Peritoneal fluid concentrations of interleukin-8 in women with endometriosis: relationship to stage of disease

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1 Human Reproduction vol.13 no.7 pp , 1998 Peritoneal fluid concentrations of interleukin-8 in women with endometriosis: relationship to stage of disease M.Rafet Gazvani 1,3, Stephen Christmas 2, Siobhan Quenby 2, John Kirwan 1, Peter M.Johnson 2 and Charles R.Kingsland 1 1 Liverpool Women s Hospital, Liverpool, L8 7SS and 2 University of Liverpool, Liverpool, L69 3BX, UK 3 To whom correspondence should be addressed at: University of Aberdeen, Department of Obstetrics and Gynaecology, Forester Hill, Aberdeen AB9 2ZD, UK There is increasing evidence that immunological mechanisms play a role in the pathogenesis and pathophysiology of endometriosis. It was therefore of interest to study interleukin-8 (IL-8), a chemokine, in the peritoneal fluid and peripheral blood of women undergoing laparoscopic procedures. The presence and concentrations of IL-8 in relation to endometriosis, infertility and abdominal pain were evaluated. Samples of peritoneal fluid (n 49) and peripheral blood (n 50) were obtained from 50 consecutive patients undergoing laparoscopic surgery for various gynaecological indications (abdominal pain, infertility, sterilization). IL-8 was present in the peritoneal fluid of most women (87%). The concentration of IL-8 in the peritoneal fluid was higher in women with endometriosis compared to women without (P 0.02). This difference was more pronounced in early (stage 1) endometriosis (P 0.001). IL-8 concentrations in the peritoneal fluid were also higher in women with early endometriosis compared to women with later stages of the disease (P 0.003). Peripheral blood concentrations did not correlate with peritoneal fluid concentrations of IL-8 and/or the presence of endometriosis. We conclude that IL-8 is an important factor that may contribute to the pathogenesis of endometriosis possibly by promoting neovascularization. This information can be a guide in the development of new therapeutic approaches for the treatment of endometriosis. Key words: endometriosis/interleukin-8/peritoneal fluid Introduction Endometriosis is an important benign gynaecological disease, associated with pain and infertility. It has a multitude of appearances, ranging from isolated peritoneal deposits to dense pelvic adhesions (Shaw, 1993). Its diagnosis, staging and treatment continues to be debated. Endometriosis is generally seen as an anatomical lesion; however, it is well recognized that it is frequently only microscopic in nature, and therefore practically undiagnosable by the naked eye (Murphy et al., 1986). The American Fertility Society classification of endometriosis (1985) has a poor correlation with pain; it weights for ovarian disease and attempts to predict fertility (Overton et al., 1996). It has been shown that peritoneal fluid of women with endometriosis contains soluble factors with angiogenic activity (Oosterlynck et al., 1993). There is increasing evidence that immunological mechanisms play a role in the pathogenesis and pathophysiology of endometriosis. It has been widely suggested that the peritoneal fluid microenvironment may contribute to endometriosis and/or endometriosis-associated infertility. Cytokines are potent immunomodulatory glycoproteins that may mediate these processes (Keenan et al., 1994). It is suggested that cytokine products of activated T-cells may be involved in regulating cellular processes of endometriosis tissue. Elevated concentrations of interleukins IL-1, IL-2, IL-10 and tumour necrosis factor alpha (TNF-α) have been observed in the peritoneal fluid of subfertile women with endometriosis as compared to fertile women without endometriosis (Halme et al., 1983; Fakih et al., 1987; Eiserman et al., 1988; Hill and Anderson, 1989; Mori et al., 1991; Kupker et al., 1996; Rana et al., 1996), suggesting that these cytokines may be involved in the progression of disease and infertility. More recently, IL-13, an interleukin with a capacity to inhibit pro-inflammatory cytokine synthesis, has been reported to be found in significantly lower concentrations in the peritoneal fluid of women with endometriosis compared to normal women (McLaren et al., 1997). The relatively recent appreciation of a new class of cytokines, the chemokines, has done much to enhance our understanding of the extracellular signals involved in the movement of various populations of white blood cells. Investigation of the molecular underpinnings of chemokine function and their involvement in inflammatory processes of all kinds is beginning to yield information about the mechanisms of pathogenesis of a number of conditions, as well as providing hope for new therapeutic insights. Interleukin-8 (IL-8), a chemokine, is a potent angiogenic, pro-inflammatory, growth promoting factor, properties which may be shared by other chemokines (Koch et al., 1992). IL-8 is also a chemoattractant for neutrophils and induces expression of several cell adhesion molecules (Koch et al., 1992). It can also lead to neutrophil activation (Peveri et al., 1988) and hence might contribute to the pathogenesis of inflammatory diseases such as endometriosis. The presence of inflammation and neovascularization observed in and around ectopic endometrial implants, and the presence of inflammatory neutrophils in these lesions (Khorram et al., 1993), is compatible with the biological actions of IL-8 (Van Deuren et al., 1992). European Society for Human Reproduction and Embryology 1957

2 M.R.Gazvani et al. Previous reports from two small studies have shown elevated concentrations of IL-8 in peritoneal fluid from women with endometriosis (Ryan et al., 1995; Rana et al., 1996), of which a substantial amount is derived from peritoneal macrophages (Rana et al., 1996). However, this was not related to disease progression or stage. A study was therefore organized to evaluate the role of IL-8 in the pathogenesis of endometriosis in relation to the stage of disease. The presence of IL-8 in peritoneal fluid and peripheral blood throughout the menstrual cycle was investigated. It was determined if differences in concentration of IL-8 existed between women with and without endometriosis, between different stages of the disease and the phases of the menstrual cycle. Materials and methods Fifty consecutive patients undergoing laparoscopic surgery for benign gynaecological indications, abdominal pain (n 21), sterilization (n 11) or infertility (n 18), were recruited to take part in the trial. Infertility was defined as delay in conception of 12 months. The study was approved by the local ethics committee and informed consent was obtained prior to the operation. None of the patients had been on medication at least 1 month prior to the laparoscopy and none was on any long-acting drugs. Operative findings were recorded regarding the presence of endometriosis, location, volume and degree. The condition of tubes, ovaries, pouch of Douglas, and bowels were inspected and endometriosis was graded according to the American Fertility Society scoring system. Peritoneal fluid was obtained at the beginning of the operation, soon after the abdominal cavity was entered, and immediately processed. The fluid was centrifuged at 400 g for 10 min, and the supernatant removed and frozen at 20 C. The venous blood (10 ml) was allowed to clot, also centrifuged at 400 g for 10 min, serum was separated and frozen. Chemokine concentrations in peritoneal fluid and serum were measured using an enzyme-linked immunosorbent assay (CYTokit Red ; CYTimmune Sciences, College Park, MD, USA) designed specifically to measure immunoreactive IL-8 in body fluids, according to the manufacturer s instructions. Samples were analysed in duplicate both neat and diluted 1:10 and optical density values falling within the linear portion of the standard curve were used to estimate sample IL-8 concentrations by interpolation. The detection limit was ~0.2 ng/ml. Peritoneal fluid and blood concentrations of IL-8 were correlated to clinical and operative findings and complaints as well as the phase of the menstrual cycle and parity. Statistical analysis Non-parametric data were described as median (interquartile range, IQR) and parametric data as mean (SD). Unpaired t-test was used for the comparison of means and Mann Whitney U-test for medians. Kendall s rank correlation test was used to assess the correlation between two variables. Differences between groups were analysed using Fisher s exact test for non-parametric and χ 2 test for parametric data. Confidence intervals (CI) were calculated at 95% level. Results Peritoneal fluid was obtained from 49 patients, as there was no peritoneal fluid in one patient. The samples from two patients were heavily blood stained with possible retrograde menstruation; therefore, samples from 47 patients were pro- cessed and evaluated in this study. The study group (n 25) consisted of patients who had evidence of endometriosis. The control group consisted of patients with no evidence of endometriosis (n 22). In the vast majority of the study group, the primary location of endometriosis was the pouch of Douglas (n 21), followed by ovaries (n 3) and lateral pelvic wall (n 1). In the endometriosis group, eight patients had stage 1 disease, six patients stage 2, seven patients stage 3 and four patients stage 4 disease according to the American Fertility Society classification. There was no difference between the groups regarding their age, parity and the phase of menstrual cycle; 23 women were in the follicular phase and 24 women were in the luteal phase. Of the 22 women, eight were in the early, six were in the mid and nine were in the late follicular phase, and of the 25 women, eight were in the early, nine were in the mid, and seven were in the late luteal phase. Women in the control group and women with differing stages of endometriosis were evenly distributed regarding the phase of the cycle. Mean age in the endometriosis group was 28 years (8.1), and 29 years (6.9) in the group without endometriosis (P 0.56, CI 5.9 to 3.3). Median parity was 0 (0 3) in both groups. Concentrations of IL-8 in the peritoneal fluid and peripheral blood of both groups are shown in Table I. IL-8 was detectable in the peritoneal fluid of 41 (87%) women tested and its concentrations did not show a significant difference depending on the phase of the menstrual cycle; follicular phase 4.6 ng/ml ( ), luteal phase 7 ng/ml (5 10.9) (P 0.22, CI 4.6 to 1.6). IL-8 concentration in the peritoneal fluid of women with endometriosis [6.4 ng/ml ( )] was significantly higher compared to women without [2.4 ng/ml (0 6.6)] (P 0.02, CI 5.4 to 0.8), as shown in Table I. The difference between women with early endometriosis (stage 1) [9 ng/ml ( )], and women without endometriosis (2.4 ng/ml) was more significant (P 0.001, CI 9.4 to 3). Women with stage 1 endometriosis also had significantly higher concentrations of IL-8 (9 ng/ ml) compared to women with later stages (2, 3 and 4) of endometriosis (median 4.6 ng/ml, ; P 0.003, CI 1.6 to 8.4). IL-8 concentrations were lower as the disease progressed; 6.4 ng/ml ( ) for stage 2, 4.6 ng/ml ( ) for stage 3, and 2 ng/ml ( ) for stage 4 (Figure 1). There was no correlation between the blood concentrations of IL-8 and the presence of endometriosis (P 0.27, CI 2.8 to 1). The comparison of blood and peritoneal fluid concentrations of IL-8 also showed no correlation (P 0.78). Peritoneal fluid IL-8 concentrations were not correlated with the complaints of infertility (P 0.96, CI 2.6 to 3.4) or abdominal pain (P 0.66, CI 2.2 to 4.2). There was no significant difference in IL-8 concentrations between parous and nulliparous women (P 0.10, CI 0.8 to 6.4). No correlation was found between the visual presence of endometriosis and abdominal pain (P 0.38), infertility (P 0.37) or being parous (P 0.88). Discussion IL-8 is detectable in the peritoneal fluid of most women with an active ovarian cycle and it is a normal constituent of 1958

3 Interleukin-8 concentrations in endometriosis Table I. Interleukin-8 (IL-8) concentrations in peritoneal fluid and peripheral blood of women with and without endometriosis Endometriosis No endometriosis P-value 95% CI (n 25) (n 22) Age a 28 (8.1) 29 (6.9) to 3.3 Parity b 0 (0 3) 0 (0 3) IL-8 in peritoneal fluid (ng/ml) c 6.4 ( ) 2.4 (0 6.6) to 0.8 IL-8 in peripheral blood (ng/ml) c 2.5 ( ) 1.5 (1 1.9) to 1 CI confidence interval. a Mean (SD), b median (range), c median (IQR). Figure 1. Interleukin-8 concentrations in the peritoneal fluid of 22 patients with no evidence of endometriosis and 25 patients who had evidence of endometriosis at various stages (stage 1, n 8; stage 2, n 6; stage 3, n 7; stage 4, n 4). Boxes represent the distance between the first and third quartiles, the diamonds (r) mark the median values, with the minimum as the origin of the trailing whisker, and the maximum as the limit of the leading whisker. peritoneal fluid in women with and without endometriosis. The concentration of IL-8 in the peritoneal fluid was higher in women with endometriosis compared to women without and that difference was statistically significant as has been reported previously (Ryan et al., 1995; Rana et al., 1996). Ryan et al. (1995) studied 27 subjects in total (patient and control), in 22 of whom reliable dates for the phase of the cycle were available. They studied all stages of endometriosis and suggested that the IL-8 concentrations were positively related to the stage of the disease, which was completely opposite to our findings. Rana et al. (1996), in another small study of 25 subjects, eight control and 17 patients with only stage 3 or 4 endometriosis, found no relationship to the stage of the disease. They suggested, however, that peripheral blood concentration of IL-8 was related to peritoneal fluid concentrations, again completely opposite to our findings. In our study, peripheral blood concentrations were not associated with the peritoneal fluid concentrations of IL-8 or the presence of endometriosis. Therefore measurement of peripheral blood concentrations of IL-8 is not useful, although it has been found that peripheral blood macrophages from endometriosis patients produced increased concentrations of IL-8 (Braun et al., 1996). The most intriguing part of the findings in this study is that in women with early endometriosis (American Fertility Society Stage 1) IL-8 concentrations were much higher compared to women with later stages of the disease. It can be speculated that this may implicate IL-8 in the induction of the disease and it is conceivable that other chemokines participate in the chronic phase of endometriosis. In contrast, concentrations of the chemokine gro α were higher in peritoneal fluid from patients with severe endometriosis compared to those without the disease (Oral et al., 1996). Presenting complaints of infertility or abdominal pain did not correlate with the IL-8 concentrations in the peritoneal fluid. It is important to note that there was also no correlation between the visual presence of endometriosis and abdominal pain. This is in line with previously published data, as laparoscopic findings are negative in anywhere from 10 to 90% of women with chronic pelvic pain (Howard, 1996). It is important that gynaecologists appreciate that laparoscopy is only one of many possible methods of evaluation and recognize its diagnostic limitations and pitfalls. Peritoneal fluid concentrations of IL-8 did not show a correlation to the phase of the menstrual cycle, which supports the findings of an in-vitro study by Kelly et al. (1994) which showed that both proliferative and secretory endometrial biopsies demonstrate positive immunological staining for IL-8. There are a number of potential cellular sources of IL-8 in 1959

4 M.R.Gazvani et al. endometriosis. Enhanced production by peritoneal macrophages has been found (Rana et al., 1996) but normal endometrial gland cells (Saito et al., 1994) and stromal cells (Arici et al., 1996) also produce IL-8 that can be enhanced by proinflammatory mediators. In normal non-pregnant endometrium, IL-8 was found to be localized perivascularly (Critchley et al., 1994), suggestive of a direct role upon endothelial cells as well as a function in presenting a fixed chemotactic stimulus to circulating leukocytes. However, other angiogenic factors, including vascular endothelial growth factor (VEGF) (McLaren et al., 1996) and TNF-α (Kupker et al., 1996), have also been shown to be present in increased concentrations in peritoneal fluid from endometriosis patients. Studies of expression of chemokines have provided correlative evidence of their involvement in disease. The evidence that members of the chemokine family contribute to the development of inflammation and concomitant tissue injury in at least some settings is incontrovertible. Because chemokines appear relatively selective in their action compared with many other inflammatory mediators, they have been regarded as promising targets for development of antiinflammatory therapies. However, inhibiting the functions of chemokines in vivo may lead to unwanted and even unanticipated secondary effects. It is only recently that more direct evidence for the role of chemokines in vivo has been obtained by using antibodies to chemokines to suppress inflammation in various animal models (Furie and Randolph, 1995). Animal studies in which antibodies are used to neutralize the activity of individual members of the chemokine family confirm that these mediators contribute to the development of both acute and chronic inflammation. Therapies that target chemokines directly or enhance the body s mechanisms for controlling their activity may prove to be reasonable approaches for treatment of inflammatory diseases. Production of anti-chemokine antibodies may constitute a second mechanism for limiting the activity of chemokines in vivo. Polyclonal antibodies against IL-8 (Peichl et al., 1992; Reitamo et al., 1993) have been described. Anti-IL-8 antibodies are found in normal human serum, predominantly in complex with its antigen. The antibody against IL-8, which binds to IL-8 with an affinity of ~10 pmol/l (Sylvester et al., 1992), is apparently present in sufficient quantity to prevent accumulation of free IL-8 in the blood (Peichl et al., 1992; Sylvester et al., 1992; Reitamo et al., 1993; Sylvester et al., 1993; Ida et al., 1994). We conclude that IL-8 may be an important factor that contributes to the pathogenesis of endometriosis possibly by promoting neovascularization. This information can be a guide in the development of new therapeutic approaches for the treatment of endometriosis. Whether chemokines such as IL-8 can be exploited therapeutically to limit the extent of injury is less certain. The feasibility of targeting these mediators for treatment of inflammatory diseases will no doubt be determined as a more complete understanding of the biology of the chemokines emerges. Acknowledgement We would like to thank Sister M. Wood and all theatre staff for their co-operation in the collection of specimens References American Fertility Society (1985) Revised American Fertility Society classification of endometriosis. Fertil. Steril., 143, Arici, A., MacDonald, P.C. and Casey, M.L. (1996) Progestin regulation of interleukin-8 mrna levels and protein synthesis in human endometrial stromal cells. J. Steroid Biochem. Mol. Biol., 58, Braun, D.P., Gebel, H., House, R. et al. (1996) Spontaneous and induced synthesis of cytokines by peripheral blood monocytes in patients with endometriosis. Fertil. Steril., 65, Critchley, H.O., Kelly, R.W. and Kooy, J. (1994) Perivascular location of a chemokine interleukin-8 in human endometrium: a preliminary report. Hum. Reprod., 9, Eiserman, J., Gast, M.J., Pineda, J. et al. (1988) Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery. Fertil. Steril., 50, Fakih, H., Baggett, B., Holtz, G. et al. (1987) Interleukin-1: a possible role in the infertility associated with endometriosis. Fertil. Steril., 47, Furie, M.B. and Randolph, G.J. (1995) Chemokines and tissue injury. Am. J. Pathol., 146, Halme, J., Becker, S., Hammond, M.G. et al. (1983) Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am. J. Obstet. Gynecol., 145, Hill, J.A. and Anderson, D.J. (1989) Lymphocyte activity in the presence of peritoneal fluid from fertile women and infertile women with and without endometriosis. Am. J. Obstet. Gynecol., 161, Howard, F.M. (1996) The role of laparoscopy in the evaluation of chronic pelvic pain: pitfalls with a negative laparoscopy. J. Am. Assoc. Gynecol. Laparosc., 4, Ida, N., Sakurai, S. and Kawano, G. (1994) Detection of monocyte chemotactic and activating factor (MCAF) in normal blood and urine using a sensitive ELISA. Cytokine, 6, Keenan, J.A., Chen, T.T., Chadwell, N.L. et al. (1994) Interferon-gamma and interleukin-6 (IL-6) in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am. J. Reprod. Immunol., 32, Kelly, R.W., Illingworth, P., Baldie, G. et al. (1994) Progesterone control of interleukin-8 production in endometrium and chorio decidual cells underlines the role of the neutrophil in menstruation and parturition. Hum. Reprod., 9, Khorram, O., Taylor, R.N., Ryan, I.P. et al. (1993) Peritoneal fluid concentrations of the cytokine RANTES correlate with severity of endometriosis. Am. J. Obstet. Gynecol., 169, Koch, A.E., Polverini, P.J., Kunkel, S.L. et al. (1992) Interleukin 8 as a macrophage derived mediator of angiogenesis. Science, 258, Kupker, W., Felberhaum, R., Bauer, O. and Diedrich, K. (1996) Significance of tumor necrosis factor alpha (TNF-alpha) in endometriosis. Geburtshilfe- Frauenheilkd, 56, McLaren, J., Prentice, A., Charnock-Jones, D.S. and Smith, S.K. (1996) Vascular endothelial growth factor (VGEF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum. Reprod., 11, McLaren, J., Dealtry, G., Prentice, A. et al. (1997) Decreased levels of the potent regulator of monocyte/macrophage activation, interleukin-13, in the peritoneal fluid of patients with endometriosis. Hum. Reprod., 12, Mori, H., Sawairi, M., Makagawa, M. et al. (1991) Peritoneal fluid interleukin 1-beta and tumor necrosis factor in patients with benign gynecologic disease. Am. J. Reprod. Immunol., 26, Murphy, A.A., Green, W.R., Bobbie, D. et al. (1986) Unsuspected endometriosis documented by scanning electron microscopy in visually normal peritoneum. Fertil. Steril., 46, Oosterlynck, D.J., Meuleman, C., Sobis, H. et al. (1993) Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil. Steril., 59, Oral, E., Seli, E., Bahtiyar, M.O. et al. (1996) Growth-regulated alpha expression in the peritoneal environment with endometriosis. Obstet. Gynecol., 88, Overton, C., Fernandez-Shaw, S., Hicks, B. et al. (1996) Peritoneal fluid cytokines and the relationship with endometriosis and pain. Hum. Reprod., 11, Peichl, P., Ceska, M., Broell, H. et al. (1992) Human neutrophil activating peptide/interleukin 8 acts as an autoantigen in rheumatoid arthritis. Ann. Rheum. Dis., 51, Peveri, P., Walz, A., Dewald, B. and Baggiolini, M. (1988) A novel neutrophilactivating factor produced by human mononuclear phagocytes. J. Exp. Med., 167,

5 Interleukin-8 concentrations in endometriosis Rana, N., Braun, D.P., House, R. et al. (1996) Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil. Steril., 65, Reitamo, S., Remitz, A., Varga, J. et al. (1993) Demonstration of interleukin 8 and autoantibodies to interleukin 8 in the serum of patients with systemic sclerosis and related disorders. Arch. Dermatol., 129, Ryan, I.P., Tseng, J.F., Schriock, E.D. et al. (1995) Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil. Steril., 63, Saito, S., Kasahara, T., Sakakura, S. et al. (1994) Detection and localization of interleukin-8 mrna and protein in human placenta and decidual tissues. J. Reprod. Immunol., 27, Shaw, R.W. (1993) An Atlas of Endometriosis. Parthenon Publishing Group, New York. Sylvester, I., Yoshimura, T., Sticherling, M. et al. (1992) Neutrophil attractant protein-1 immunoglobulin G immune complexes and free anti-nap-1 antibody in normal human serum. J. Clin. Invest., 90, Sylvester, I., Suffredini, A.F., Boujoukos, A.J. et al. (1993) Neutrophil attractant protein-1 and monocyte chemoattractant protein-1 in human serum. Effects of intravenous lipopolysacchride on free attractants, specific IgG autoantibodies and immune complexes. J. Immunol., 151, Van Deuren, M., Dofferhoff, A.S.M. and Vander Meer, J.W.M. (1992) Cytokines and the response to infection. J. Pathol., 168, Received on November 10, 1997; accepted on March 30,