Expression of interleukin-10 in patients with adenomyosis

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1 Expression of interleukin-10 in patients with adenomyosis Fei Wang, M.D., Ph.D., Hui Li, Ph.D., Zhongli Yang, M.D., Xuelian Du, M.D., Ph.D., Min Cui, M.D., Ph.D., and Zeqing Wen, M.D. Department of Obstetrics and Gynecology, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, People s Republic of China Objective: To investigate the expression of interleukin-10 (IL-10) in adenomyosis. Design: Laboratory study using human tissue. Setting: University hospital. Patient(s): Thirty-four patients with adenomyosis and 30 women without adenomyosis who underwent hysterectomy for nonendometrial pathology. Intervention(s): Tissue sections were immunostained with murine monoclonal antihuman IL-10 antibodies. Main Outcome Measure(s): Microscopic evaluation to assess the presence and localization of IL-10 throughout the menstrual cycle in both eutopic endometrial and adenomyotic tissues of women with adenomyosis and compare it with IL-10 expression in the normal endometrium. Result(s): In the eutopic and ectopic endometrium of women with adenomyosis, epithelial cells showed higher staining intensity than the normal controls. However, no significant differences were found in the epithelial IL-10 immunostaining H score values between the eutopic endometrium and adenomyosis foci. Nonetheless, we observed a cyclic variation in the eutopic epithelial IL-10 immunoreactivity throughout the menstrual cycle with higher H score values in the secretory phase than in the proliferative phase. Conclusion(s): These findings suggest that an abnormality of inflammatory response may be present in the eutopic and ectopic endometrium of women with adenomyosis and that IL-10 may contribute to the pathogenesis and pathophysiology of adenomyosis. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Adenomyosis, endometriosis, endometrium, interleukin-10, immunology Adenomyosis is a common but complex gynecological syndrome of unknown pathogenesis; it is defined as the presence of islands of endometrial glands and stroma within the myometrium. Although adenomyosis is traditionally regarded to be closely related to endometriosis, obvious differences exist between the two diseases with respect to clinical characteristics and likely pathogenetic mechanisms. Endometriosis is believed to be an important cause of infertility among reproductive-age women (1), whereas adenomyosis is more common in parous women and can cause a high incidence of early miscarriage (2). Many studies have shown that alterations in the immune system might contribute to the pathogenesis of endometriosis (3 5); however, to date, there have been no similar reports on the inflammatory nature of adenomyosis, particularly with regard to immunosuppression. Received September 20, 2007; revised and accepted February 26, 2008; published online April 25, F.W. has nothing to disclose. H.L. has nothing to disclose. Z.Y. has nothing to disclose. X.D. has nothing to disclose. M.C. has nothing to disclose. Z.W. has nothing to disclose. Reprint requests: Zeqing Wen, Department of Obstetrics and Gynecology, Shandong Provincial Hospital Shandong University, 324 Jingwu Road, Jinan, Shandong , People s Republic of China (FAX: ; wzqdoctor@163.com). Interleukin-10 (IL-10) is an important immunomodulatory cytokine produced by many cell populations. It was first described as a cytokine synthesis inhibitory factor for T lymphocytes produced by T helper 2 (Th2) cell clones and was proved to inhibit interferon-g synthesis in Th1 cell clones (6). Numerous research studies suggest that IL-10 is one of the major anti-inflammatory cytokines and plays important roles in several chronic inflammatory diseases and cancers. Furthermore, uterine IL-10 has been demonstrated to have a dichotomous effect on human leukocyte antigen expression on trophoblast cells by inducing human leukocyte antigen G expression while down-regulating the expression of classical class I and class II antigens (7). Therefore, we postulate that IL-10 expression may contribute to the establishment and maintenance of immunosuppression and might explain the persistence of the ectopic foci within the peritoneal cavity or myometrium without elimination by the immune system of the host in women with endometriosis or adenomyosis. It has been reported that the peritoneal IL-10 level is significantly higher in women with endometriosis than in healthy women (8). Yet there are no similar reports on the expression of IL-10 in patients with adenomyosis. Therefore, we designed this study to determine whether patients with adenomyosis also express IL-10 in the ectopic and/or eutopic endometrium and to compare IL-10 expression levels among the normal endometrium, eutopic endometrium, and ectopic endometrium of women with adenomyosis /09/$36.00 Fertility and Sterility â Vol. 91, No. 5, May doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 TABLE 1 H score values for epithelial IL-10 expression in the endometrium. No. of samples H score (mean ± SEM) P Normal endometrium Proliferative phase Secretory phase <.01 a Eutopic endometrium <.01 b Proliferative phase Secretory phase <.01 c Adenomyosis d a Normal secretory endometrium versus normal proliferative endometrium. b Eutopic endometrium versus normal endometrium. c Eutopic secretory endometrium versus normal proliferative endometrium. d Adenomyosis versus eutopic endometrium. MATERIALS AND METHODS Tissue Collection This study was approved by the Institutional Review Board of Shandong University, and written informed consent was obtained from all participants. Adenomyosis tissue samples with their homologous eutopic endometrium were collected from 34 women (mean age, 44.5 years; range, years) during hysterectomy. The initial indications for hysterectomy were hypermenorrhea (n ¼ 17), leiomyoma (n ¼ 13), and benign adnexal mass (n ¼ 4). Samples of normal endometrial tissues were obtained from 30 fertile women (mean age, 43.6 years; range, years) who had undergone hysterectomy for nonendometrial pathologies such as leiomyoma (n ¼ 26) or benign ovarian cysts (n ¼ 4); these patients were included in the control group. All patients in both study and control groups had normal menstruation cycles, and none of them had received any hormone therapy within 6 months before surgery. Adenomyosis was confirmed by histological examination. The day of the menstrual cycle was established from the women s menstrual history and was confirmed by endometrial dating using the criteria of Noyes et al. (9). All endometrial samples were categorized according to the menstrual cycle phases: proliferative (days 1 14 of the cycle) and secretory (days of the cycle). Thirteen and 21 samples were in the proliferative and secretory phases, respectively, among the eutopic endometrium of women with adenomyosis, whereas in the control group, 10 and 20 samples were in the proliferative and secretory phases, respectively. Immunohistochemistry Formalin-fixed tissues were embedded in paraffin and cut into 5-mm sections. The tissue sections on glass slides coated with poly-l-lysine were deparaffinized in turpentine, hydrated in a graded series of ethanol, and placed in phosphate-buffered saline (PBS; ph ¼ 7.6). Antigen retrieval was performed by boiling for 15 minutes in 0.01 mol/l citrate buffer (ph ¼ 6.0). Sections were treated with 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity, rinsed with deionized water, and washed with PBS. Sections were first incubated with the primary murine monoclonal anti-human IL-10 antibody (Boster, Wuhan, Hubei, China) at 1:200 dilution at 37 C in a moist chamber for 30 minutes and then washed 3 times with PBS. Antibody detection was performed using a nonbiotin horseradish peroxidase detection system, PV9000 Polymer Detection System (ZSGB-BIO, Beijing, China) with a secondary antibody, conjugated Fabhorseradish peroxidase polymer, which can increase the detection sensitivity. After incubation with polyperoxidaseanti-mouse IgG for 30 minutes at room temperature, the tissue sections were washed with PBS. Diaminobenzidine tetrahydrochloride chromogen (ZSGB-BIO) was added for 3 minutes at room temperature. The chromogen causes brown granules to appear in IL-10-positive cells, wherever IL-10 exists. Sections were counterstained with hematoxylin, dehydrated, and then coverslipped with Permount. For negative control specimens, all the above-mentioned steps were followed, except for the addition of the primary antibody. The sections were viewed using a Leica DM4000B microscope (Leica, Wetzlar, Germany), and pictures were acquired using the IM50 image analysis system (Leica). Endometrial epithelial and stromal cells were separately counted. Expression and localization of IL-10 in the glands and stroma of the normal, eutopic, and ectopic endometrium were compared using the H score system. Each slide was scored by two different observers blinded to the tissue origin. The H score was calculated using the equation: H score ¼ P PiðI þ 1Þ; where I represents the intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi represents the percentage of stained cells for each intensity, varying from 0 to 100% (16, 17). The intraobserver variation was <5%, and the interobserver variation was <10%. The average score from each observer was calculated and expressed as a mean Wang et al. IL-10 in adenomyosis Vol. 91, No. 5, May 2009

3 FIGURE 1 Representative micrograph of IL-10 in normal endometrial samples in the proliferative phase (100 magnification). FIGURE 2 Representative micrograph of IL-10 in normal endometrial samples in the secretory phase (100 magnification). Statistical Analysis The Kolmogorov-Smirnoff test was used to determine whether the epithelial and stromal IL-10 H score values showed normal distribution. The Mann-Whitney rank sum test was used to analyze the differences between proliferative and secretory phase samples as well as differences in epithelial and stromal IL-10 H score values among the normal endometrium, eutopic endometrium, and adenomyosis samples; pairwise multiple comparisons were analyzed using nonparametric analysis of variance on ranks (Kruskal-Wallis test). The statistical software SPSS 13.0 for Windows (SPSS, Inc., Chicago) was used for all statistical analyses. Data are expressed as mean SEM. P<.05 was considered statistically significant. RESULTS IL-10 Expression in the Normal Endometrium In all the normal endometrial samples, epithelial and stromal IL-10 expression was observed throughout the menstrual cycle. The epithelial cells of the endometrium showed higher staining intensity than the stromal cells in both the proliferative- and secretory-phase samples. The epithelial and stromal staining was mostly cytoplasmic. The H score values of epithelial IL-10 immunostaining were significantly higher in the secretory phase than in the proliferative phase (P<.01; Table 1), whereas no difference was observed in stromal IL-10 immunoreactivity throughout the menstrual cycle (P¼0.62; Figs. 1 and 2). with adenomyosis in both the proliferative and secretory phases. Similar to the normal endometrial samples, the epithelial cells of the eutopic endometrium and adenomyosis foci showed higher staining intensity than the stromal cells. The epithelial and stromal staining was mostly cytoplasmic (Figs. 3, 4, and 5). In the eutopic and ectopic endometrium of women with adenomyosis, epithelial cells showed higher staining intensity than those of the normal controls (both P<.01). However, no significant difference was observed in the epithelial FIGURE 3 Representative micrograph of IL-10 in the eutopic proliferative phase endometrium of patients with adenomyosis (100 magnification). IL-10 Expression in Adenomyosis Foci and in Their Corresponding Eutopic Endometrium Epithelial and stromal IL-10 immunoreactivity was observed in all eutopic endometrium and adenomyosis foci of women Fertility and Sterility â 1683

4 FIGURE 4 Representative micrograph of IL-10 in the eutopic secretory phase endometrium of patients with adenomyosis (00 magnification). FIGURE 5 Representative micrograph of IL-10 in adenomyosis foci (100 magnification). IL-10 immunostaining H score values between the eutopic endometrium and adenomyosis foci (P ¼.08). In addition, no differences were observed in stromal IL-10 expression among adenomyosis foci, their eutopic endometrium, and normal controls (P¼.37). We observed a cyclic variation in eutopic epithelial IL-10 immunoreactivity throughout the menstrual cycle, with higher H score values in the secretory phase than in the proliferative phase (P<.01; Table 1). However, no significant difference was observed in eutopic stromal IL-10 immunoreactivity throughout the menstrual cycle in eutopic endometrium (P¼.22). DISCUSSION In the present study, we found that in the eutopic endometrium of women with adenomyosis, the epithelial staining intensity of IL-10 was significantly higher in the secretory phase than it was in the normal endometrium, whereas no statistical difference was observed in stromal IL-10 expression between the normal endometrial samples and eutopic endometrial samples of women with adenomyosis. The elevated expression of IL-10 in the eutopic and ectopic endometrium of patients with adenomyosis indicates that this molecule may play an important role in the pathogenesis and development of this disease by altering the immune system. No related literature is available with regard to the mechanism of IL-10 interference with local and total immune response in patients with adenomyosis. However, the role of IL-10 has been extensively studied in some cancers, and the results suggest that IL- 10 exerts a variety of biological effects, thereby creating an immunosuppressive microenvironment favorable for the development of tumors (10 13). Moreover, IL-10 is one of the cytokines implicated in the induction of HLA-G, which is suggested to be a part of the strategies used by tumor cells to escape from the immunosurveillance of the host (14). Additionally, in some chronic inflammatory diseases, IL-10 can inhibit the secretion of Th1 cell derived cytokines (15, 16) and reduce peritoneal T-cell activation (17). Although there is no direct evidence for the role of IL-10 in the regulation of immune response in patients with adenomyosis, based on our present results and previous findings on the mechanism through which IL-10 exerts its effect on the immune system in the tumor microenvironment, we assume that the elevated expression level of IL-10 may involve the immune tolerance of ectopic and eutopic endometrium. Furthermore, we conclude that before the infiltration of the myometrium, the eutopic endometrium may develop an ability to escape the immunosurveillance of the host by expressing IL-10. Based on this hypothesis, it is suggested that once the endometrium infiltrates the myometrium, it uses IL-10 to increase its chance of survival in the myometrium without being eliminated by the components of the immune system. However, the exact molecular mechanisms through which IL-10 exerts its effect in the local area and/or on the entire immune system remain unclear, and further studies are required to clarify these mechanisms. Therefore, the next steps would be to determine the presence of cytokines, immune cells, and other unexpected molecules in the eutopic and ectopic endometrium of patients with adenomyosis and to compare the levels of these molecules or cells between normal and abnormal endometrium. In the present study, we observed that IL-10 expression in the eutopic endometrium of patients with adenomyosis shows cyclic variation, with higher levels in the secretory phase. This cyclic variation indicates that sex steroid hormones may regulate the expression of IL-10, and this hypothesis is supported by the results of an in vitro study (18). This 1684 Wang et al. IL-10 in adenomyosis Vol. 91, No. 5, May 2009

5 previous study showed that estrogen increased the intracellular expression of IL-10 in mouse spleen dendritic cells at both mrna and protein levels. In agreement with the conclusion of this previous study, it was revealed that the pregnancy E 2 concentrations (and higher) enhance the production of IL- 10 in stimulated whole blood cells, whereas no effect of P on IL-10 production was observed (19). Both the eutopic and ectopic endometrium of women with endometriosis and adenomyosis were found to express aromatase and estrone sulfatase, which catalyzes the local production of estrogen (20). Furthermore, estrogen and P receptors have been detected in adenomyotic tissues (21). Based on the abovementioned evidence, we can conclude that adenomyosis is an estrogen-dependent disease and that IL-10 expression may be regulated by a cyclic variation in estrogen levels. In summary, we found that IL-10 expression was significantly increased in the eutopic and ectopic endometrium of patients with adenomyosis compared with that in subjects with normal endometrium. To our knowledge, our study is the first to investigate differences in the endometrial expression of IL-10 between healthy women and women with adenomyosis. In addition, we demonstrate that glandular cells are the main source of IL-10 in both eutopic and ectopic endometrium and that IL-10 expression exhibits cyclic variation. Based on all these results, we hypothesize that IL-10 may play an important role in the pathogenesis of adenomyosis, and further studies on this molecule will be helpful to clarify the inflammatory nature of adenomyosis. REFERENCES 1. Gianetto-Berrutti A, Feyles V. Endometriosis related to infertility. Minerva Ginecol 2003;55: Kano T, Furudono M, Nabetani H. The incidence of endometriosis and adenomyosis in patients with habitual abortion in relation to immunological abnormalities. Jpn J Fertil Steril 1997;42: Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75: Seli E, Berkkanoglu M, Arici A. Pathogenesis of endometriosis. Obstet Gynecol Clin N Am 2003;30: Bischoff F, Simpson JL. Genetics of endometriosis: heritability and candidate genes. Best Pract Res Clin Obstet Gynaecol 2004;18: Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 1989;170: Moreau P, Adrian-Cabestre F, Menier C, Guiard V, Gourand L, Dausset J, et al. IL-10 selectively induces HLA-G expression in human trophoblasts and monocytes. Int Immunol 1999;11: Vigano P, Somigliana E, Mangioni S, Vignali M, Vignali M, Di Blasio AM. Expression of interleukin-10 and its receptor is up-regulated in early pregnant versus cycling human endometrium. J Clin Endocrinol Metab 2002;87: Noyes RW, Hertig AT, Rock JR. Dating the endometrial biopsy. Fertil Steril 1950;1: Yue FY, Dummer R, Geertsen R, Hofbauer G, Laine E, Manolio S, et al. Interleukin-10 is a growth factor for human melanoma cells and downregulates HLA class-i, HLA class-ii and ICAM-1 molecules. Int J Cancer 1997;71: Geertsen R, Yue FY, Pavlovic J, Laine E, Dummer R. Interleukin-10 inhibits the immune stimulatory potential of melanoma cells retrovirally transduced with human B7.1 or B7.2. Adv Exp Med Biol 1998;451: Khong HT, Restifo NP. Natural selection of tumor variants in the generation of tumor escape phenotypes. Nat Immunol 2002;3: Seliger B, Cabrera T, Garrido F, Ferrone S. HLA class I antigen abnormalities and immune escape by malignant cells. Semin Cancer Biol 2002;12: Thellin O, Coumans B, Zorzi W, Igout A, Heinen E. Tolerance to the foeto-placental graft : ten ways to support a child for nine months. Curr Opin Immunol 2000;12: Li MC, He SH. IL-10 and its related cytokines for treatment of inflammatory bowel diseases. World J Gastroenterol 2004;10: Nu~nez C, Alecsandru D, Varade J, Polanco I, Maluenda C, Fernandez- Arquero M, et al. Interleukin-10 haplotypes in celiac disease in the Spanish population. BMC Med Genet 2006;7: Ho HN, Wu MY, Chao KH, Chen CD, Chen SU, Yang YS. Peritoneal interleukin-10 increases with decrease in activated CD4þ T lymphocytes in women with endometriosis. Hum Reprod 1997;12: Yang L, Hu Y, Hou Y. Effects of 17beta-estradiol on the maturation, nuclear factor kappa B p65 and functions of murine spleen CD11c-positive dendritic cells. Mol Immunol 2006;43: Matalka KZ. The effect of estradiol, but not progesterone, on the production of cytokines in stimulated whole blood, is concentration-dependent. Neuro Endocrinol Lett 2003;24: Yamamoto T, Noguchi T, Tamura T, Kitawaki J, Okada H. Evidence for estrogen synthesis in adenomyotic tissues. Am J Obstet Gynecol 1993;169: Ferenczy A. Pathophysiology of adenomyosis. Hum Reprod 1998;4: Fertility and Sterility â 1685