1 II Department of Obstetrics and Gynecology, University of Milano, 2 Department of Pathology, Istituti Clinici di Perfezionamento, 3 Roche Milano Ricerche and 4 Istituto Auxologico Italiano, Milano, Italy
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Abstract |
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Key words: ectopic implantation/endometriosis/interleukin-12/murine model
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Introduction |
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During menstruation, part of the menstrual effluent is regurgitated through the Fallopian tubes into the peritoneal cavity in 90% of women with patent tubes (Oral and Arici, 1997). This phenomenon is nowadays considered a necessary condition for ectopic endometrial implantation and endometriosis establishment in the pelvis. Other more unusual endometriosis locations are explained by different, more rare, ways of dissemination (lymphatic, haematogenic, iatrogenic). Consistent with this pathogenic model, both the ability of the endometrium to implant in ectopic sites and the failure of the immune surveillance to counteract this phenomenon have been claimed as potentially responsible for the expression of the disease (Ho et al., 1997
; Sharpe-Timms, 1997
). Endometrial characteristics critical to implantation include the microscopic and molecular structure of refluxed endometrial fragments (Starzinski-Powitz et al., 1998
), the amount of regurgitated endometrial cells (Cramer et al., 1986
), their ability to proliferate (Wingfield et al., 1995
), to secrete angiogenetic factors (Healy et al., 1998
) and to discontinue peritoneal surface by producing metalloproteinases (Sillem et al., 1998
). On the other hand, the inability of ectopic endometrial antigens to stimulate effectively an immune response may result from defects in antigen recognition, absence of costimulatory molecules and effect of locally released cytokines (Somigliana et al., 1999
). Treatment of endometriosis should be directed not only towards complete ablation of active endometriotic tissue but also towards the prevention of recurrences without interfering with fertility potential. Indeed, since one of the chief complaints associated with the disease is infertility (Evers et al., 1995
), the strategy to prevent endometriosis should not be based on inhibition of ovulation and induction of amenorrhoea.
Experimental evidence indicates that interleukin (IL)-12 may be involved in the development of endometriosis (Mazzeo et al., 1998). IL-12 is a heterodimeric cytokine composed of co-valently linked 40 kDa (p40) and 35 kDa (p35) subunits (Wolf et al., 1991
, 1992
; D'Andrea et al., 1992
). IL-12 acts on T and natural killer (NK) cells inducing cytokine production, primarily interferon (INF)-
, enhancing NK cell cytotoxic activity and favouring the generation of T-helper (Th)1 response (Kobayashi et al., 1989
; Wysoka et al., 1995
; Kelly et al., 1997
). Both subunits are required for IL-12 biological activity while the free p40 chain alone acts as a natural antagonist of IL-12 (Mattner et al., 1993
; Gillessen et al., 1995
; Ling et al., 1995
). Interestingly, the free p40/IL-12 ratio has been shown to be significantly increased in peritoneal fluid of patients with endometriosis and to correlate with the severity of the disease (Mazzeo et al., 1998
). Heterodimeric IL-12 promotes NK cell-mediated response toward endometrial antigens while the free p40 subunit of the molecule is able to abrogate the IL-12-induced cytotoxicity. Therefore, while the excess of the free p40 subunit in peritoneal fluid of patients with endometriosis may be related to the immune defect associated with the disease, heterodimeric IL-12 is able to correct the p40-related alteration in vitro (Mazzeo et al., 1998
).
In this study, we created a model of endometriosis in immunocompetent mice to evaluate both the critical phase in endometriosis development which is the ability of endometrium to implant in ectopic sites and the potential application of IL-12 in preventing this ectopic implantation. This procedure modelled a massive retrograde menses and did not consist in a mere surgical transplantation of endometrium which has been criticized as not conforming with the pathogenic mechanism for the disease (Ramey et al., 1996). Endometriosis in mice derived by this technique has the major macroscopic and histopathologic features (endometriosis-like lesions and cysts) found in humans. Administration of IL-12 to these mice induces a strong prevention of endometriosis establishment underscoring the role of IL-12 in stimulating an immune response against ectopic endometrium.
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Materials and methods |
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Induction of endometriosis
Endometriotic lesions were induced at day 0 by inoculating finely chopped endometrial fragments corresponding to about 50% (about 15 mg) of the endometrial tissue from both uterine horns of syngenic mice as described below through a small vertical midline laparotomic incision into the peritoneal space, just below the umbilicus. Mice challenged with endometrium have been subjected to ovariectomy at day 7 and injected with oestrogens depot in castor oil (Progynon; Schering, Milan Italy; 100 µg/kg i.m.) at days 7, 0, +7, +14. Ovarectomy plus oestrogen supplementation was decided in order to abrogate differences related to the stage of the oestrous cycle.
Uterine samples to be inoculated were obtained from syngenic mice used as donor mice. Similarly to challenged mice, donor mice were ovariectomized and oestrogen-treated at day 7. At day 0, one donor mouse was killed for every two mice to be challenged with endometrium. Both uterine horns were removed using an aseptic technique and subsequently placed in a sterile Petri dish containing sterile normal saline. Then, uterine samples were gently peeled in order to detach the uterine muscle from the endometrium. Following this procedure, they were transferred to a glass slide and finely chopped using two blunt scalpels. Endometrial fragments were suspended in 0.8 ml of sterile normal saline per challenged mice and inoculated into the peritoneal cavity. At histological examination performed in preliminary experiments, no muscle layers were detected in fragments to be inoculated. At day +21 after endometrium challenge, mice were sacrificed, the abdomen was inspected and endometriotic lesions were carefully excized from the surrounding tissue in order to assess their weight and surface area. Weight assessment was performed 24 h after extraction of lesions in order to abrogate differences due to water content. The surface areas were measured with a calliper and calculated. When the lesion had a cyst appearance, measures were taken after aspiration of the cyst. The operator (E.S.) was blinded for the different conditions. A schematic diagram of the overall procedure is shown in Figure 1. Each group of mice was initially composed of 1012 animals.
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Induction of ectopic implantation of small bowel mucosa
Two different groups of BALB/c mice were challenged with similar amounts of small bowel mucosa (n = 12) and endometrium (n = 9) in order to evaluate the ability to implant in ectopic locations of these two tissues.
Endometriosis was established as described above. Ectopic implants of small bowel mucosa were similarly induced. At day 0, finely chopped fragments corresponding to about 20% of the small bowel mucosa were inoculated into the peritoneal space, just below the umbilicus, through a small vertical midline laparotomic incision. Mice challenged with small bowel mucosa have been subjected to ovariectomy at day 7 and injected with oestrogens (100 µg/kg i.m.) at days 7, 0, +7, +14 as endometriotic mice. Samples of small bowel mucosa to be inoculated were obtained from syngenic mice used as donor mice. Small bowel was removed using an aseptic technique and subsequently placed in a sterile Petri dish containing sterile normal saline. Then, samples of intestines were gently peeled to detach the muscle from the mucosa and washed several times in order to reduce micro-organisms contamination. Following this procedure, they were transferred to a glass slide and finely chopped using two blunt scalpels. Fragments of small bowel mucosa were suspended in 0.8 ml of sterile normal saline per challenged mice and inoculated into the peritoneal cavity. At day +21 after small bowel mucosa challenge, mice were killed, the abdomen was inspected and ectopic lesions were carefully excized from the surrounding tissue in order to assess their weight and surface area.
Recombinant murine IL-12 administration
Murine recombinant IL-12 was provided by Dr M.Gately (Hoffman-La Roche, Nutley). For administration to mice, IL-12 was diluted in PBS containing 100 µg/ml mouse serum albumin (Sigma-Aldrich, Milano, Italy). In both C57BL/6 and BALB/c mice, IL-12 was administrated i.p. by daily injections of 0.15 µg/0.4 ml for 5 days, from 2 through +2. Control mice received only the vehicle following the same procedure.
Histological examination
The histological diagnosis of endometriosis was based on the morphological identification of endometrial glandular tissue and stroma. Specimens corresponding to gross lesions and to apparently disease-free peritoneum were fixed in formalin, dehydrated through graded ethanol, cleared in xylene and embedded in paraffin. Tissue sections (5 µm) were stained using a routine haematoxylin and eosin-phloxine staining procedure. All slides were evaluated by an experienced pathologist (S.C.) who was blind to the study.
Statistical analysis
Data are expressed as mean ± SEM. The unpaired Student's t-test and the Fisher's exact test were used as appropriate to test for statistical significance of differences between groups. Probability P < 0.05 was considered as statistically significant.
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Results |
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Discussion |
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Progress in endometriosis research has been limited so far by the lack of a valuable and promptly available model of the disease (Sharpe-Timms, 1997). Indeed, endometriosis occurs spontaneously only in monkeys, which happen to be the only non-human animal that has cyclic menstrual periods and retrograde menses (Sharpe-Timms, 1997
). In non-primates, models of the disease developed in the past consisted essentially in the surgical transplantation of a uterine square in the peritoneal cavity which is by far the most common site of the disease (Vernon, 1990
). However, while this type of model is helpful in investigating the effects of medications on ectopic endometrial growth, its results are inadequate to understand the aetiologic and predisposing factors involved in endometriosis development and to establish novel therapeutical interventions able to prevent the disease. Few experiments consisted in intraperitoneal injection of uterine fragments and, in most cases, these studies were restricted to nude mice who were implanted with human endometrium (Zamah et al., 1984
; Bruner et al., 1997
). We are currently unaware of any previous studies that have successfully documented the implantation and growth of endometrial tissue in immunocompetent mice following intraperitoneal challenge of syngenic endometrial fragments. Previously, Ramey et al. failed to establish endometriosis lesions in mice by injecting endometrial scrapings into the peritoneum (Ramey et al., 1996
). The reason for the discrepancy with our results might be in the amount of the challenged endometrium, the procedure of administration and the oestrogen supplementation.
The fact that 100% of the animals tested showed evidence of peritoneal endometriosis supports the concept that endometrium displays a marked ability to implant in ectopic sites. This is further confirmed by experiments in which small bowel mucosa instead of endometrium was used as intraperitoneal insult. Indeed, intestines are very efficiently eliminated when inoculated into the peritoneum.
Endometrial characteristics critical to ectopic implantation could not be assessed in our study. Furthermore, our results are not able to clarify definitely whether the major mechanism in endometriosis establishment is the ability of the endometrium to implant in ectopic sites or the failure of the immune surveillance to counteract this phenomenon. However, our results may be of clinical relevance. Indeed, medical therapeutical interventions used so far have been strongly limited by the drugs' inability to differentiate adequately between eutopic and ectopic endometrium. Hence, present chemicals for the disease (oral contraceptives, progestins, gonadotrophin-reducing hormone (GnRH) analogues, danazol) do alter normal reproductive cyclicity and interrupt fertility (Kettel and Hummel, 1997). In our study, we investigated the possibility of using a rationale immunologically-based therapy for endometriosis that does not interfere with endometrial physiology.
The interest in IL-12 was based on the observation that an unbalanced secretion of IL-12 and its free p40 subunit is present in peritoneal fluid of patients affected by endometriosis (Mazzeo et al., 1998). Administered purified recombinant murine IL-12 is able to induce a strong prevention of the disease in a mouse model. Therapeutical intervention by intraperitoneal administration of IL-12 was initiated 2 days before endometriosis induction and lasted for 5 days in order to assess its ability to limit ectopic implantation of endometrium. The low dose and the short-term therapy as opposed to the unnatural massive retrograde menstruation realized in our model were chosen considering the benign nature of the disease. The administrated dose was at least 67-fold lower than that proved for anti-tumour efficacy in B16F10 melanoma tumour-bearing mice or used to induce a dramatic decrease in diabetic incidence in NOD (non-obese diabetic) mice (Brunda et al., 1996
; O'Hara et al., 1996
). Indeed, a side effect-free dose which is efficacious in extreme disease conditions represents an inevitable requisite for testing new therapeutical strategies for this disease.
Based on IL-12-mediated well-established in-vivo actions, several potential hypotheses for this effect can be suggested. A direct growth inhibitory effect on endometrial cells seems unlikely since endometrial cells do not express receptors for IL-12 (data not shown). Another potential explanation for these results is that IL-12 may enhance the growth and augment the cytolytic activity of both NK/LAK (lymphokine-activated killer) and T cells. Thus, the IL-12-mediated prevention of endometrial implantation might be mediated through stimulation of one or both these lymphocyte populations. In the recent past, the role of the immune system in the aethiopathogenesis of endometriosis has been largely debated (Braun and Dmonski, 1998). In particular, some studies focused on the ability of NK cells to recognize and eliminate ectopic endometrial cells (Somigliana et al., 1999
). Aoki et al. heterotransplanted human endometrium in nude and SCID (severe combined immunodeficiency) mice (Aoki et al., 1994
). Acceptance rate of human endometrium was higher in SCID (100%) than in nude (40%) mice. Moreover, endometrial transplant size correlated negatively with NK cell function and tended to be greater in nude mice treated with a monoclonal antibody to asialoGM1. Since IL-12 is known to promote enhancement of both NK and cytotoxic T cell activity (Kobayashi et al., 1989
; Wolf et al., 1991
; D'Andrea et al., 1992
), our results are in line with these findings. However, no general consensus exists on the specific lymphocyte subtype implicated in this recognition. The contribution of these different mechanisms in mediating the IL-12-dependent endometrial rejection in vivo needs to be addressed in future experiments.
The activity of a number of cytokines demonstrated in animal models has not always translated to successful use in human therapy. The data presented here clearly establish IL-12 as a cytokine able to prevent endometriosis establishment. Furthermore, this finding supports the idea that such effects on the immune system could become a novel management approach to controlling the disease. Future clinical trials will determine if the effect demonstrated in animals can be translated into efficacy against human endometriosis.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 1, 1999; accepted on September 13, 1999.