Endometrial ability to implant in ectopic sites can be prevented by interleukin-12 in a murine model of endometriosis

E. Somigliana1, P. Viganò1,4,5, G. Rossi1, S. Carinelli2, M. Vignali1 and P. Panina-Bordignon3

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immune dysfunctions in endometriosis are widely documented but the effectiveness of immunotherapies for the management of the disease is still debated. Progress in this field has also been limited by the lack of an appropriate animal model of the disease. In this study, we created a model of endometriosis in immunocompetent mice to verify the ability of endometrium to implant in ectopic sites and to investigate the potential application of the cytokine interleukin (IL)-12 in preventing this ectopic implantation. Endometriotic lesions were induced in both C57BL/6 and BALB/c mice by inoculating syngenic endometrial fragments through a small laparotomic incision into the peritoneal space. All the animals challenged with syngenic endometrium showed evidence of peritoneal endometriosis at 3 weeks. Histologically, endometriotic lesions consisted of cystic endometrial glands surrounded by a stroma. Intraperitoneal injection of IL-12 was able to reduce total weight and total surface area of endometriotic lesions respectively of 77 and 61% in C57BL/6 and of 42 and 28% in BALB/c mice. These results demonstrate that IL-12 is able to induce a significant prevention of ectopic endometrial implantation in an in-vivo model of endometriosis. These findings support the possibility of using the immune system to generate novel therapies for the management of the disease.

Key words: ectopic implantation/endometriosis/interleukin-12/murine model


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General estimates indicate that at least 25% of all women in their thirties and forties may be suffering from endometriosis and that the disease involves 6.9% of all diagnoses for hospitalizations for genito-urinary problems (Monteforte et al., 1995Go; Eskenazi and Warner, 1997Go). No drug has been demonstrated to be effective in eradicating the disease or in preventing it without inadmissible side effects (Adamson and Nelson, 1997Go; Kettel and Hummel, 1997Go). Surgery is the first-line treatment to eradicate endometriotic lesions; nevertheless, patients who undergo surgical procedures are threatened by a percentage of recurrence up to 47% (Evers et al., 1995Go).

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, 1997Go). 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., 1997Go; Sharpe-Timms, 1997Go). Endometrial characteristics critical to implantation include the microscopic and molecular structure of refluxed endometrial fragments (Starzinski-Powitz et al., 1998Go), the amount of regurgitated endometrial cells (Cramer et al., 1986Go), their ability to proliferate (Wingfield et al., 1995Go), to secrete angiogenetic factors (Healy et al., 1998Go) and to discontinue peritoneal surface by producing metalloproteinases (Sillem et al., 1998Go). 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., 1999Go). 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., 1995Go), 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., 1998Go). IL-12 is a heterodimeric cytokine composed of co-valently linked 40 kDa (p40) and 35 kDa (p35) subunits (Wolf et al., 1991Go, 1992Go; D'Andrea et al., 1992Go). IL-12 acts on T and natural killer (NK) cells inducing cytokine production, primarily interferon (INF)-{gamma}, enhancing NK cell cytotoxic activity and favouring the generation of T-helper (Th)1 response (Kobayashi et al., 1989Go; Wysoka et al., 1995Go; Kelly et al., 1997Go). 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., 1993Go; Gillessen et al., 1995Go; Ling et al., 1995Go). 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., 1998Go). 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., 1998Go).

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., 1996Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Female, 6 to 8 week old, C57BL/6 and BALB/c mice 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 anaesthetized by 1.6 mg sodium thiopental in 0.65 ml sterile physiological saline 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.

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 1Go. Each group of mice was initially composed of 10–12 animals.



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Figure 1. Schematic diagram of the mouse model of endometriosis. Endometriotic lesions were induced at day 0. Mice challenged with endometrium were subjected to ovariectomy at day –7 and injected with oestrogens at days –7, 0, +7, +14. Uterine samples to be inoculated were obtained from syngenic mice that were ovariectomized and oestrogen-treated at day –7. At day +21 after endometrium challenge, mice were sacrificed and endometriotic lesions were evaluated.

 
In some experiments performed using BALB/c mice (n = 5) for each group, ectopic endometrial implantation was evaluated as a function of the amount of endometrium inoculated and the time after inoculation. Firstly, endometriosis was induced in mice using two different amounts of challenged endometrium. Lesions were evaluated after 3 weeks from tissue challenge. Secondly, endometriosis was induced using ~15 mg of endometrium and lesions were evaluated at 1, 3 and 5 weeks after tissue challenge.

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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of endometriosis
A hundred per cent of the animals (n = 64) challenged with syngenic endometrium showed evidence of peritoneal endometriosis at time of abdominal inspection. No differences in gross and microscopic appearance or in the number of endometriotic lesions were observed between C57BL/6 (n = 15) and BALB/c mice (n = 49). On gross examination, lesions consisted of pink to tan multicystic nodules 1–8 mm in diameter bulging from and loosely attached to the serosal coat (Figure 2Go). Evidence of neovascularization was observed on their surface. Similarly to humans (Jenkins et al., 1986Go), lesions were influenced by the effect of gravity as the most frequent location was the anterior abdominal wall. Other locations in decreasing order of frequency were the omentum, the posterior abdominal wall, the caudal abdominal fat and the mesentery. Microscopic examination confirmed that all the gross lesions analysed were endometriotic in character (Figure 3Go). Cystically dilated glands were lined by columnar to cuboidal endometrial epithelium, and surrounded by a thin layer of endometrial stroma. In the adjacent tissues, there were scattered small granulomas with haemosiderin-laden macrophages related to repeated haemorrhages and some lymphocytes. New blood vessel growth was also observed in the endometriotic lesions. Sections of randomly chosen peritoneum showed no evidence of microscopic endometriosis.



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Figure 2. Gross findings of ectopic endometrial implants. The black arrow points to a single lesion attached to the posterior abdominal wall. The white arrows indicate grouped lesions appearing as tan multicystic areas bulging under the serosal coat of the anterior abdominal wall. U = uterus; B = bladder; Bw = bowels.

 


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Figure 3. Microscopic aspect of ectopic endometrial implants. The upper panel shows an endometriotic cystic lesion attached on the abdominal wall (original magnification x2.5). The picture in the middle shows multicystic areas resulted from grouped lesions (original magnification x2.5). The lower panel indicates a detail of the surface endometrial epithelium of an endometriotic cystic lesion (original magnification x40).

 
As shown in Figure 4Go (left panel), the extent of induced endometriosis was dependent upon the quantity of challenged endometrium. Total weight and surface area of the lesions increased up to about 15 mg of tissue inoculated which corresponds approximately to 50% of the whole endometrial tissue of an oestrogen-stimulated host. For a higher quantity of endometrium introduced, a decrease in survival rate and an increase in infective processes were observed. In the different animals, the ratio between weight of the resultant endometriotic lesions and weight of the inoculated endometrium varied between 0.26 and 0.46. A slight increase in endometriosis extent with time was also found (Figure 4Go right panel). After the inoculation, total weight and total surface area slightly increased from weeks 1–3 and then tended to remain stable up to 5 weeks.



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Figure 4. Ectopic endometrial implantation as a function of the amount of endometrium inoculated (left panel) and the time after inoculation (right panel). Data are represented as mean ± SEM of five mice per group.

 
Prevention of endometriosis induction by IL-12
In both C57BL/6 and BALB/c mice, intraperitoneal administration of IL-12 during endometrial challenge was able to induce a significant prevention of endometriosis establishment (Figures 5–6GoGo). As endometriosis is not a life-threatening disease, drug safety was considered a primary requirement for the strategy of treatment. Therefore, preliminary experiments were performed to detect the minimal effective dose which was shown to be 0.15 µg. At this dose, no differences in gross toxicity or survival rate were observed between IL-12-treated and non-treated mice. In the C57BL/6 strain, total weight of the lesions, evaluated after 3 weeks from inoculation of endometrium, were significantly lower in IL-12-treated animals (0.60 ± 0.17 mg) than in non-treated control mice (2.66 ± 0.45 mg; P < 0.005) (Figure 5Go left panel). Similarly, total surface area resulted 9.91 ± 1.40 mm2 in IL-12-treated animals and 25.64 ± 2.87 mm2 (P < 0.001) in non-treated control mice (Figure 5Go right panel). In the BALB/c strain, total weight of the lesions was 2.26 ± 0.31 mg and 3.87 ± 0.64 mg for IL-12-treated and non-treated mice respectively (P < 0.05) (Figure 6Go left panel). Total surface area resulted 19.7 ± 2.7 mm2 for IL-12-treated and 27.54 ± 3.45 mm2 for non-treated mice (P = 0.09) (Figure 6Go right panel). This corresponds to an overall reduction in weight and surface area of endometriosis lesions induced by IL-12 treatment respectively of 77 and 61% in C57BL/6 and of 42 and 28% in BALB/c mice.



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Figure 5. IL-12 prevents endometriosis in C57BL/6 mice. Endometriosis was induced in C57BL/6 (n = 15) mice by inoculating syngenic endometrial fragments. Seven mice were treated with IL-12 while eight mice were used as controls. Total weight of the lesions (left panel) and total surface area (right panel) were significantly reduced in IL-12 treated animals versus controls (P < 0.005 and P < 0.001 respectively).

 


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Figure 6. IL-12 prevents endometriosis in BALB/c mice. Endometriosis was induced in 15 BALB/c mice by inoculating syngenic endometrial fragments. Eight mice were treated with IL-12 while seven mice were used as controls. Total weight of the lesions (left panel) was significantly reduced in IL-12 treated animals versus controls (P < 0.05); difference in total surface area (right panel) was not significant (P = 0.09).

 
Failure to induce peritoneal implantation of small bowel mucosa
In order to verify whether implantation in ectopic locations can be aspecifically induced for every tissue inoculated in peritoneum or represents a more intrinsic property of endometrium, we compared lesions obtained by intraperitoneal inoculation of endometrium with those resulting using a control tissue. In this contest, organs that are both embryogenically and structurally similar to endometrium are not available in adequate amount. Therefore, small bowel mucosa was chosen as a control tissue since it shares with endometrium a grossly similar anatomical structure. Weights of small bowel mucosa and of endometrium challenged in this experiment were 18.99 mg and 12.12 mg respectively. Only seven out of 12 (58%) of the BALB/c animals challenged with syngenic bowels showed evidence of peritoneal lesions at 3 weeks whereas endometriosis was demonstrated in 100% of the nine mice challenged with endometrium (P < 0.05 compared to BALB/c mice challenged with endometrium). On gross examination, lesions, when present, consisted of white nodules on the peritoneum. The ratio between weight of the resultant lesions and weight of challenged small bowel mucosa was 0.07. Size of the lesions were also significantly decreased when compared to endometriotic implants (Figure 7Go). Indeed, total surface area and total weight of lesions were reduced in mice challenged with intestines (6.06 ± 2.12 mm2 versus 31.62 ± 2.71 mm2; P = 0.0001 and 1.30 ± 0.54 mg versus 5.52 ± 0.34 mg; P = 0.0001). Furthermore, no small bowel mucosal structures were clearly detectable at histological examination. Most of the lesions consisted of fibrotic materials.



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Figure 7. Ectopic implantation of small bowel mucosa in BALB/c animals. Small bowel mucosa represents a control tissue. A similar amount of small bowel mucosa (18.99 mg) and endometrium (12.12 mg) were used to challenge 12 and nine mice respectively. Total weight and total surface area of the lesions are shown (*significantly different from endometriosis; P = 0.0001).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, normal immunocompetent mice were intraperitoneally challenged with syngenic endometrium to induce endometriosis. All the animals challenged with endometrium showed evidence of peritoneal disease at 3 weeks. Moreover, the cytokine IL-12 has been shown to be effective in preventing endometrial ectopic implantation in two different strains of mice.

Progress in endometriosis research has been limited so far by the lack of a valuable and promptly available model of the disease (Sharpe-Timms, 1997Go). 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, 1997Go). 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, 1990Go). 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., 1984Go; Bruner et al., 1997Go). 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., 1996Go). 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, 1997Go). 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., 1998Go). 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 6–7-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., 1996Go; O'Hara et al., 1996Go). 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, 1998Go). In particular, some studies focused on the ability of NK cells to recognize and eliminate ectopic endometrial cells (Somigliana et al., 1999Go). Aoki et al. heterotransplanted human endometrium in nude and SCID (severe combined immunodeficiency) mice (Aoki et al., 1994Go). 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., 1989Go; Wolf et al., 1991Go; D'Andrea et al., 1992Go), 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.


    Acknowledgments
 
The work was supported in part by a grant from Centro di Studi Cronobiologici dell' Endometriosi, University of Milano, Milano, Italy.


    Notes
 
5 To whom correspondence should be addressed at: II Dept. Obstetrics and Gynecology, Via Commenda 12, 20122 Milan, Italy Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on July 1, 1999; accepted on September 13, 1999.