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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 3 911-916
Copyright © 1998 by The Endocrine Society

Original Studies

Interleukin-12 and Its Free p40 Subunit Regulate Immune Recognition of Endometrial Cells: Potential Role in Endometriosis1

D. Mazzeo2, P. Viganó2, A. M. Di Blasio, F. Sinigaglia, M. Vignali and P. Panina-Bordignon

Roche Milano Ricerche (D.M., F.S., P.P-.B.); II Department of Obstetrics and Gynecology, University of Milan (P.V., M.V.); and Istituto Auxologico Italiano (A.M.D.), Milan, Italy

Address all correspondence and requests for reprints to: Dr. Paola Panina-Bordignon, Roche Milano Ricerche, 20132 Milan, Italy. E-mail: paola.panina{at}roche.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An alteration of immune recognition and killing of misplaced endometrial cells, refluxed with menstrual debris in ectopic sites, has been claimed to be responsible for the initiation and progression of endometriosis. In particular, current evidence emphasizes the role of natural killer (NK) cells as potential effectors of peritoneal immune surveillance. Interleukin-12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, has potent regulatory effects on NK cell growth and function. The purpose of this study was to evaluate whether this cytokine may also have a role in the specific cytolytic NK cell response toward endometrial antigens. To this aim, concentrations of IL-12 and its free p40 subunit were determined in peritoneal fluid of 33 patients with endometriosis and 40 women without laparoscopic evidence of the disease. Similar concentrations of IL-12, but significantly higher levels of free p40, were present in peritoneal fluid of patients with endometriosis compared to those in women without the disease. We also observed that the IL-12 plus free p40/IL-12 ratio increased with the severity of the disease. Moreover, we investigated whether incubation of NK cells with heterodimeric IL-12 and/or p40 has any effect on NK cell-mediated lysis of endometrial cells. NK cells pretreated with heterodimeric IL-12 exhibited an enhanced cytotoxic response toward endometrial targets. This IL-12-induced cytotoxicity could be abrogated by the p40 subunit in a specific and dose-dependent manner. The p40 inhibitory effect was mediated by down-regulation of IL-12 high affinity binding sites on NK cells, as we observed inhibition of surface IL-12 receptor ß1-chain expression, a decrease in IL-12-binding capacity, and inhibition of phosphorylation of STAT4 (signal transducer and activator of transcription) protein. These data suggest that the excess of p40 present in peritoneal fluid of patients with endometriosis may be related to the NK cell defect associated with the disease. Moreover, IL-12 could be a potential specific agent able to correct the p40-induced defect in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GENERAL estimates indicate that endometriosis is the most frequent diagnostic entity in gynecology and one of the most common surgical conditions in young women. Thus, it represents per se a relevant public health problem (1, 2). It has been postulated that endometrial cells and fragments, desquamated during the menstrual period, are transported through the fallopian tubes into the peritoneal cavity where they implant, proliferate, and develop into endometriotic lesions (3). Transport of shed fragments of endometrium to the pelvis seems to be a phenomenon common to most menstruating women, but it is presently unclear why in only some women do endometrial cells implant in ectopic locations and give rise to endometriosis (4, 5).

Currently available data suggest an association between endometriosis and alterations in cell-mediated immunity (4). In particular, recent in vitro findings provide evidence that an immunological clearance is responsible for protection against outgrowth of endometrial cells in ectopic sites (6, 7, 8, 9, 10). These studies support the existence of a physiological natural killer (NK) cell-mediated cytotoxicity toward autologous endometrial cells. In patients with endometriosis, this phenomenon has been reported to be defective, and this defect correlates with disease severity.

Interleukin-12 (IL-12), produced by myelomonocytic cells, plays a major role in the regulation of NK cell cytotoxic activity (11, 12). It exerts pleiotropic effects on NK and T cells, including induction of transcription and secretion of cytokines, enhancement of cytotoxicity, and induction of proliferation of T and NK cells. IL-12 is a heterodimer composed of two disulfide-linked chains, p35 and p40, encoded by separate genes. Simultaneous expression of the two genes is required for the production of the biologically active IL-12 heterodimer. Although the p35 gene is constitutively expressed in most cell types, the presence of p40 gene transcripts is restricted to those cells able to produce IL-12 (13, 14). Although the expression of p40 and p35 genes is regulated by activation of the producing cells, the expression of the p40 gene is much more finely regulated than that of the p35 gene (15, 16). All cell types producing the biologically active p75 heterodimer also produce a large excess of the free p40 chain (12, 13). The physiological significance of the overproduction of free p40 still needs to be elucidated. Free homodimeric p40 and, to a lesser extent, monomeric p40 act as an IL-12 receptor (IL-12R) antagonist (17, 18, 19). However, the possibility exists that free p40 has as yet unknown biological activities.

Given the variety of effects exerted on NK cells by IL-12, we reasoned that IL-12 may play a role in NK cell-mediated lysis of endometrium. Therefore, we determined levels of IL-12 and its free p40 subunit in peritoneal fluid of women with and without endometriosis. We report herein that IL-12 plus free p40/IL-12 ratios are higher in women with endometriosis than in women without the disease. IL-12-treated NK cells exhibit enhanced recognition of endometrial cells, whereas its p40 subunit counteracts the IL-12 effect by down-regulation of IL-12Rß1 chain on NK cells. As a consequence, high levels of free p40 subunit may lead to a reduction of NK cell responsiveness to IL-12 and decreased clearance of ectopic endometrial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human samples

Samples of peritoneal fluid, endometrium, and heparinized blood were obtained from reproductive age women undergoing laparoscopy. The criterion for inclusion were 1) that the day of the last menstrual period was certain; 2) that the patients were cycling normally, and none had received hormones for at least 3 months before surgery; and 3) that there was no evidence of either endometritis or previous autoimmune or neoplastic disorders. Endometriosis was documented based on the results of diagnostic laparoscopy and staged according to the revised criteria of the American Fertility Society (20). Women undergoing tubal sterilization after achieved parenthood or laparoscopy for benign ovarian cysts or adhesions served as the control group. No evidence of endometriosis was found in any of these cases. Peritoneal fluid was aspirated immediately before laparoscopy, centrifuged, and frozen until assayed. Endometrial biopsies (10 secretory and 9 proliferative) were performed using an endometrial biopsy curette. The patients were informed that the tissues would be used for research purposes, and they gave written consent.

Cytokines

Affinity-purified human recombinant IL-12 was provided by Dr. M. Gately (Hoffmann-La Roche, Nutley, NJ) (18). A plasmid encoding the human p40 subunit complementary DNA for expression in insect cells was provided by Dr. U. Gubler (Hoffmann-La Roche). A baculovirus expressing the human p40 subunit protein was generated using the BaculoGold transfection kit (PharMingen, San Diego, CA), and a single recombinant virus was isolated by plaque purification. Human p40 was produced and purified on a 4D6-conjugated protein G-Sepharose immunoaffinity column as previously described (18, 19). The baculovirus-expressed human p40 was characterized by SDS-PAGE and Western blot analysis. Under nonreducing conditions, the purified human p40 revealed a single immunoreactive band migrated at the 40-kDa position.

ELISA for IL-12 and p40

ELISAs for p40 and IL-12 were performed as previously described (21). Rat monoclonal antibodies (mAb) to human IL-12 and p40 were supplied by Dr. M. Gately (Hoffmann-La Roche). The IL-12-specific capture antibody, 20C2, is an IgG1 that reacts with a conformational epitope on the 75-kDa IL-12 heterodimer and, thus, was used to measure concentrations of heterodimeric IL-12. The p40-specific capture antibody, 4A1, is an IgG1 that detects both heterodimeric IL-12 and free p40.

Cells

The establishment of primary cultures of endometrial stromal cells was described in detail previously (22, 23). Briefly, after removal of all blood clots, the remaining tissue was minced and incubated in complete medium containing 0.1% collagenase (Boehringer Mannheim Biochemicals, Milan, Italy) for 2 h at 37 C in a shaking water bath. At the end of the incubation, single stromal cells were separated from large clumps of epithelium by differential sedimentation at unity gravity and selective adhesion to tissue culture dishes. Endometrial stromal cells were cultured in Ham’s F-10 medium supplemented with 10% FCS, 4 mg/mL D-glucose, 2 mmol/L L-glutamine, and 50 µg/mL gentamicin. The Kit225/K6 cell line was provided by Dr. D. Presky (Hoffmann-La Roche). The NK3.3 cell line was provided by Dr. M. Malnati (Dibit, Milan, Italy).

Isolation of CD3-CD56+ cells

Peripheral blood lymphocytes from women with and without endometriosis were enriched by incubation in RPMI 1640 10% FCS in tissue culture-treated plastic petri dishes for 2 h twice, and the nonadherent cells were saved. This fraction was further depleted of contaminating T and B lymphocytes by panning with anti-CD3 and anti-CD5 mAbs (Immunotech, Marseille, France). Nondepleted cells were then stained with anti-CD56 and anti-CD3 mAbs (Becton Dickinson, Mountain View, CA) and sorted on a FACStar Plus (Becton Dickinson). Sorted CD3- CD56+ NK cells were more than 95% pure by cytofluorometric analysis.

Cytotoxicity assay

The cytolytic activity of NK cell-enriched preparations, previously treated with the indicated combination of cytokines for 24 h, was tested against that of endometrial stromal cells derived from autologous donors at different effector/target (E:T) cell ratios as previously described (10). Target cells (1 x 106) were labeled with 100 µCi sodium-51Cr and used at 103 cells/well. After a 4-h incubation, supernatants were harvested and counted in a {gamma}-counter to determine the isotope release. Specific lysis was obtained as the mean value of three replicates.

Flow cytofluorometric analysis

IL-12Rß1 expression was detected by incubating 2 x 103 cells for 30 min on ice with 2B10 mAb (rat anti-human IL-12Rß1; provided by Dr. M. Gately, Hoffmann-La Roche), followed by fluorescein isothiocyanate mouse anti-rat Ig (Jackson ImmunoResearch Laboratories, West Grove, PA). Rat IgG2a (Becton Dickinson, Mountain View, CA) was used as the isotype control. Stained cells were analyzed using a FACScan flow cytometer gated to exclude nonviable cells.

IL-12 binding assay

Binding of IL-12 to NK3.3 cells was detected as previously described (24). Briefly, cells were incubated overnight at 37 C with p40 at 30 ng/mL. Before starting the experiment, cells were washed for 30 s with ice-cold complete RPMI 1640, pH 3, to remove bound p40 and then washed twice with large volumes of complete RPMI 1640, pH 7.5. Cells were incubated with 500 pmol/L [125I]IL-12 for 2 h at 4 C, washed three times at 4 C, and then shifted to 37 C. At different time points, aliquots were collected, and the cell pellet was resuspended in glycine-Cl-, pH 3, and 0.15 mol/L NaCl for 5 min at 4 C. Membrane-associated radioactivity was separated by centrifugation of cell suspension through 0.1 mL of an oil mixture (1:2 mixture of Thomas silicone fluid (A.H. Thomas, Chicago, IL) and silicone oil). Nonspecific binding was determined, including 25 nmol/L unlabeled IL-12 in the assay.

Detection of STAT4 (signal transducer and activator of transcription) phosphorylation

T and NK cells were washed to remove IL-2 and incubated overnight with 30 ng/mL p40. After three washes, 5 x 106 cells/mL were induced for 15 min at 37 C with IL-12 (4 ng/mL) or p40 (20 ng/mL). Cells were washed once with cold PBS and then lysed in 400 µL immunoprecipitation buffer [10 mmol/L Tris/Cl- (pH 7.4), 150 mmol/L NaCl, 1 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (pH 8.0), 1 mmol/L ethylenediamine tetraacetate (pH 8.0), 1% Nonidet P-40, 0.25% sodium deoxycholate, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride, hydrochloride (AEBSF), 1 mmol/L sodium orthovanadate, and 10 mmol/L NaF] on a shaker for 1 h at 4 C. After centrifugation (30 min, 13,000 rpm at 4 C), extracts were immunoprecipitated for 1 h at 4 C using an anti-STAT4 mAb (Santa Cruz Biotechnology, Santa Cruz, CA). Immunocomplexes were absorbed onto Protein-A Fast Flow (Pharmacia, Uppsala, Sweden) for 1 h at 4 C. The beads were washed four times with immunoprecipitation buffer and, after the addition of 2.5 x SDS-sample buffer, boiled at 95 C for 3 min. SDS-PAGE was performed using the Laemmli method. After electrophoresis, Western blot was performed, transferring proteins onto nitrocellulose membranes. The membranes were blocked by incubating overnight at 4 C with TBS-T (20 mmol/L Tris base, 137 mmol/L NaCl, and 0.1% Tween-20, pH 7.6) and 5% nonfat dry milk and then probed at room temperature with antiphosphotyrosine (Upstate Biotechnology, Lake Placid, NY), followed by antimouse horseradish peroxidase (Amersham Life Science, Arlington Heights, IL) or with a anti-STAT4 mAb (Santa Cruz Biotechnology) followed by antirabbit horseradish peroxidase. Proteins were detected by the enhanced chemiluminescence detection system (Amersham Life Science).

Statistical analysis

Data are expressed as the mean ± SEM. Significance between groups was determined using the Mann-Whitney nonparametric test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-12 and p40 levels in peritoneal fluid

Levels of p40 and IL-12 were measured in peritoneal fluid of 33 patients with endometriosis and 40 women without laparoscopic evidence of the disease. The ELISA detecting both heterodimeric IL-12 and free p40 revealed a mean concentration of 59.1 ± 8.7 pg/mL in endometriosis patients, which was significantly higher than the mean concentration in control women (32.9 ± 5.4 pg/mL; P < 0.02). The ELISA detecting only heterodimeric IL-12 revealed similar IL-12 concentrations in the peritoneal fluid of the endometriosis group and the control group (16.3 ± 6.6 and 13.7 ± 4.7 pg/mL, respectively). The results obtained indicate the presence of significantly higher levels of free p40 in the peritoneal fluid of patients with endometriosis than those in women without evidence of the disease. Consistent with these findings, IL-12 plus free p40/IL12 ratio correlated with the severity of the disease, being 2.6 in stage I and increasing to 4.2 in stage II, 6.2 in stage III, and 13.3 in stage IV endometriosis, respectively (Fig. 1Go).



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  		Figure 1. Concentrations of heterodimeric IL-12 and p40 in peritoneal fluid of 33 patients affected by endometriosis and 40 women without evidence of the disease. Results are presented as the mean ± SEM and divided according to the stage of endometriosis (13 cases at stage I, 7 cases at stage II, 9 cases at stage III, and 4 cases at stage IV). p40/IL-12 ratios are indicated above the bars.

 
p40 antagonizes the effect of IL-12 on NK-mediated lysis of endometrial stromal cells

The addition of IL-12 to NK cell-enriched fractions caused an enhancement of cytotoxicity against autologous endometrial stromal cells in a dose-dependent fashion in both women with endometriosis and women without the disease (Fig. 2Go). The estimated percentage increase of NK cell lysis induced by IL-12 (10 ng/mL) at an E:T cell ratio of 40:1 was 128.8 ± 14.3 in endometriosis patients and 64.2 ± 7.1 in women without the disease. Preincubation of CD56+ NK cells from both women without (data not shown) and those with endometriosis (Fig. 3Go) with p40 alone did not alter NK cell-mediated lysis of autologous endometrial antigens. However, p40 inhibits the IL-12 (10 ng/mL)-induced enhancement of NK cell activity toward endometrial cells (Fig. 3Go). Although the effect of p40 on IL-12-induced NK cell activity was maximal at 6 ng/mL, 28% was already detected when p40 was added at 0.6 ng/mL (Fig. 4Go). The observed inhibition was IL-12 specific, as IL-2-induced NK cell activity was not affected by the addition of p40.



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  		Figure 2. IL-12 enhances NK cell-mediated lysis of in vitro cultured autologous endometrial stromal cells. NK cells and autologous endometrial samples were obtained from six women with endometriosis (stages I, II, and III), and five women without evidence of the disease. Purified CD56+ NK cells were incubated with different concentrations of IL-12 for 24 h. Cytotoxicity at various E:T cell ratios was then tested in a standard 4-h 51Cr release assay using autologous endometrial stromal cells as targets. Results obtained at an E:T cell ratio of 40:1 are presented as the percent increase in lysis. Values represent the mean ± SEM.

 


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  		Figure 3. p40 inhibits NK cell-mediated lysis of in vitro cultured autologous endometrial stromal cells. NK cells and autologous endometrial samples were obtained from three women with endometriosis (stages I, II, and III). Purified CD56+ NK cells were incubated with the indicated combination of IL-12 (10 ng/mL) and p40 (6 ng/mL) for 24 h and then tested against endometrial stromal cells at an E:T cell ratio of 40:1 in a standard 4-h 51Cr release assay. Addition of p40 (6 ng/mL) alone had no effect on unstimulated lysis. Results represent the mean ± SEM.

 


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  		Figure 4. p40 specifically inhibits NK cell-mediated lysis of in vitro cultured autologous endometrial stromal cells. Purified CD56+ NK cells from a woman with endometriosis (stage II) were incubated for 24 h with IL-12 (10 ng/mL) together with various dilutions of p40 ({circ}) or PBS buffer (•). In the same experiments, CD56+ NK cells were also incubated with IL-2 (500 U/mL) together with various dilutions of p40 ({triangleup}) or PBS buffer ({blacktriangleup}). Activated NK cells were tested against endometrial stromal cells at an E:T cell ratio of 40:1 in a standard 4-h 51Cr release assay. Unstimulated lysis is indicated on the y-axis. Values represent the mean of three replicates of a representative experiment.

 
Down-regulation of IL-12R by p40

Kit225/K6 cells or a NK cell-enriched fraction from the peripheral blood of healthy donors were stimulated overnight with IL-12 (10 ng/mL) in the presence or absence of increasing concentrations of p40 and then stained with a mAb recognizing the ß1-chain of the IL-12R. When cells were preincubated with p40, the IL-12Rß1 chain was down-regulated (Fig. 5Go, a and c). Preincubation with mouse (p40)2, which is known to bind to the human IL-12R, gave a similar result (Fig. 5dGo). p40 induces specific down-regulation of the IL-12Rß1 chain, as preincubation of cells with human p40 had no effect on the expression of the IL-2R{alpha} chain (data not shown). Other lymphokines, such as IL-2 (Fig. 5bGo), do not affect surface IL-12Rß1 expression, as previously reported (25, 26).



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  		Figure 5. Expression of surface IL-12Rß1 expression in T and NK cells. Kit225/K6 and purified NK cells from a healthy donor were incubated overnight with medium alone (black line), IL-12 at 10 ng/mL (dashed line), or IL-12 at 10 ng/mL and p40 at 10 ng/mL (dotted line; a and c). Kit225/K6 cells were also incubated with 10 U/mL IL-2 (dotted line; b). NK cells were also incubated with m(p40)2 (dotted line; d). Surface IL-12Rß1 expression was detected by FACS analysis of cells stained with the IL-12Rß1-specific mAb 2B10. The logarithm of fluorescence intensity is plotted on an arbitrary scale from 1–200. One representative experiment of five is shown.

 
Similar to other cytokines, IL-12 is internalized after binding to the IL-12R. To determine whether p40 affects the binding of IL-12 to the IL-12R, NK3.3 cells were either left untreated or were treated with p40 overnight, then acid washed to remove bound p40, and incubated with [125I]IL-12 (500 pmol/L) sufficient to saturate receptor sites and achieve equilibrium binding (24). Cells were washed to remove unbound [125I]IL-12, resuspended in binding buffer, and shifted to 37 C for the designated times. The binding at time zero was measured after the cells were resuspended in binding buffer, but before shifting to 37 C. We demonstrated that at time zero, bound IL-12 decreased by 62% in cells that were pretreated with p40 (Fig. 6Go). Treatment of cells with acid medium resulted in a rapid and nearly complete dissociation of surface-bound [125I]IL-12. When the cells were shifted to 37 C, there was a time-dependent increase in the proportion of cell-bound radioactivity that was resistant to acid treatment. The amount of [125I]IL-12 internalized in p40-treated cells reached a maximum at 60 min and then declined, with similar kinetics in cells treated with p40 and those left untreated (data not shown).



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  		Figure 6. p40 inhibits binding of [125I]IL-12 to IL-12R on NK3.3 cells. NK3.3 cells were cultured overnight with medium alone ({circ}) or with p40 (10 ng/mL; {blacktriangleup}), then incubated in the presence of [125I]IL-12 (500 pmol/L) for 2 h at 4 C, washed, resuspended in binding buffer, and transferred to 37 C. The data represent specific binding. Nonspecific binding was between 5–10% of total binding at each time point. The experiment shown is representative of three that gave similar results.

 
p40 inhibits tyrosine phosphorylation of STAT4

Down-regulation of the high affinity binding sites by p40 suggests that responsiveness to IL-12 may be altered after ligand-receptor interaction. To address this question, we measured tyrosine phosphorylation of STAT4 proteins in p40-treated NK3.3 cells, as STAT4 phosphorylation is known to be one of the earliest events after IL-12 stimulation (27). Stimulation of NK3.3 cells with IL-12 induces a rapid tyrosine phosphorylation of STAT4 proteins. However, no tyrosine phosphorylation of STAT4 was observed after the cells had been cultured with p40 (Fig. 7Go). These results indicate that IL-12 responsiveness in NK3.3 cells decreases markedly after they are cultured with p40.



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  		Figure 7. p40 inhibits IL-12-induced phosphorylation of STAT4 proteins. NK3.3 cells were incubated overnight with medium alone or p40 (30 ng/mL), and then induced with IL-12 (10 ng/mL) for 15 min at 37 C. The cell lysates were immunoprecipitated with anti-STAT4 mAb, separated by SDS-PAGE, and transferred to a membrane. Phosphotyrosine was detected by antiphosphotyrosine antibody. The filters were then reprobed with anti STAT4 mAb to determine the amount of STAT4 in each lane. The experiment shown is representative of three that gave similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Endometriosis has an obscure etiology and pathogenesis, an extremely variable clinical presentation, an unpredictable course, and, except for physiological or surgical menopause, no known cure (1). Earlier studies focused on alterations in both cell-mediated and humoral immunity as central events during the development of the disease (4, 28, 29). Subsequently, we and others demonstrated that the NK cell-mediated activity is significantly reduced in patients with endometriosis (6, 7, 8, 9, 10). Thus, it has been proposed that a decreased immunological clearance of endometrial fragments shed into the peritoneal cavity may facilitate the implantation of endometrial cells in ectopic locations and result in endometriosis.

To gain further insight into the immunological mechanisms leading to the development and/or maintenance of endometriosis and explore the possibility of using regional immunotherapy as a potential therapeutical approach to the disease, we investigated the role of IL-12 in regulating NK-endometrial cell interaction. The results presented herein demonstrate that 1) IL-12 and p40 are both present in peritoneal fluid of women with and without the disease, but in patients with endometriosis, levels of free p40 are significantly higher and IL-12 plus free p40/IL-12 ratios correlate with the stage of the disease; 2) IL-12 enhances the NK cell-mediated cytotoxicity toward autologous endometrial cells; 3) p40 is a potent and specific inhibitor of IL-12-induced NK activity against endometrial cells; 4) p40 induces down-regulation of surface IL-12R expression on NK cells.

IL-12 is particularly effective during the first hours of an immune response. During this time, macrophages begin to produce IL-12, and this burst stimulates the proliferation of NK and T cells, enhances their killing capacity, and triggers a surge of interferon-{gamma} from both cell types (11, 16, 26). It is noteworthy to consider that from the results obtained in this study, IL-12 seems to be a normal constituent of peritoneal fluid. Thus, its presence in peritoneum would suggest a physiological role of this cytokine in the control of local immune processes. This concept is strongly supported by the evidence that IL-12 possesses the ability to modulate the NK cell-mediated recognition of endometrial targets.

As we could not detect secretion of IL-12 or free p40 by shed endometrial cells (data not shown), they probably derive from peritoneal macrophages. Indeed, peritoneal macrophages from women with endometriosis can be induced to produce IL-12 in vitro (data not shown). Our data suggest that in patients affected by endometriosis, peritoneal macrophage secretion of IL-12 might be altered. This results in the production of a substantial excess of the free p40 subunit that acts by down-regulating IL-12R on NK cells. As a consequence, NK cells may become less responsive to IL-12, thus accounting at least in part for the decreased NK cell-mediated lysis of refluxed endometrium observed in patients with endometriosis. However, the precise nature of the events that induce the secretion of abnormal levels of these cytokines by peritoneal macrophages from women with endometriosis has yet to be elucidated.

This is the first report showing that an unbalanced production of IL-12 and its free p40 subunit is present in a pathological condition. It has been proposed that p40 docks to the ß1-subunit of IL-12R, whereas the ß2-subunit of IL-12R acts as a signal-transducing element (30). Therefore, the antagonistic activity of p40 has been believed up to now to result from blocking the binding of IL-12 to the ß1-subunit of the IL-12R (18, 19). We show that free p40 inhibits IL-12 activity not only by competitive binding to IL-12R, but also via down-regulation of its ß1-chain, which has been shown to be an essential component of the functional IL-12R (31). As a functional consequence of the down-regulation of high affinity IL-12-binding sites after culture with p40, tyrosine phosphorylation of STAT4 protein was inhibited. These findings indicate a novel mechanism by which the free p40 subunit acts as a physiological regulator of IL-12 and suggest that p40 may have biological activities different from those mediated by the heterodimer.

Finally, it is of utmost interest that in endometriosis patients, heterodimeric IL-12 greatly enhances NK cell-mediated cytotoxicity toward endometrial targets. The key role of IL-12 in immune regulation has suggested its use in patients with tumors, allergy, or immunodeficiencies (32, 33). The data presented here indicate that the immunostimulatory capacity of IL-12 might be exploited as a novel therapeutic strategy to control the cytolytic arm of the initial immune surveillance to ectopic endometrial antigens.


   Acknowledgments
 
We thank Dr. M. Gately for providing many reagents used in this study, Drs. L. Adorini, and F. Mattner for reviewing the manuscript, Prof. M. Busacca for helpful discussion, and Ms. R. Lang for technical support.


   Footnotes
 
1 This work was supported in part by a grant from Centro di Studi Cronobiologici dell’Endometriosi, University of Milan, Milan, Italy. Back

2 D.M. and P.V. contributed equally and are listed in alphabetical order. Back

Received September 15, 1997.

Revised November 5, 1997.

Accepted November 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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