Induction of endometriosis in the mouse inhibits spleen leukocyte function

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1 Acta Obstet Gynecol Scand 2001; 80: Copyright C Acta Obstet Gynecol Scand 2001 Printed in Denmark All rights reserved Acta Obstetricia et Gynecologica Scandinavica ISSN ORIGINAL ARTICLE Induction of endometriosis in the mouse inhibits spleen leukocyte function EDGARDO SOMIGLIANA 1, PAOLA VIGANÒ 1,2, BEATRICE ZINGRILLO 1, STEFANIA RANIERI 1, PAOLA FILARDO 1, MASSIMO CANDIANI 1 AND MARIO VIGNALI 2 From the 1 II Department of Obstetrics and Gynecology, Clinica L. Mangiagalli, University of Milano, and the 2 IRCCS Centro Auxologico Italiano, Milano, Italy Acta Obstet Gynecol Scand 2001; 80: C Acta Obstet Gynecol Scand 2001 Background. It is still unclear whether the immunologic perturbation observed in women with endometriosis represents an intrinsic defect of the immune system or it is consequent to the presence of endometrium in ectopic sites. The present study was aimed to evaluate the immunoregulatory properties of ectopic endometrial implants in a model of experimental endometriosis in mice. Methods. Endometriosis was induced in nω10 mice by inoculating endometrial fragments obtained from syngenic donor mice into the peritoneal space. Twelve mice were similarly treated but were not inoculated with endometrium and were used as control mice. At an explorative laparotomy performed in all the animals after three weeks, the extent of peritoneal lesions, when present, was evaluated by weight assessment and surface area measurement. In both mice that were inoculated with endometrium and control animals, spleens were removed. The effect of endometriosis induction on concanavalin A-induced spleenocyte proliferation was investigated. Results. A significant inhibition of spleenocyte growth was demonstrated in mice in which endometriosis was induced ( cpm) compared to control animals ( cpm; p 0.05). Moreover, a significant inverse correlation was found between spleenocyte proliferation and weight and surface area of the peritoneal endometriotic lesions (rω0.76; p 0.02 and rω0.75; p 0.02, respectively). Conclusion. These findings suggest that the presence of ectopic endometrial implants within the peritoneal cavity leads to substantial changes of the immune response in vivo. These changes may have significant implications for the understanding of the etiopathogenesis of endometriosis. Key words: endometriosis; mouse model; spleen Submitted 16 August, 2000 Accepted 17 October, 2000 The etiology and pathogenesis of endometriosis, which is the ectopic occurrence of endometrial tissue, has been enigmatic from its very first description until today. Several decades of investigation have documented specific immunological changes in women with endometriosis. A Abbreviations: NK: natural killer; Th: T helper; IL: interleukin; CPM: count per minute; mg: milligram; mm: millimeter; 1mCi: 1 microcurie; ml: milliliter; ml: microliter. dampening of T lymphocytes and natural killer (NK) cell function in endometriosis occurs coincident with an increase in peritoneal macrophage number and activation status. This is associated with a very particular balance between T helper (Th)1-type and Th2-type cytokines present in the peritoneal cavity of women with the disease. Finally, eutopic endometrial expression of specific mediators of the immune response is somehow aberrant in women with endometriosis (1, 2). Therefore, according to a recent model, both the auto-

2 Spleen leukocytes and endometriosis in mice 201 transplantable nature of the endometrial cells and the failure of the immune surveillance to counteract this phenomenon are thought to induce the expression of the disease (1). Consistent with these observations, it has been recently demonstrated that in vivo administration of substances able to stimulate the immune system can reduce experimentally induced endometriosis. Specifically, interleukin (IL)-12 and interferon (IFN)-a-2b are able to limit implantation and progression of the disease in two different murine models (3, 4). Notwithstanding the consistent burden of literature in favor of an immunologic perturbation in patients with endometriosis, it is still controversial whether the immune response is intrinsically abnormal in these women, or whether these changes are a secondary effect due to the presence of ectopic endometrium in the peritoneal cavity. Indeed, it has been hypothesized that, in endometriosis women, the alterations of the immune parameters that contribute to endometrial cell implantation in ectopic sites may be consequent to the largely documented endometrial ability to modulate the immune system. To address this issue, we utilized a model for the experimental induction of endometriosis in mice and investigated the immunomodulatory properties of endometriotic implants. Specifically, we compared the proliferative response of spleen leukocytes in animals in which endometriosis was or was not induced and correlated the observed findings to the extent of the disease. Materials and methods Female, 6- to 8-week old, C57BL/6 mice (nω22) were purchased from Charles River (Calco, Como, Italy). Mice were fed on mouse diet and water ad libitum and kept on a light/dark cycle of 16/8 hours under controlled conditions. Prior to any invasive procedure, the mice were anesthetized by 1.6 mg sodium thiopental in 0.65 ml sterile NaCl physiological solution by intraperitoneal injection. Laparotomies were performed by a standardized midline incision and microsurgical technique under clean but not aseptic conditions. A closed carbon dioxide chamber was used for euthanasia. Endometriotic lesions were induced in nω10 mice using a procedure which has been described in detail elsewhere (4). Briefly, at day 0, chopped endometrial fragments corresponding to about 75% of the endometrial tissue from both uterine horns were inoculated into the peritoneal space through a small vertical midline laparotomic incision. Mice challenged with endometrium have been subjected to bilateral adnexiectomy at day ª7 and injected with estrogens depot at days ª7, 0, π7, π14. Adnexiectomy plus estrogen supplementation was decided in order to abrogate differences related to the stage of the estrous cycle. Moreover, due to estrogen stimulation, detection of endometriotic lesions was facilitated. Uterine samples to be inoculated were obtained from syngenic mice used as donor mice. Similarly to challenged mice, donor mice were ovariectomyzed and estrogen-treated at day ª7. At day 0, donor mice were sacrificed; both uterine horns were removed and gently peeled in order to detach the uterine muscle from the endometrium. They were, then, finely chopped, suspended in sterile normal saline and inoculated into challenged mice. At day π21 after endometrium challenge, mice were sacrificed, the abdomen was finally inspected and endometriotic lesions were carefully excised from the surrounding tissue in order to assess their weight and surface area. Lesions consisted of multicystic nodules 1 to 8 mm in diameter bulging from and loosely attached to the serosal coat. Weight assessment was performed 24 hours after lesions extraction in order to abrogate differences due to water content. The surface areas were measured with a caliper and calculated. When the lesion had a cyst appearance, measures were taken after aspiration of the cyst. Weight assessment was performed 24 hours after lesions extraction in order to abrogate differences due to water content. The operator (E.S.) was blinded for the different conditions. The endometriotic character of the lesions were histologically confirmed in preliminary experiments based on the identification of endometrial glandular tissue and stroma. Control animals (nω12) were subjected to bilateral adnexiectomy at day ª7, injected with estrogens depot at days ª7, 0, π7, π14, laparotomically incised and inoculated with sterile normal saline at day 0, sacrificed and inspected at day π21 but were not inoculated with endometrial fragments. From both control animals and mice challenged with endometrium, spleens were removed on day π21 and used that day in the proliferation assay. Freshly isolated whole spleen leukocytes (erythrocytes lysed) ( ) were resuspended in RPMI 1640 medium (Euroclone, UK) supplemented with 10% Fetal Calf Serum (Euroclone, UK) and antibiotics. They were then plated in 96-well U-bottom plates in a total volume of 200 ml per well and incubated at 37æC per 48 hours in presence of 1 mg/ml of Concanavalin-A (Con-A) (Sigma, Milano Italy). During the last 6 hours of the culture period, cells were pulsed with 1 mci methyl- [ 3 H]thymidine (Amersham Life Science, Milano, Italy). The cells were then harvested onto glass fiber filters and the radioactivity on the dried filters was measured by liquid scintillation counting. Re-

202 E. Somigliana et al. sults represent the mean of triplicate wells and are expressed as counts per minute (CPM) of methyl- [ 3 H]thymidine incorporation s.e. mean. Difference between groups was compared, as appropriate, by unpaired Student s t-test or regression analysis.

3 202 E. Somigliana et al. sults represent the mean of triplicate wells and are expressed as counts per minute (CPM) of methyl- [ 3 H]thymidine incorporation s.e. mean. Difference between groups was compared, as appropriate, by unpaired Student s t-test or regression analysis. Probability 0.05 was considered as statistically significant. Results A hundred percent of the animals challenged with syngenic endometrium showed evidence of peritoneal endometriosis at three weeks. As previously reported, lesions consisted of pink to tan multicystic nodules 1 to 8 mm in diameter bulging from and loosely attached to the serosal coat. Interestingly, macroscopic aspect of the lesions could sometimes be identical to classical human black lesions (Fig. 1). Microscopic examinations performed in preliminary experiments confirmed that all the gross lesions analyzed were endometriotic in character. Total weight of the lesions as evaluated after three weeks from inoculation of endometrium was mg while total surface area was mm 2. As shown in Fig. 2 (upper panel), the number of spleen cells recovered was similar in animals in which endometriosis was induced ( ) and control mice ( ). In contrast, the proliferation of spleenocytes induced by Con-A was significantly inhibited in mice challenged with syngenic endometrium Fig. 2. Effect of endometriosis induction on spleen leukocyte number (upper panel) and proliferation induced by Con-A (lower panel). Results represent the number of cells (upper panel) and counts per minute (CPM) of methyl-[ 3 H]thymidine incorporation (lower panel) SEM (*p 0.05). Fig. 1. Gross findings of ectopic endometrial implants. The white arrow points to a single lesion attached to the anterior abdominal wall. ( cpm) compared to control animals ( cpm; p 0.05) (Fig. 1 lower panel). In order to better clarify whether the actual presence of endometrial implants in the peritoneal cavity could interfere with spleen cell function, we correlated the extent of the lesions with spleen leukocyte proliferative response. A significant inverse correlation was found when spleenocyte proliferation was correlated with surface area (rω0.75; p 0.02) (Fig. 2 upper panel) and weight (rω0.76; p 0.02) (Fig. 3 lower panel) of the peritoneal endometriotic lesions.

4 Spleen leukocytes and endometriosis in mice 203 Fig. 3. Correlation between proliferation of spleen leukocytes induced by Con-A and the extent of endometriosis indicated as total surface area (upper panel) and total weight (lower panel) of peritoneal lesions. Discussion An immunologic defect in removing the endometrial cells, once they have reached the peritoneal cavity by retrograde menstruation, might be an independent factor or might depend on specific characteristics of effluent to eliminate. These characteristics are likely to include the amount of retrograde menstruation and the ectopic expression of regulatory molecules and factors able to circumvent the immune response (1). To investigate this issue, the present report evaluated the immunoregulatory properties of peritoneal lesions induced experimentally in a mouse model. The results of this study demonstrate that, in presence of peritoneal endometriotic lesions, mitogen-stimulated spleen lymphocyte proliferation is suppressed. Moreover, the entity of this suppression significantly correlates with the size of the lesions. The concept that ectopic endometrial tissue may be immunosuppressive is not particularly surprising, given that its site of origin, the uterus, must be immunosuppressive or at least immunomodulatory to allow pregnancy to occur (5). Several studies demonstrated that both cycling endometrium and decidua secrete molecules able to interfere with lymphocyte proliferation and NK cell activity as a prerequisite for pregnancy maintenance (6, 7). Some of these factors have been already characterized. A specific unresponsiveness of lymphocytes at the maternal-fetal interface seems to be related to the anergy induced by Th2-like cytokines such as IL-10 (5). Placental protein 14 has been shown to inhibit various immunological reactions in vitro and its immunosuppressive actions may be instrumental during implantation and early pregnancy (8, 9). Other molecules that merit special attention for their ability to play immunoregulatory roles are transforming growth factor b2 and progesteroneinduced blocking factor (5). These cytokines and factors are likely to be secreted also by endometrial cells when they implant outside the uterus. In agreement with these findings, both ectopic and eutopic endometrial cells were shown to affect mitogenic activity of autologous lymphocytes (10). Furthermore, medium conditioned by endometrial and endometriotic tissue was demonstrated to have a marked inhibitory effect on NK cell activity (11, 12). Finally, it cannot be excluded that other cell populations present in the peritoneal cavity might also be involved; macrophages, for example, activated by the presence of endometriotic implants could secondarily secrete inhibitory factors (13). In this context, it has to be noted that our study is not able to clarify whether the immune perturbation would be the consequence of the mere presence of syngenic tissue in ectopic site or whether this effect should be specifically attributed to endometrium. On the other hand, differently from other tissues, endometrium displays a unique ability to implant in ectopic sites. Indeed, we failed to provide evidence of ectopic implants in experiments in which mice were challenged with small bowel mucosa instead of endometrium (4). Therefore, although the injection of other tissues in control mice would have been helpful in clarifying whether this effect was endometrium-specific or more generalized, this control condition was essentially unrealizable. In any case, the results of the present study represent an in vivo confir-

5 204 E. Somigliana et al. mation of the hypothesized role of the ectopic endometrium to directly or indirectly influence specific immune functions. It is noteworthy that our model of endometriosis induction implies a massive inoculation of endometrium in the peritoneal cavity. The quantity of endometrial fragments inoculated per each mouse corresponded to about 75% of the endometrial tissue from both uterine horns. This may explain the observed strong influence on the immune parameters. In humans, it is unlikely that this influence might be so relevant and biologically important systemically rather than only locally. Indeed, patients with endometriosis do not show an increased incidence of malignancies and immune-dependent tumors as should be expected in patients with a compromised immune function (14). Moreover, Oosterlynck et al. demonstrated that the defect of peritoneal NK cell activity in endometriosis was 2.4 times higher than the defect found in peripheral blood (13). These findings could also be considered indirect evidences supporting, once again, the secondary nature of the endometriosisdependent immunosuppression. On the other hand, it has been shown that the decreased NK cell activity observed in women with endometriosis persisted after surgical excision of the endometriotic lesions thus suggesting that NK cells could be inhibited before the development of the disease (15). Some explanations may be proposed to justify this discrepancy with our findings. Endometriotic lesions cannot always be fully excised during laparoscopy and microscopic implants have been demonstrated on macroscopically disease-free peritoneum of patients with endometriosis (16). Finally, it might be speculated that endometrial cells might affect the peritoneal immune system also in the absence of endometriotic lesions. Indeed, it should be underlined that the suppression of peritoneal NK cells is particularly evident during the first days of the cycle which exactly correspond to the arrival in the peritoneum of endometrial cells refluxed with retrograde menstruation (13). In conclusion, the results of this study support the idea that specific immunological changes associated with endometriosis are actually a secondary effect of the presence of endometrium in the peritoneal cavity. On the other hand, the fact that in vivo therapeutic manipulation with substances that stimulate the immune response can reduce the extent of the lesions or prevent endometriosis development strongly indicates that a causative relationship exists between specific immunological events and the etiology of the disease (3, 4). Thus, taken together, these observations suggest that the immunological defect detected in endometriosis women may depend on the endometrial ability to directly or indirectly modulate the immune function and that this property could be critical for the development of the disease. Acknowledgment This work was supported in part by a grant from Centro di Studi Cronobiologici dell Endometriosi, University of Milan, Milan, Italy. References 1. Braun DP, Dmowski WP. Endometriosis: abnormal endometrium and dysfunctional immune response. Curr Opin Obstet Gynecol 1998; 10: Somigliana E, Viganò P, Vignali M. Endometriosis and unexplained recurrent spontaneous abortion: pathological states resulting from aberrant modulation of natural killer cell function? Hum Reprod Update 1999; 5: Ingelmo JMR, Quereda F, Acien P. Intraperitoneal and subcutaneous treatment of experimental endometriosis with recombinant human interferon-a-2b in a murine model. Fertil Steril 1999; 71: Somigliana E, Viganò P, Rossi G, Carinelli S, Vignali M, Panina-Bordignon P. Endometrial ability to implant in ectopic sites can be prevented by Interleukin-12 in a murine model of endometriosis. Hum Reprod 1999; 14: Raghupathy R. Th1-type immunity is incompatible with successful pregnancy. Immunol Today 1997; 18: Vassiliadou N, Searle RF, Bulmer JN. Immunoregulatory activity of decidua in spontaneous early pregnancy loss. Hum Reprod 1999; 14: Wang HS, Kanzaki H, Yoshida M, Tokushige M, Mori T. Suppression of lymphocyte reactivity in vitro by supernatants of explants of human endometrium. Am J Obstet Gynecol 1987; 157: Dalton CF, Laird SM, Estdale SE, Saravelos HG, Li TC. Endometrial protein PP14 and CA-125 in recurrent miscarriage patient; correlation with pregnancy outcome. Hum Reprod 1998; 13: Okamoto N, Uchida A, Takakura K, Kariya Y, Kanzaki H, Riittinen L et al. Suppression by human placental protein PP14 of natural killer cell activity. Am J Reprod Immunol 1991; 26: Helvacioglu A, Aksel S, Peterson RDA. Endometriosis and autologous lymphocyte activation by endometrial cells: are lymphocyte or endometrial cell defect responsible? J Reprod Med 1997; 42: Hirata J, Kikuchi Y, Imaizumi E, Tode T, Nagata I. Endometriotic tissues produce immunosuppressive factors. Gynecol Obstet Invest 1994; 37: Somigliana E, Viganò P, Gaffuri B, Candiani M, Busacca M, Di Blasio AM et al. Modulation of NK cell lytic function by endometrial secretory factors: potential role in endometriosis. Am J Reprod Immunol 1996; 36: Oosterlynck DJ, Meuleman C, Waer M, Vanderputte M, Koninckx PR. The natural killer cell activity of peritoneal fluid is decreased in women with endometriosis. Fertil Steril 1992; 58: Brinton LA, Gridley G, Persson I, Baron J, Bergqvist A. Cancer risk after a hospital discharge diagnosis of endometriosis. Am J Obstet Gynecol 1997; 176: Oosterlynck DJ, Waer M, Koninckx PR, Vanderputte M. CO2-laser excision of endometriosis does not improve the decreased natural killer activity. Acta Obstet Gynecol Scand 1991; 73: Murphy AA, Green WR, Bobbie D, De la Cruz ZC, Rock

6 Spleen leukocytes and endometriosis in mice 205 JA. Unsuspected endometriosis documented by scanning electron microscopy in visually normal peritoneum. Fertil Steril 1986; 46: Address for correspondence: Paola Viganò, D.Sc. II Department of Obstetrics and Gynecology Clinica L. Mangiagalli Via Commenda Milano Italy

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