Interleukin 1, interleukin-6, and tumor necrosis factor- in endometriotic tissue and in endometrium

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1 FERTILITY AND STERILITY VOL. 75, NO. 3, MARCH 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Interleukin 1, interleukin-6, and tumor necrosis factor- in endometriotic tissue and in endometrium Agneta Bergqvist, M.D., Ph.D., Christine Bruse, M.D., Magdalena Carlberg, Ph.D., and Kjell Carlström, Ph.D. Department of Obstetrics and Gynecology, Huddinge University Hospital, Huddinge, and Karolinska Institutet, Stockholm, Sweden Received June 20, 2000; revised and accepted September 5, The study was supported by grant B95-17X A from the Swedish Medical Research Council, Stockholm and by the Karolinska Institutet, Stockholm, Sweden. Reprint requests: Agneta Bergqvist, M.D., Ph.D., Department of Obstetrics and Gynecology, Huddinge University Hospital, S Huddinge, Sweden (FAX: ; obgyn.hs.sll.se) /01/$20.00 PII S (00) Objective: To compare the levels of IL-1, IL-6, and TNF in endometriotic tissue and in endometrium from women with endometriosis and healthy controls. Design: Open. Setting: Department of Obstetrics and Gynecology at a university hospital. Patient(s): Twenty-six women with endometriosis and 22 controls operated on for clinical indications. Intervention(s): ELISA in homogenized tissue samples collected during surgery. Main Outcome Measure(s): Levels of IL-1, IL-6, and TNF in tissue homogenates. Result(s): The three types of tissue differed significantly with respect to all three cytokines. Endometriotic tissue had significantly higher concentrations of IL-1 than endometrium from both patients with endometriosis and healthy controls. Both endometriotic tissue and endometrium from patients had significantly higher concentrations of IL-6, and endometriotic tissue had significantly lower concentration of TNF than did endometrium from controls. IL-1 showed a cycle phase dependence that was significant in endometrium from patients, being higher in the secretory than in the follicular phase. IL-1 was significantly higher in endometrioma than in lesions of other localizations. Concentrations of IL-1 and IL-6 were positively correlated in endometriotic tissue and in endometrium from controls. No other significant correlations were found. Conclusion(s): This study has shown a significant production of IL-1, IL-6, and TNF in endometriotic tissue and endometrium, with significant differences between the tissue types, indicating a deviating cytokine pattern in both endometriotic tissue and endometrium from women with endometriosis compared with that from healthy controls. (Fertil Steril 2001;75: by American Society for Reproductive Medicine.) Key Words: IL-1, IL-6, TNF, endometriosis, endometrium The adhesion of endometrial fragments to other tissues is regarded as the incitement of a local inflammatory reaction that, over time, develops into a chronic inflammation (1). The inflammatory reaction in the pelvic cavity involves, to a high degree, activation of macrophages, which produce a number of growthregulating substances. There are indications that some of the inflammatory factors involved also stimulate the growth of ectopic endometrial cells in early stages of endometriosis (2, 3). These compounds may also have effects on fertility as well as on nociceptors, thus causing infertility and pain. Cytokines are regulatory peptides or glycoproteins that can be produced by virtually every nucleated cell type in the body and that have pleiotropic regulatory effects on many cell types. Unlike hormones, cytokines usually act both as paracrine and/or autocrine signals, only occasionally coming into the circulation, where they may act as endocrine mediators (4). Macrophages are among the major producers of cytokines, especially interleukins-1 and -6 (IL-1, IL-6) and tumor necrosis factor- (TNF ); this is probably not the case under normal conditions but after stimulation with a variety of substances (2). The interleukins are 489

2 regarded as modulators of cellular proliferation and as inducers of other cytokines, like a cascade in acute inflammation (5). Cytokines produced within the uterine environment participate in the growth regulation of the endometrium by a steroid-cell and intercellular interaction (6). Cytokines may also contribute to the pathophysiology of endometriosis in at least two ways, namely by enhancing the establishment and proliferation of ectopic endometrial implants and by influencing the secretion of cytokines by macrophages, which may lead to adverse changes in the hormonal, chemical, and/or cellular milieu (7). The cytokines IL-1, IL-6, and TNF are of high interest because they are partly hormonally regulated and they play an important role as inflammatory mediators. IL-1 is involved in the regulation of immune response and inflammation. There are two distinct forms of IL-1, and, with similar biological activities (8). IL-1 is present in endometrium, both in epithelial and stromal cells, at least in the late secretory phase. IL-1 has the same distribution but appears normally in lower amounts. IL-1 mrna is expressed in the endometrium in the late secretory phase and parallels serum levels of IL-1 that vary during the cycle, with maximum values during the secretory phase (9). IL-6 is a multifunctional cytokine that stimulates cell proliferation and is involved in creation of adhesions. It is found in endometrium, both in stromal and epithelial cells but mainly in the latter, and the secretion is hormonally regulated (6). Thus IL-6 may play a central role in epithelial stromal cell interaction, governing the regulation of normal uterine function. IL-6 is a normal constituent in peritoneal fluid, and the concentration is increased in women with endometriosis (10). TNF has different effects, both stimulating and inhibiting proliferation, immunomodulation, and angiogenesis, and it is cytotoxic and proinflammatory. Both its mrna and the protein itself increase in endometrial epithelial cells during the proliferative phase, decline in the early secretory phase, and rise again during the late secretory phase, when they appear both in epithelial and stromal cells (11, 12). TNF has been shown to induce the production of IL-1 and other growth factors (5). Although a production of different cytokines from endometriotic stromal and epithelial cells in vitro has been observed (13, 14), we have found no data on tissue concentrations. To gain more information about the possible roles of cytokines in the autocrine and paracrine growth regulation of endometriosis, we have measured the levels of IL-1, IL-6, and TNF in tissue homogenates of endometrium and endometriotic tissue from women with endometriosis and in endometrium from healthy women. MATERIALS AND METHODS Clinical Material Endometriotic tissue samples were obtained during surgery from 26 women, 14 of whom were in the follicular phase and 12 in the luteal phase. Uterine endometrium was obtained by curettage from 25 women with endometriosis, 18 of whom also contributed with endometriotic tissue. Thirteen women were in the follicular phase, and 12 were in the luteal phase. Normal endometrium was obtained by curettage from 22 controls, 9 in the follicular phase and 13 in the luteal phase, who were operated on because of sterilization or myomas. All women were regularly menstruating, and none of them were on or had received any hormonal treatment or hormonal contraception or had been breast feeding for the last 3 months before surgery. The study was approved by the local ethics committee, and the women gave their oral consent to the tissue sampling. To obtain endometriotic tissue that was as pure as possible and to make the samples as similar and compatible as possible, we preferred endometriotic tissue scraped with the back edge of a knife from the inside of ovarian endometriomas after the cyst had been rinsed in cold physiological saline. Nineteen samples were obtained in this way. Moreover, in two cases, a small lesion was extirpated from the ovarian surface; in one case, the endometriotic sample was obtained from a peritoneal lesion; in one case, it was obtained from submucous vaginal endometriosis; in two cases, from scar endometriosis in the abdominal wall; and in one case, from the omentum. The tissue samples were prepared immediately after extirpation in the operation theatre. Part of the endometriotic tissue was carefully separated from inappropriate surrounding tissue, snap-frozen in liquid nitrogen within 30 minutes, and transported for storage to a 80 C freezer within 2 hours until assays were performed. The rest of the sample was fixed in 4% formalin for histological evaluation after staining with hematoxylin and eosin. The same procedure was performed for the endometrial samples. The menstrual cycle phase of each woman was assessed according to date of latest menstrual bleeding; the histologic cycle phase in the endometrial samples was determined according to the method of Noyes et al. (15). The samples were classified into proliferative or secretory patterns. Tissue Preparation The samples were thawed, washed in phosphate-buffered saline (PBS, Dulbecco s Gibco BRL, Life Technologies LT, Paisley, Scotland), blotted on filter paper, and weighed. Thereafter, they were homogenized at 4 C in plastic tubes (15 ml, Falcon, Becton-Dickinson, Lincoln Park, NJ) containing PBS plus sodium chloride adjusted to a final concentration of 0.5 M, using a Polytron homogenizer (International Laboratory App, GmbH, 7801 Dottingen, Germany). The homogenates were centrifuged at 10,000 g for 10 minutes at 4 C. The supernatants were collected and diluted with 490 Bergqvist et al. Cytokines in endometriosis and endometrium Vol. 75, No. 3, March 2001

3 PBS plus sodium chloride to 0.5 M to contain cytosol from 0.01 g tissue/ml and were stored at 80 C until assayed. Analytical Methods IL-1 in the tissue homogenates was determined by ELISA using a commercial kit from R&D Systems (Minneapolis, MN). IL-6 and TNF were determined by immunoradiometric methods using commercial kits from Medgenix SA (Fleurus, Belgium). Detection limits and within- and between-assay variations were as follows: for IL-1, 1 ng/l: 3% and 7%; for IL-6, 5 ng/l: 6% and 8%; and for TNF, 5 ng/l: 6% and 7%, respectively. Values below the detection limits were set to 0.5 ng/l for IL-1 and to 2 ng/l for IL-6 and TNF in the statistical calculations. The values are expressed as picograms of protein per milligram of tissue wet weight because sufficient homogenate for protein determination (16) was not available in all samples. However, excellent correlations (P , linear regression) were found between cytokine concentrations expressed per milligram of wet weight and per milligram of protein: for IL-1, r 0.96, n 47; for IL-6, r 0.72, n 51; and for TNF, r 0.83, n 50. We therefore considered expression per milligram of wet weight sufficiently adequate, and we have used it to permit inclusion of our total clinical material. Statistical Methods The values were not normally distributed and are given as median and range. The occurrence of significant differences between the three groups of tissue was tested by Kruskal- Wallis test; post hoc statistical analysis of difference between two groups was made by Mann-Whitney U test. Correlations were tested by Spearman s rank correlation test. The significance level was set at P FIGURE 1 Tissue concentrations per milligram wet weight of IL-1 (A), IL-6 (B), and TNF (C) in endometriotic tissue (EOS, n 26), in uterine endometrium from patients with endometriosis (EM PAT, n 25) and from healthy controls (EM CTR, n 22). Box plots showing the 10th, 25th, 50th, 75th, and 90th percentiles. RESULTS Bergqvist. Cytokines in endometriosis and endometrium. Fertil Steril Light-microscopic examination of the hematoxylin and eosin stained endometriotic sections confirmed endometriotic tissue in all samples, consisting of glands and surrounding cytogenic stroma. Concentrations of IL-1, IL-6, and TNF in homogenates of endometriotic tissue and of endometrium from patients with endometriosis and from healthy controls are given in Figure 1a c. Because of lack of sufficient sample material, determination of IL-1 and IL-6 could not be performed in 10 samples each, and neither could we determine TNF in two control endometrial samples. The three types of tissue differed significantly in the total material with respect to IL-1 (P 0.01), to IL-6 (P 0.001), and to TNF (P 0.05; Kruskal-Wallis test). In unpaired comparisons, both endometriotic tissue and endometrium from patients with endometriosis had significantly higher concentrations of IL-6, and endometriotic tissue had significantly lower concentrations of TNF, than did endometrium from healthy controls (Table 1). Paired comparisons in patients with endometriosis revealed a significantly higher level of IL-6 in endometriotic tissue than in uterine endometrium, 5.10 pg/mg wet weight (range, ) versus 1.89 (range, ) pg/mg wet weight, respectively (P 0.05, n 17). Otherwise, no significant differences were found. Concerning the type of endometriotic lesion, there were 19 ovarian endometriomas and 7 of other locations. Ovarian endometriomas had significantly higher IL-1 content than did lesions from other locations: 0.62 pg/mg wet weight (range, ) versus 0.10 ( ) pg/mg wet weight, respectively (P 0.01). There were no differences in the concentration of other cytokines. The subgroups of nonovarian localization were too small for a statistical comparison. When tissue concentrations in different cycle phases were FERTILITY & STERILITY 491

4 TABLE 1 Tissue concentrations of IL-1, IL-6, and TNF- in endometriotic tissue and in endometrium from women with endometriosis and in endometrium from healthy controls. Cytokine (pg/mg wet weight) Endometriotic tissue Endometrium, patients Endometrium, controls IL ( ) a,c (17/20) 0.11 ( ) (12/21) 0.05 ( ) (10/22) IL ( ) c (23/26) 1.90 ( ) c (22/25) 0.20 ( ) (9/22) TNF ( ) b (17/26) 1.11 ( ) (17/25) 2.08 ( ) (19/20) Note: Values are given as median (range). Numbers of samples within parentheses are given as number of samples with measurable concentration/total samples. a P 0.05 (statistical significance of difference versus endometrium from patients with endometriosis). b P 0.01 (statistical significance of difference versus endometrium from healthy controls). c P (statistical significance of difference versus endometrium from healthy controls). Bergqvist. Cytokines in endometriosis and endometrium. Fertil Steril compared, a significantly higher concentration was found only for IL-1 in the secretory phase compared with in the proliferative phase in endometrium from patients with endometriosis: 0.36 pg/mg wet weight (range, ) versus 0.04 (range, ) pg/mg wet weight (P 0.01). In proliferative phase, the level of IL-1 was much higher in endometriotic tissue than in endometrium, both from the same patient and from controls (P 0.01 and P 0.001, respectively). In secretory phase, the only significant difference was found for IL-6; that was significantly higher in endometrium from women with endometriosis compared with control endometrium (P 0.01). No other significant differences between cycle phases were found. In the total material, the tissue concentrations of IL-1 and IL-6 showed significant positive correlations in endometriotic tissue (r s 0.65, P 0.01) and in endometrium from healthy controls (r s 0.44, P 0.05). No statistically significant correlation between IL-1 and IL-6 concentrations was found in endometrium from patients with endometriosis (r s 0.42, P 0.06). No significant correlations were found between IL-1 and IL-6 on the one hand and TNF on the other. A positive correlation of borderline significance was found for TNF in paired samples of endometriotic tissue and uterine endometrium (r s 0.48, P 0.055). No correlation between the two paired tissue types was found for IL-1 or IL-6. There were no differences in cytokine content between endometriotic tissue from the inside of ovarian endometriotic cysts (n 20) and from other locations (n 6; data not shown). DISCUSSION This study showed above all that endometriotic tissue differed significantly from normal endometrium concerning tissue concentrations of IL-1, IL-6, and TNF. This is interesting but not entirely surprising in view of the different cellular surroundings and different morphological response to ovarian steroids. More interesting, however, is that endometrium from women with endometriosis also differed from endometrium from healthy women. The most pronounced differences concerned IL-6, which was significantly higher, and TNF, which was somewhat lower, although not statistically significantly so. Regarding endometrium from women with endometriosis, all three cytokines appeared to occur at intermediate levels compared with levels in endometriotic tissue and healthy endometrium. IL-6 and TNF concentrations were similar in endometriotic tissue and endometrium from women with endometriosis, and TNF concentrations in the two types of tissues obtained from the same patients were positively correlated. This suggests that pathological changes in the uterine endometrium in certain women may contribute to the development of endometriosis. In some samples, mainly endometrium from healthy women, the cytokines were not even detectable. This might indicate that the contamination of macrophages is greater in tissue samples from women with endometriosis. The basal synthesis of IL-1, IL-6, and TNF in peripheral blood monocytes and peritoneal fluid macrophages is reported to be increased in women with endometriosis (7, 12). It may therefore be argued that differences in contamination by macrophages may be one reason for the higher tissue concentrations of IL-1 and IL-6 found in our study. This is contradicted by the significantly lower levels of TNF found in the tissue samples from the same patients. In addition, a study by us has shown that both stromal and epithelial cells from endometrium and endometriotic tissue are capable of producing IL-1, IL-6, and TNF in vitro (14). It may therefore be concluded that most, if not all, of the cytokines found in the present study are related to the tissue cells studied and not to migrating monocytes or macrophages. It is not possible to evaluate the macrophage content in tissue samples that are homogenized. It is known that macrophages are spread heterogeneously in tissues (17), which is why it would not have been relevant to estimate by immunohistochemistry the number of macrophages in a 492 Bergqvist et al. Cytokines in endometriosis and endometrium Vol. 75, No. 3, March 2001

5 neighboring tissue sample. Jones et al. (18) did not find any significant differences in leukocyte populations between endometrium from women with endometriosis and from controls in any phase of the menstrual cycle. The findings of significantly higher tissue concentrations of IL-6 in endometriotic tissue and endometrium from patients compared with endometrium from healthy controls is in line with findings from Rier and co-workers (19). They found that stromal cells, isolated from endometriotic implants, secreted large amounts of IL-6 in vitro but stained weakly for IL-6 receptors and did not respond to IL-6 by growth inhibition. A dose-dependent stimulation of IL-6 production by IL-1 has been shown in endometrial stromal cells, endometriotic cells, and decidua (6, 9, 20, 21), and this may certainly explain our finding of positive correlations between IL-6 and IL-1 in endometriotic tissue and in endometrium. Taken together, our results indicate an active production of the proinflammatory cytokines IL-1 and IL-6 and an autocrine stimulatory mechanism in endometriotic tissue. The facts that IL-6 is involved in different aspects of inflammation and that the endometrium is a tissue in which continuous damage and repair is taking place have led to the suggestion that IL-6 may play a central role in the cyclic shedding of endometrial tissue (6). The consequences of the significantly higher tissue concentrations of IL-6 in endometrium from women with endometriosis compared with that in endometrium from healthy women have to be studied further. Estradiol-17, and even more so, progesterone, is reported to down-regulate cytokine expression, especially the production of IL-6, in endometrial as well as in endometriotic stromal cells (21 23). Estrogen and progesterone receptor levels are lower in endometriotic tissue than in endometrium and do not vary according to the menstrual cycle (24 26). This may result in a different hormonal regulation and in different production of cytokines in endometriotic tissue than in endometrium, and the steroid effect on IL-6 may be reduced in women with endometriosis. This is interesting from a clinical point of view, as gestagen treatment of endometriosis has varying and, to some degree, dose-dependent effects (27). Another reason for the elevated IL-6 levels in endometriotic tissue and endometrium from patients may be that they reflect a general increase in the production of IL-6 in this disease. Pellicer and coworkers have demonstrated distinctly elevated circulating concentrations of IL-6 in patients with endometriosis, compared with levels in healthy women (28). The concentration of TNF in endometriotic tissue was significantly lower than in endometrium from healthy controls. Also, the level of TNF in endometrium from patients was somewhat lower than in endometrium from controls, although not significantly, but there is an interesting trend from lowest median level in endometriotic tissue over a slightly higher level in endometrium from patients to significantly higher level in endometrium from controls. A trend in the opposite direction was found for IL-1 and IL-6. TNF influences the adhesion of human endometrial cells to peritoneum (29). The low TNF concentrations found in endometrium from women with endometriosis suggest that this cytokine is rapidly consumed, not activated, or down-regulated in endometriotic tissue. Thus, although the level of TNF in endometriotic tissue and in endometrium from women with endometriosis is low, TNF from other sources as well as other factors in the peritoneal fluid might instead regulate the process of adhesion of endometrial fragments to peritoneum (29). The basal synthesis of TNF and other cytokines in macrophages in the peritoneal fluid has indeed been found elevated in patients with endometriosis (7). One reason for low levels of TNF in endometriotic tissue might be the high IL-6 content also demonstrated in this study because IL-6 has been shown under certain circumstances to down-regulate TNF (30). Thus, a high production of IL-6 in endometriotic tissue may in a similar manner suppress the local production and action of TNF. Although a benign disease, endometriosis has some tumorlike features, such as the capacity of invasiveness. Production of TNF-suppressing substances might be another such similarity. Tissue concentrations of IL-1 were significantly higher in endometriotic tissue than in endometrium from patients and from controls, and ovarian endometriomas had significantly higher levels than endometriotic lesions of other locations. The endometrium from the two patient groups did not differ in this respect. Our finding of low IL-1 concentrations in endometrium confirms previous results of Tabibzadeh and Sun (31) that demonstrated weak immunohistochemical staining of IL-1 in endometrium. IL-1 mrna in endometrium is mainly localized to macrophages and endothelial cells but also to stromal cells and even more so to epithelial cells (4). The significantly higher IL-1 levels found in the secretory phase compared with in the proliferative phase in endometrium from women with endometriosis may reflect a larger volume of epithelial cells in relation to the volume of stromal cells in the secretory phase. In control endometrium, the difference between proliferative and secretory phase did not reach statistical significance (P 0.075). The explanation for the difference in the level of IL-1 in secretory related to proliferative endometrium from women with endometriosis and controls can only be speculated on. One explanation might be the limited number of cases. It might also depend on the higher number of activated macrophages in women with endometriosis. However, a third explanation might be that it is related to the different local steroid environment found in endometrium of women with endometriosis compared with healthy women (32). This is interesting because a higher production of IL-1 by a progesterone-primed endometrium has previously been reported (33). FERTILITY & STERILITY 493

6 What impact the high IL-1 level in endometriotic tissue may have on surrounding cells remains to be elucidated. Endometrial stromal cells have often been compared with fibroblasts, but unlike fibroblasts, which proliferate when exposed to IL-1, the proliferation of normal endometrial stromal cells is inhibited by IL-1, at least in vitro (34). IL-1 is a potent stimulator of collagenase production by several cell types, including fibroblasts and endometrial epithelium, and it has been proposed to be a major mediator of tissue degradation and remodeling in inflammatory diseases (35). Our results are therefore of interest in view of the compact hard fibrotic tissue that is often created around endometriotic implants during the chronic inflammatory process. IL-1 and IL-6 have been shown to induce changes in vascular permeability (12, 36). It may be speculated that the elevated levels of these cytokines in endometriotic tissue may contribute to increased vascular permeability at the adhesion site, thus increasing the inflammatory reaction. Our findings of elevated tissue concentrations of IL-1 in endometriotic tissue and of IL-6 in endometriotic tissue and endometrium from patients with endometriosis are also interesting in view of the dysmenorrhoea often related to endometriosis. Dysmenorrhoea is referred to as a pronounced local hypertension in the uterine cavity due to a hypercontraction of the uterine muscle. This is probably stimulated by prostaglandins and has been shown to cause ischemia in the myometrium (36). IL-1 stimulates PGE2 production by endometrial epithelial cells (31), and a local release of IL-1 may contribute to this reaction. 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