Increased levels of interleukin-15 in the peritoneal fluid of women with endometriosis: inverse correlation with stage and depth of invasion

Aydin Arici1,4, Ioannis Matalliotakis1,2, Anastasia Goumenou2, Georgios Koumantakis2, Simon Vassiliadis3, Belgin Selam1 and Neal G. Mahutte1

1 Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8063, USA, 2 Department of Obstetrics and Gynaecology, University Hospital of Heraklion Crete and 3 Department of Biology, University of Crete, Greece

4 To whom correspondence should be addressed. e-mail: aydin.arici{at}yale.edu


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Interleukin (IL)-15 is a novel cytokine with immunoregulatory and angiogenic properties. We compared IL-15 levels in the peritoneal fluid (PF) of women with and without endometriosis. METHODS: PF samples were obtained from 55 women with endometriosis (23 with superficial peritoneal implants, 19 with deep endometriotic implants and 13 with ovarian endometriomas). Eighteen women with normal pelvic anatomy undergoing tubal sterilization served as controls. RESULTS: PF IL-15 concentrations were increased in women with endometriosis (2.7 ± 0.5 pg/ml) versus controls (2.1 ± 0.3 pg/ml; P < 0.001). However, IL-15 levels were higher in women with superficial peritoneal implants (2.9 ± 0.5 pg/ml) than women with deep endometriotic implants (2.6 ± 0.4 pg/ml; P = 0.01) or ovarian endometriomas (2.2 ± 0.4 pg/ml; P < 0.001). IL-15 was also higher in women with deep implants than in those with endometriomas (P < 0.05). PF IL-15 correlated inversely with both depth of invasion (r = –0.52) and the stage of endometriosis (r = –0.42). PF IL-15 levels demonstrated little variation during the menstrual cycle, and did not discriminate between women with infertility or pelvic pain. CONCLUSION: PF IL-15 levels are increased in women with endometriosis. However, IL-15 levels are inversely correlated with the depth of invasion and disease stage, suggesting a possible role for this cytokine in the early pathogenesis of endometriosis.

Key words: angiogenesis/cytokines/endometriosis/interleukin-15/peritoneal fluid


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alterations in the intraperitoneal immune environment play a role in the pathogenesis of endometriosis (Senturk and Arici, 1999Go; Starzinski-Powitz et al., 2001Go; Witz, 2002Go). In particular, cytokines are believed to have major regulatory and effector functions (Harada et al., 2001Go). Interleukin (IL)-6, IL-8, IL-10, leptin, monocyte chemotactic protein (MCP)-1 and vascular endothelial growth factor (VEGF) are all increased in the peritoneal fluid (PF) of women with endometriosis (Arici et al., 1996Go; Punnonen et al., 1996Go; Arici et al., 1997Go; Harada et al., 1997Go; Mahnke et al., 2000Go; De Placido et al., 2001Go). In some cases (IL-6, MCP-1, VEGF) levels correlate with the severity of disease, whereas in others (leptin) an inverse correlation with endometriosis staging has been reported.

IL-15 is a recently described cytokine (Grabstein et al., 1994Go) that activates the beta and gamma chains of the IL-2 receptor (Giri et al., 1994Go). There is increasing evidence that IL-15 may play an important role in diseases where mononuclear cell infiltration is a hallmark feature (Perera, 2000Go). IL-15 activates human natural killer (NK) cells (Carson et al., 1994Go) and also promotes lymphocyte proliferation and migration (Armitage et al., 1995Go; McInnes et al., 1996Go). Although IL-15 plays a critical role in normal, early immune responses, there is evidence that abnormal IL-15 production may contribute to a variety of immune disorders including rheumatoid arthritis (McInnes et al., 1996Go), ulcerative colitis (Sakai et al., 1998Go) and sarcoidosis (Kirman et al., 1998Go).

IL-15 is produced by a wide variety of cells and tissues. Among these are epithelial cell lines and monocytes. Human endometrium also expresses IL-15, most abundantly in the secretory phase of the cycle (Kitaya et al., 2000Go; Okada, H. et al., 2000Go). Progesterone is a potent inducer of IL-15 mRNA expression by human endometrial stromal cells (Okada, S. et al., 2000Go), although this effect may be mediated by prostaglandin E2 and interferon-gamma (Dunn et al., 2002Go).

To our knowledge, no previous studies have investigated PF IL-15 levels in women with endometriosis. Given the expression of IL-15 by endometrial tissue and monocytes, the important immunoregulatory roles of IL-15, and the dysregulation of IL-15 in many immune-mediated diseases, we measured IL-15 in the PF of women with and without endometriosis. We sought to assess the correlation, if any, between PF IL-15, the phase of the menstrual cycle, and the depth/stage of endometriosis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peritoneal fluid samples were collected from women undergoing laparoscopy at Yale–New Haven Hospital between 1997 and 2000. Indications for laparoscopy included evaluation of infertility, pelvic pain, pelvic mass or elective tubal sterilization. The Human Investigation Committee approved the study prior to its onset, and informed consent was obtained from each participant before surgery. The day of the menstrual cycle was established from the woman’s menstrual history and verified by histological examination of the endometrium in 14/18 controls and 43/55 cases.

Participants were classified according to the findings observed during laparoscopy. All 18 controls were healthy, fertile women with normal pelvic anatomy undergoing tubal sterilization. The 55 women with endometriosis consisted of 23 with superficial peritoneal implants, 19 with deep endometriotic implants, and 13 with ovarian endometriomas. The depth of invasion was based on the visual impression of the excised lesion(s) at the time of surgery. A threshold of 5 mm was used to distinguish superficial from deep lesions, and all case assessments were performed by reproductive endocrinologists with extensive experience in endometriosis surgery. Implants included all types of endometriosis, including black, red and white lesions. The diagnosis of endometriosis was confirmed by pathological examination following resection of the lesions. Staging of the disease was performed according to the revised American Fertility Society (1985) classification. All ovarian endometriomas were >3 cm in diameter.

None of the controls, and only eight of the women with endometriosis, had used any hormonal medication in the 3 months prior to surgery. In all eight cases the hormonal medication used was leuprolide acetate. Six of these women had stage III endometriosis (two with deep implants, four with endometriomas), and the other two had stage IV endometriosis (both with endometriomas).

Peritoneal fluid was aspirated from the pelvic cavity during the initial observation period of the laparoscopy, and before any operative manipulation was carried out. Fluid from the anterior and posterior cul-de-sacs was collected into a sterile syringe and centrifuged at 600 g for 10 min at 4°C to remove cells, aliquoted, and frozen at –80°C until assayed.

Measurement of IL-15
IL-15 levels in the PF of women with and without endometriosis were measured by enzyme-linked immunosorbent assay (ELISA). The sensitivity of the method (the minimum detectable dose) was 0.5 pg/ml. The primary antibody used in this work was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The affinity-purified mouse monoclonal IgG2a antibody sc-8437 reacts with IL-15 from the mouse, rat and human. The secondary antibody used for the primary antibody was rabbit anti-goat IgG, (Fab')2 fragment-specific, horse-radish peroxidase (HRP)-conjugated. This was purchased from Pierce (Rockford, IL, USA).

The ELISA experiments were performed as follows. PF fluid specimens were used at a concentration of 1:100 in carbonate buffer pH 9.6. These were coated in 96-well flat bottom plates (Sarstedt, Numbrecht, Germany), incubated overnight at 4°C and washed four times in 5% Tween-20 (Sigma, St Louis, MO, USA). The remaining protein-free sites in the plate were blocked by 2% phosphate-buffered saline (PBS)–bovine serum albumin (BSA) solution after an incubation of 2 h at room temperature. After washing four times, 100 µl of test (primary) antibody diluted in 0.1% PBS–BSA was added and incubated for 1 h at room temperature. Extensive washing of the plates was followed by addition of 100 µl of secondary IgG antibodies coupled to HRP (1:1000 dilution; Sigma) and incubated for 1 h at room temperature in the dark. Finally, the reaction was developed by adding 100 µl/well of tetramethyl benzidine–H2O2 (Sigma) for 20 min. The enzymatic reaction was stopped with 50 µl H2SO4 (2 mol/l). The optical density was measured at 450 nm using a Titertec ELISA photometer (Digiscan; ASYS Hitech GmbH, Engendorf, Austria). The results were derived from corresponding standard curves expressed as factor concentration ± SEM.

Statistical analysis
Non-parametric data were described as median and parametric data as mean ± SD. Unpaired t-tests were used for the comparison of means and Mann–Whitney U-test for medians. Differences between groups were analysed using Fisher’s exact test for non-parametric data. Correlation analysis was performed by Spearman’s rank test. Note that in the correlation analysis, both stage of disease and depth of invasion (superficial = 1, deep = 2, endometrioma = 3) were treated as ordinal variables. Statistical significance was defined as a P value < 0.05.


    Results
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 Materials and methods
 Results
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There were no statistically significant differences between the study (mean ± SD: 33.3 ± 6.5 years) and control groups (33.6 ± 6.3 years) with respect to age. In women with endometriosis, the primary indication for surgery was pelvic pain in 32 cases and infertility in 23 cases. In each group, half the patients were in the proliferative phase of the menstrual cycle, and half in the secretory phase of the cycle.

Women with endometriosis were found to have significantly higher PF IL-15 concentrations than controls (2.7 ± 0.5 versus 2.1 ±0.3 pg/ml, P < 0.001; Figure 1). The mean PF levels of IL-15 were significantly higher in women with superficial endometriosis (2.9 ± 0.5 pg/ml) than women with deep endometriotic implants (2.6 ± 0.4 pg/ml; P = 0.01) or ovarian endometriotic cysts (2.2 ± 0.4 pg/ml; P < 0.001) Women with deep implants also had higher IL-15 concentrations than those with endometriomas (P = 0.04). No significant difference in IL-15 levels was found between women undergoing tubal ligation and women with endometriotic cysts. There was an inverse correlation between PF IL-15 levels and the depth of invasion of endometriosis (r = –0.52, P < 0.001).



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Figure 1. Levels of IL-15 (pg/ml) in the peritoneal fluid (PF) of women undergoing tubal ligation (controls) and in women with endometriosis. Data are presented as mean ± SD. IL-15 levels in PF of women with endometriosis were significantly higher than the controls (aP < 0.001). Moreover, among women with endometriosis, those with only superficial lesions had significantly higher PF IL-15 levels than those with deep implants or ovarian endometriomas (bP = 0.01 and cP < 0.001 respectively). Finally, women with deep implants had higher PF IL-15 levels than women with ovarian endometriomas (dP = 0.04).

 
The group of women with endometriosis was further classified into subgroups according to the stage of endometriosis (Table I). There was a statistically significant difference in IL-15 levels between women with stage I and stage III or IV endometriosis (P = 0.01 and P = 0.006 respectively). Moreover, a significant inverse correlation was found between endometriosis staging and IL-15 levels (r = –0.42, P < 0.001).


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Table I. Peritoneal fluid interleukin (IL)-15 levels (pg/ml) in women with endometriosis
 
Levels of IL-15 did not show a difference according to the phase of the cycle in either group. In the women with endometriosis, the median concentration of IL-15 was 2.70 pg/ml in the proliferative phase and 2.65 pg/ml in the secretory phase. In women with no endometriosis, mean concentrations were 2.10 pg/ml in both the proliferative and secretory phase. However, the levels of IL-15 in PF were significantly higher in proliferative and secretory phase in women with endometriosis than in controls (P = 0.002 and P = 0.01 respectively).

In neither cases nor controls did PF IL-15 levels correlate with the day of the menstrual cycle or with the age of the patient. Furthermore, among women with endometriosis, no significant difference was seen between women with infertility (2.56 pg/ml) and women with pelvic pain (2.64 pg/ml).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytokines are believed to play a major role in the pathogenesis of endometriosis. Cytokines may serve as immunomodulators, angiogenic factors, or agents promoting endometrial cell growth. In some cases, they may serve all of these functions, whereas in others they may have apparently competing effects on nascent endometriotic implants.

IL-8 is a good example, as it is believed to have a major facilitatory role in the development of endometriosis. IL-8 has been shown to stimulate adhesion of endometrial stromal cells to fibronectin (Garcia-Velasco and Arici, 1999Go), to up-regulate matrix metalloproteinases that may allow invasion through the peritoneal surface, and to induce local angiogenesis (Garcia-Velasco and Arici, 1999Go). IL-8 is also an autocrine growth factor for endometrial stromal cells (Arici et al., 1998Go). However, IL-8 is also a leukocyte chemotactic factor, and an inhibitor of neutrophil apoptosis (Kwak et al., 2002Go). Thus, IL-8 may facilitate attachment of endometrial cells to the peritoneal surface, invasion of the extracellular matrix, local angiogenesis and endometrial proliferation, while simultaneously augmenting local immune responses.

IL-15 has some similarities to IL-8. IL-15 inhibits neutrophil apoptosis (Girard et al., 1996Go). IL-15 also stimulates the production of matrix metalloproteinases (Constantinescu et al., 2001Go). A role in angiogenesis has also been demonstrated, as IL-15 up-regulates VEGF secretion by endometrial NK cells (Li, X.C. et al., 2001Go).

This study demonstrates for the first time that IL-15 is present in the PF, and that women with endometriosis have higher PF IL-15 concentrations than fertile women with normal pelvic anatomy. Moreover, levels of IL-15 were significantly higher in women with superficial peritoneal and deep endometriotic implants than women with endometriomas. Similarly, significantly higher PF IL-15 levels were present in early stage versus advanced stage endometriosis.

The inverse correlation between the stage of disease and PF IL-15 levels is distinct from other PF cytokines such as IL-6, VEGF and MCP-1, all of which have been shown to correlate positively with the stage of endometriosis. Although the reason for this is unclear, it suggests a possible role for IL-15 in the early pathogenesis of endometriosis.

Such a role for Il-15 in endometriosis would be consistent with the recognized role of IL-15 in other early immune responses. For example, although both IL-2 and IL-15 stimulate T-cell proliferation in vitro, there is evidence in vivo that IL-2 renders T-cells susceptible to apoptosis, whereas IL-15 initiates T-cell division (Li, X.F. et al., 2001Go). Similarly, although both IL-2 and IL-15 activate NK cells, IL-2 renders them susceptible to apoptosis whereas IL-15 prevents apoptosis (Rodella et al., 2001Go). Such data suggest that early in the immune response, prior to activation of T-cells, IL-15 generates activated NK cells as a first-line defence. However, once T-cells become activated, they produce sufficient IL-2 to keep the NK and T-cell response in check. Thus, physiologically IL-15 may have a more prominent role in early, as opposed to late, immune responses to endometriosis.

An alternative explanation for the inverse correlation between IL-15 and the extent of endometriosis may be that the pathogenesis of peritoneal endometriosis is different from the pathogenesis of deep endometriosis or endometriomas. Whereas PF factors may play an important role in peritoneal disease, deeper lesions may be more influenced by haematological or ovarian factors (Koninckx et al., 1991Go, 1998). Indeed, morphological, morphometric and histochemical evidence has been presented suggesting that peritoneal and ovarian endometriosis are different entities (Nisolle and Donnez, 1997Go).

In either case, our results clearly demonstrate that PF IL-15 concentrations are elevated in women with early stage, peritoneal endometriosis. Furthermore, it is well established that IL-15 may alter normal immune surveillance, stimulate the production of matrix metalloproteinase, and up-regulate VEGF. All of these properties are consistent with a role for IL-15 in the development of endometrial peritoneal implants, and suggest that the relationship between PF IL-15 and endometriosis merits further study.


    Acknowledgements
 
This study was supported by grants from the Department of Obstetrics and Gynecology, University of Crete. All IL-15 assays were performed in Department of Biology, University of Crete. We would like to thank the Chief of Biology Department, I.Athanassakis, for her invaluable contribution to this work. In addition, we would like to thank Mrs E.Dionyssopoulou and L.Papadimitriou for their technical assistance. The co-operation of Nacile Mulayim, MD from the Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut, USA, was appreciated.


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Submitted on August 12, 2002; accepted on November 5, 2002.