations were significantly lower in women with moderate/severe endometriosis when compared with those in women with minimal/mild endometriosis, and in controls (P < 0.05). In addition, peritoneal IL-15 concentrations did not correlate with the phase of menstrual cycle in endometriosis or control women. Conclusions: Our results suggest that the decreased peritoneal IL-15 concentrations in women with moderate/severe endometriosis imply a role of IL-15 in the pathogenesis of advanced endometriosis as compared to those with minimal/mild endometriosis and fertile disease-free controls. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Endometriosis; Interleukin-15; Cytokine; Peritoneal fluid
1. Introduction There is increasing evidence that peritoneal cytokines play a critical role in the pathogenesis of endometriosis [1]. Recent studies have shown that the level of many cytokines including interleukin (IL)-6, IL-8, IL-10, leptin, tumour necrosis factor (TNF)-a and vascular endothelial growth factor (VEGF) is increased in peritoneal fluid for women with endometriosis [2–6]. These cytokines may be involved in the development and progression of this disorder by mediating the local inflammatory and cellular immune response to retrograded endometrial cells within the pelvic cavity or increasing the neo-angiogenesis of pelvic peritoneum. * Corresponding author. Tel.: +86 571 88854260 (home)/+7061501 2112 (office); fax: +86 571 87061878. E-mail address: [email protected] (J. Lin).
The aetiology of endometriosis may be multifactorial; yet, abnormal immune response has been considered a key factor in the pathophysiology of this disease. In women with endometriosis, there is an impaired activation of peritoneal T cells predominant by Th1 (T cell helper type 1) inflammatory cells and a decreased natural killer (NK) cell activity in peritoneal fluid. These could lead to the implantation and growth of regurgitated endometrial cells, and the formation of endometriotic lesions [7–10]. Interestingly, the activity of peritoneal T and NK cells in women with endometriosis can be restored by the treatment of the standard gonadotropin-releasing hormone agonists (GnRH-a) [8] or Danazol [9]. IL-15 has been recently described as a cytokine that activates the beta and gamma chains of IL-2 receptor, and also a key regulator of local innate tissue inflammatory and adaptive immunity [11]. Although IL-15 shares similar biological activity and receptor component with IL-2, it
0301-2115/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2006.01.005
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J. Lin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 129 (2006) 169–173
facilitates the generation and persistence of NK and NK-T cells, favors persistence of memory phenotype CD8+ T cells in vivo, and assists maintenance of a long-lasting immune response to pathogens and vaccines [12]. As a T cell growth factor, IL-15 has also been shown to promote NK cell and lymphocyte proliferation, to induce the production of interferon (INF)-g, and to enhance lymphocyte cytotoxic killing ability [13]. It has been reported that IL-15 is produced by a wide variety of cells and tissues including epithelial cell lines, monocytes, macrophages, decidual tissue, eutopic and ectopic endometrial tissues [14–17]. In normal human endometrial cells, the production of IL-15 is regulated by ovarian steroid hormones, suggesting an important role that IL-15 plays in human reproductive physiology [18]. Two recent studies have demonstrated a trend toward higher peritoneal IL-15 concentrations in women with endometriosis as compared to healthy controls [19,20]. However, in one study, the concentrations of IL-15 in peritoneal fluid were not significantly different [19]. In the other study, peritoneal IL-15 concentrations were not related to the presence of ovarian endometriotic cyst although peritoneal IL-15 concentrations were associated with the presence and severity of endometriosis [20]. In this study, the concentrations of IL-15 in peritoneal fluid of women with endometriosis were measured, and the role of IL-15 in the pathogenesis of endometriosis was evaluated.
of Women’s Hospital, Zhejiang University School of Medicine, China approved the study protocol. Peritoneal fluid was aspirated from the cul-de-sac within the pelvic cavity at the beginning of the operation, right after the abdominal cavity was entered. The fluid was collected into a sterile syringe and centrifuged at 400 g for 10 min under 4 8C to remove cells. The samples were aliquoted, and frozen at 70 8C until assayed. Peritoneal IL-15 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using a human IL-15 ELISA kit (Human IL15 D1500, Bender Medsystems, CA, USA) designed specifically to measure IL-15 in body fluids according to the manufacturer’s instructions. The sensitivity of the assay was less than 2 pg/mL, and the intra- and interassay coefficients of variation are both less than 5%. In order to avoid subjective bias, an experienced technician blind to the status of subject at laboratory conducted the detection of peritoneal IL-15 concentrations. For each sample, duplicate peritoneal fluids were assayed. For statistical analysis, nonparametric data were described as medians (ranges) and parametric data as means (S.D.s). Unpaired t-test was used for the comparison of means. Mann–Whitney U-test was used to compare medians. Confidence intervals were calculated at the 95% level. IL-15 concentrations in peritoneal fluid were correlated with clinical and operative findings as well as the phase of the menstrual cycle and previous abortion.
3. Results 2. Materials and methods Seventy-nine consecutive patients undergoing laparoscopic surgery from July 2002 through July 2003 at Women’s Hospital were recruited for this study. Gynecologic indications for laparoscopy included evaluation of infertility, abdominal pain, pelvic mass or elective tubal sterilization. The control group consisted of 29 patients who were healthy, fertile women with normal pelvic anatomy undergoing tubal sterilization. The case group consisted of 50 patients diagnosed with different stages of endometriosis by laparoscopic and histological examinations. The day of the menstrual cycle was established from the women’s menstrual history and verified by histological examination of the endometrium in the study and control group. None of the patients had been on any medication for at least 3 months before the laparoscopy, and none was on any long-acting drugs. Besides drug use, clinical data, menstrual history and obstetric history were also collected in all study participants. Furthermore, operative findings such as the presence, location, volume, and degree of endometriosis and the condition of the pelvis, tubes, ovaries, Pouch of Douglas, and bowels were also recorded. Endometriosis was graded according to the Revised American Society for Reproductive Medicine (ASRM) (1997). Informed consent was obtained from each participant before surgery, and the ethic committee
Peritoneal fluid was obtained from all women undergoing laparoscopic assessment. The study group (n = 50) consisted of patients with evidence of endometriosis, and the control group consisted of patients who were free of endometriosis (n = 29). The primary indication for surgery was infertility (n = 21), abdominal pain (n = 13) and pelvic mass (n = 16) for women with endometriosis. All patients were parous women in the control group and had no abdominal pain. Of these 50 patients with endometriosis, 17 had early stage (stage I + II) and 33 had advanced stage (stage III + IV). In the vast majority of cases, the primary location of endometriosis was the ovaries (n = 36), followed by the Pouch of Douglas (n = 10) and the lateral pelvic wall (n = 4). Fifty-three women were in the follicular phase (of whom 36 had evidence of endometriosis and 17 were controls), and 26 women were in the luteal phase (of whom 14 had evidence of endometriosis and 12 were controls). There was no statistically significant difference between the study and control groups with respect to their age, previous abortion, and the phase of menstrual cycle. The mean age was 33.0 years (S.D. = 7.2) in the endometriosis group and 34.8 years (S.D. = 8.4) in the control group. The mean concentrations of IL-15 in peritoneal fluid were 11.17 pg/mL (S.D. = 3.89) in women with endometriosis, and 12.59 pg/ mL (S.D. = 4.11) in women without endometriosis. There was
J. Lin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 129 (2006) 169–173 Table 1 Study subjects’ demographics and peritoneal fluid concentrations of IL-15 Parameter
No endometriosis (n = 29)
Endometriosis (n = 50)
Agea, years (S.D.) Abortionb (range) Concentration of IL-15a, pg/mL (S.D.) Follicular phase concentration of IL-15a, pg/Ml (S.D.) Luteal phase concentration of IL-15a, pg/mL (S.D.)
34.8 (8.4) 1 (0–4) 12.59 (4.11)
33.0 (7.2) 1 (0–4) 11.17 (3.89)
12.38 (4.64)
11.28 (4.02)
11.89 (4.09)
10.87 (3.63)
a b
Mean. Median.
a trend toward lower peritoneal IL-15 concentration in the endometriosis group as compared to the control group, but the difference of peritoneal IL-15 concentrations between two groups was not statistically significant. Median abortion was 1 (range 0–4) in both groups (Table 1). On staging of endometriosis, the mean concentrations of IL-15 in peritoneal fluid were 13.09 4.53 pg/mL in minimal/mild stage, and 10.18 3.14 pg/mL in moderate/ severe stage of the disease. There was a significantly lower peritoneal concentration of IL-15 in the patients with moderate/severe stage endometriosis than those with minimal/mild stage endometriosis and those without endometriosis (Fig. 1).
Fig. 1. Peritoneal concentration of IL-15 in relation to the severity of endometriosis. Non-EM: no endometriosis (controls), EM: endometriosis, early: minimal/mild stage, advanced: moderate/severe stage. (*)Advanced vs. controls: P = 0.011; advanced vs. early: P = 0.01.
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The concentrations of IL-15 in peritoneal fluid did not show differences according to the phase of the cycle in either group. In the patients with endometriosis, the mean concentrations of IL-15 in peritoneal fluid were 11.28 4.02 pg/mL in the follicular phase, and 10.87 3.63 pg/mL in the luteal phase. In women without endometriosis, concentrations were 12.38 4.64 pg/mL in the follicular phase and 11.89 4.09 pg/mL in the luteal phase. In women with endometriosis, there was no significant difference in peritoneal IL-15 concentrations in terms of infertility. The mean concentrations of IL-15 in peritoneal fluid were 11.26 4.06 pg/ml in the patients with endometriosis-associated infertility and 11.80 3.84 pg/ml in the endometriosis patients without infertility. In addition, peritoneal IL-15 concentrations did not correlate with the complaints of abdominal pain and the history of previous abortion in the patients with endometriosis.
4. Comment In the present study we demonstrated that peritoneal IL15 concentrations did not show significant difference between the patients with and without endometriosis. This result is in agreement with the previous report by Chegini et al. [19]. Although a higher peritoneal IL-15 concentration in the patients with endometriosis, which was associated with the severity of this disorder, was reported in the study by Arici et al. [20], only 23.6% (13/55) of the patients with endometriosis had ovarian endometriomas. Peritoneal IL-15 concentrations did not reach statistical significance in the patients with ovarian endometriomas. In our study, there were 36 (72%) patients with ovarian endometriomas. There was a significantly lower concentration of IL-15 in peritoneal fluid from patients with moderate/severe stage endometriosis. These indicated that peritoneal IL-15 might play an important role in the pathogenesis of endometriosis. IL-15, a T cell growth factor, has been shown to induce the proliferation of NK cells from premature to mature, and to enhance and sustain lymphocyte cytotoxicity when human body is invaded by pathogens [21,22]. It is indicated that reduced activity or level of IL-15 may impair the ability of NK cells to fight against pathogens due to decreased immune function. Decreased cellular immune response in women with endometriosis has been considered as a crucial role in the pathogenesis of the disease [7–10]. Collectively, it is intriguing to speculate that a trend toward lower rather than higher peritoneal IL-15 concentration in the patients with endometriosis may suggest IL-15 playing in the pathogenesis of this disorder. It has been suggested that decreased cellular immune response including an impaired activity of NK cells mainly occurs in the advanced stage of endometriosis [23,24]. IL-15 is the physiologic NK cell hematopoietic factor, and also sustains NK cell survival [25,26]. Furthermore, exogenous administration of IL-15 could rapidly expand the production
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of NK cells, and could also activate the cytotoxicity and cytokine-producing function of NK cells [27]. Although IL15 shares similar biological activity and receptor component with IL-2 [12], cytokine therapy with IL-15 could be better or more efficient than IL-2 in expanding the NK cell compartment in immunotherapy of tumors [12,28,29]. Thus, reduced peritoneal IL-15 concentrations in the patients with moderate/severe endometriosis may imply a key role of IL15 in the pathogenesis of advanced disease stage. It has been shown that an impaired activation of peritoneal T cells predominant by Th1 inflammatory cells and a decreased NK cell activity in peritoneal fluid of women with endometriosis may facilitate the implantation and growth of regurgitated endometrial cells, and the formation of endometriotic lesions [7–10]. In this study we found that women with moderate/severe stage endometriosis had a significant lower concentration of IL-15 in peritoneal fluid as compared to women with minimal/mild stage endometriosis or healthy fertile controls. IL-15, a Th1 proinflammatory cytokine [30], can enhance the immune response of Th1 predominant proinflammatory cytokines [31], the proliferation of NK cells and lymphocyte cytotoxicity [32]. It is suggested that reduced concentrations of peritoneal IL-15 in women with moderate/severe stage endometriosis may be insufficient to activate NK and T cells and the immune response of Th1 predominant proinflammatory cytokines, thus leading to the development and progression of advanced endometriosis. Much evidence has suggested that the higher concentrations of IL-8, TNF-a, IL-6, IL-1 and monocyte chemoattractant protein (MCP)-1 in peritoneal fluid from women with endometriosis play an important role in the pathogenesis of this disease [2–4,33,34]. IL-15 can strongly inhibit the production of IL-8 and MCP-1 in colonic epithelial cells [35], and act as a potent autocrine regulator of macrophage proinflammatory cytokine production TNF-a, IL-6 and IL-1 [36]. Consequently, reduced or abnormal concentrations of peritoneal IL-15 in women with moderate/severe endometriosis may play a role in the pathogenesis of advanced stage endometriosis by inversely increasing the production of IL-8 and MCP-1 or regulating the production of TNF-a, IL-6 and IL-1. In summary, our results suggest that decreased peritoneal IL-15 concentrations in women with moderate/severe stage endometriosis may imply IL-15 playing an important role in the pathogenesis of advanced disease stage. However, due to differences between the previous reports and our study, it is necessary to validate these findings in a larger study. Further work is needed to explore the use of this potent molecule in animal and in vitro models of endometriosis.
Reference ¨ ski P, Marianowski L. Role of cytokines in pathogen[1] Barcz E, KamiA esis of endometriosis. Med Sci Monit 2000;6:1042–6.
[2] Mahnke JL, Dawood MY, Huang JC. Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril 2000;73:166–70. [3] Barcz E, Rozewska ES, Kaminski P, Demkow U, Bobrowska K, Marianowski L. Angiogenic activity and IL-8 concentrations in peritoneal fluid and sera in endometriosis. Int J Gynaecol Obstet 2002;79:229–35. [4] Punnonen J, Teisala K, Ranta H, Bennett B, Punnonen R. Increased levels of interleukin-6 and interleukin-10 in the peritoneal fluid of patients with endometriosis. Am J Obstet Gynecol 1996;174:1522–6. [5] Matarese G, Alviggi C, Sanna V, et al. Increased leptin levels in serum and peritoneal fluid of patients with pelvic endometriosis. J Clin Endocrinol Metab 2000;85:2483–7. [6] Bedaiwy MA, Falcone T, Sharma RK, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod 2002;17:426–31. [7] Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75:1–10. [8] Umesaki N, Tanaka T, Miyama M, Mizuno K, Kawamura N, Ogita S. Increased natural killer cell activities in patients treated with gonadotropin releasing hormone agonist. Gynecol Obstet Invest 1999;48:66– 8. [9] Matalliotakis I, Neonaki M, Zolindaki A, Hassan E, Georgoulias V, Koumantakis E. Changes in immunologic variables (TNF-a, sCD8 and sCD4) during danazol treatment in patients with endometriosis. Int J Fertil Womens Med 1997;42:211–4. [10] Ho HN, Wu MY, Yang YS. Peritoneal cellular immunity and endometriosis. Am J Reprod Immunol 1997;38:400–2. [11] Perera LP. Interleukin 15: its role in inflammation and immunity. Arch Immunol Ther Exp (Warsz) 2000;48:457–64. [12] Waldmann TA. IL-15 in the life and death of lymphocytes: immunotherapeutic implications. Trends Mol Med 2003;9:517–21. [13] Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood 2001;97:14–32. [14] Ohteki T. Critical role for IL-15 in innate immunity. Curr Mol Med 2002;2:371–80. [15] Okada S, Okada H, Sanezumi M, Nakajima T, Yasuda K, Kanzaki H. Expression of interleukin-15 in human endometrium and decidua. Mol Hum Reprod 2000;6:75–80. [16] Kitaya K, Yasuda J, Yagi I, Tada Y, Fushiki S, Honjo H. IL-15 expression at human endometrium and decidua. Biol Reprod 2000;63:683–7. [17] Dunn CL, Critchley HO, Kelly RW. IL-15 regulation in human endometrial stromal cells. J Clin Endocrinol Metab 2002;87:1898– 901. [18] Okada H, Nakajima T, Sanezumi M, Ikuta A, Yasuda K, Kanzaki H. Progesterone enhances interleukin-15 production in human endometrial stromal cells in vitro. J Clin Endocrinol Metab 2000;85:4765–70. [19] Chegini N, Roberts M, Ripps B. Differential expression of interleukins (IL)-13 and IL-15 in ectopic and eutopic endometrium of women with endometriosis and normal fertile women. Am J Reprod Immunol 2003;49:75–83. [20] Arici A, Matalliotakis I, Goumenou A, et al. Increased levels of interleukin-15 in the peritoneal fluid of women with endometriosis: inverse correlation with stage and depth of invasion. Hum Reprod 2003;18:429–32. [21] Yoshikai Y, Nishimura H. The role of interleukin 15 in mounting an immune response against microbial infections. Microbes Infect 2000;2:381–9. [22] Lu J, Giuntoli 2nd RL, Omiya R, Kobayashi H, Kennedy R, Celis E. Interleukin 15 promotes antigen-independent in vitro expansion and long-term survival of antitumor cytotoxic T lymphocytes. Clin Cancer Res 2002;8:3877–84. [23] Garzetti GG, Ciavattini A, Provinciali M, Muzzioli M, Di Stefano G, Fabris N. Natural killer activity in stage III and IV endometriosis: impaired cytotoxicity and retained lymphokine responsiveness of natural killer cells. Gynecol Endocrinol 1995;9:125–30.
J. Lin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 129 (2006) 169–173 [24] Garzetti GG, Ciavattini A, Provinciali M, Amati M, Muzzioli M, Governa M. Decrease in peripheral blood polymorphonuclear leukocyte chemotactic index in endometriosis: role of prostaglandin E2 release. Obstet Gynecol 1998;91:25–9. [25] Prlic M, Blazar BR, Farrar MA, Jameson SC. In vivo survival and homeostatic proliferation of natural killer cells. J Exp Med 2003; 197:967–76. [26] Cooper MA, Bush JE, Fehniger TA, et al. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood 2002;100:3633–8. [27] Naora H, Gougeon ML. Enhanced survival and potent expansion of the natural killer cell population of HIV-infected individuals by exogenous interleukin-15. Immunol Lett 1999;68:359–67. [28] Sun R, Fan J, Wei H, Zhang C, Tian Z. Use of interleukin-15 for preparation of adherent NK cells from human peripheral blood: comparison with interleukin-2. J Immunol Meth 2003;279:79–90. [29] Kobayashi H, Carrasquillo JA, Paik CH, Waldmann TA, Tagaya Y. Differences of biodistribution, pharmacokinetics, and tumor targeting between interleukins 2 and 15. Cancer Res 2000;60:3577–83. [30] Mizuno Y, Takada H, Nomura A, et al. Th1 and Th1-inducing cytokines in Salmonella infection. Clin Exp Immunol 2003;131:111–7.
173
[31] Strengell M, Sareneva T, Foster D, Julkunen I, Matikainen S. IL-21 upregulates the expression of genes associated with innate immunity and Th1 response. J Immunol 2002;169:3600–5. [32] Fehniger TA, Cooper MA, Caligiuri MA. Interleukin-2 and interleukin-15: immunotherapy for cancer. Cytokine Growth Factor Rev 2002;13:169–83. [33] Wu MY, Ho HN. The role of cytokines in endometriosis. Am J Reprod Immunol 2003;49:285–96. [34] Pizzo A, Salmeri FM, Ardita FV, Sofo V, Tripepi M, Marsico S. Behaviour of cytokine levels in serum and peritoneal fluid of women with endometriosis. Gynecol Obstet Invest 2002;54:82–7. [35] Lugering N, Kucharzik T, Maaser C, Kraft M, Domschke W. Interleukin15 strongly inhibits interleukin-8 and monocyte chemoattractant protein-1 production in human colonic epithelial cells. Immunology 1999;98:504–9. [36] Alleva DG, Kaser SB, Monroy MA, Fenton MJ, Beller DI. IL-15 functions as a potent autocrine regulator of macrophage proinflammatory cytokine production: evidence for differential receptor subunit utilization associated with stimulation or inhibition. J Immunol 1997; 159:2941–51.