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Original Article |
Istituto Auxologico Italiano (P.V., S.M., A.M.D.), 20135 Milan, Italy; Second Department of Obstetrics and Gynecology, University of Milan (E.S., M.V.), 20122 Milan, Italy; and Department of Obstetrics and Gynecology (M.V.), University of Milan Bicocca, 20122 Milan, Italy
Address all correspondence and requests for reprints to: Dr. A. M. Di Blasio, Molecular Biology Laboratory, Istituto Auxologico Italiano, V.le Monte Nero 32, 20135 Milan, Italy. E-mail: a.diblasio{at}auxologico.it.
Abstract
Previous reports suggest that systemic and/or placental presence of T helper 2-type cytokines would be supportive of normal pregnancy. Among these cytokines, IL-10 is thought to be produced at the feto-maternal interface to control fetal-ablating immune responses. However, expression of IL-10 in nonhemopoietic maternal cells of the human uterus has not been characterized in detail. Thus, we studied the expression and modulation of the cytokine and its receptor in human endometrial cells obtained in different phases of the cycle and in early pregnancy. Both cycling and pregnant endometrium express the genes for IL-10 and its receptor, but secretion of the cytokine was significantly increased in decidual cultures compared with that by endometrial cells in both proliferative and secretory phases of the cycle. Similarly, the expression of IL-10 receptor mRNA was up-regulated in early decidua compared with that in menstrual cycle-dependent endometrium. IL-1ß, but not gonadal steroid hormones, was able to directly increase endometrial/decidual IL-10 production. Based on the activity of IL-10 in other nonhemopoietic cell populations, we also evaluated its potential effects on TNF
secretion and proliferation of endometrial/decidual cells, but we were unable to demonstrate any direct role of IL-10 as a regulator of these specific functions. It is evident that IL-10 and its receptor are normal constituents of endometrium and early decidua, and their up-regulation during early pregnancy may participate in the T helper type 2 predominance at the feto-maternal interface. The inability of the cytokine to exert autocrine effects on TNF secretion and proliferation of decidual cells leads to speculation that the cytokine acts mostly as a paracrine mediator able to affect maternal immune responses.
PREGNANCY IS A unique natural condition in which the tolerance of a genetically dissimilar tissue, the fetus, implies that different mechanisms are established to both compromise and provoke the immune system (1, 2, 3, 4, 5). Any disturbance of the delicate immunological balance within the maternal-fetal interface may result in pregnancy loss or other perinatal complications. Several cytokines have been implicated in this immune system balance and may modulate maternal immune cells. In particular, in pregnancy the cytokine profile is thought to be shifted away from T helper 1 (Th1)-type responses toward Th2-type responses (6, 7). Th1-type cytokines such as IL-2, interferon-
(IFN), and TNF have been shown to induce adverse effects on the conceptus either directly or indirectly by activating local natural killer (NK) cells/macrophages that are able to damage the trophoblast (7, 8). As a consequence, a Th2-type-biased immune response that inhibits Th1-type responses would be beneficial for fetal survival and would favor immunological tolerance of the fetal allograft.
Among Th2-type cytokines, the crucial role of IL-10 in supporting murine gestation has been extensively documented (9). The cytokine has been reported to prevent fetal wastage in a resorption-prone CBA x DBA/2 mating combination characterized by increased levels of local inflammatory cytokines. On the contrary, injection of an anti-IL-10 monoclonal antibody (Ab) in these mice resulted in a pronounced increase in the rate of fetal loss (10). In a rat model of fetal growth restriction induced by lipopolysaccharide (LPS), administration of IL-10 was able to attenuate fetal resorption and demise (11).
Clinical and experimental evidence indicates that a weakening of Th1-type responses and a strengthening of Th2-type responses occur in pregnant women (12). In a successful human pregnancy, an increase in the production of IL-4 and IL-10 by in vitro mitogen- and trophoblast-stimulated peripheral blood mononuclear cells is accompanied by a parallel decrease in the production of IL-2 and IFN, with a predominant type 1 to type 2 shift observed in the third trimester (13, 14). Conversely, peripheral blood mononuclear cells from women with a history of unexplained recurrent abortions were shown to respond in vitro to trophoblast antigens by producing high levels of IFN and TNFß, but very low levels of IL-4 and IL-10 (13, 15). In general, this Th1/Th2 model of cytokine secretion during gestation could explain the specific immunological patterns of human pregnancy, namely a certain increased susceptibility to infections that require a Th1-type response for protection (16) and the gestationally induced systemic bias toward Ab production with concurrent dampening of delayed-type hypersensitivity and NK cell immunity (9).
Interestingly, these pregnancy-related systemic modifications are thought to be associated with an intrauterine Th2-type environment. Indeed, in murine gestation, placental and decidual tissues have been shown to produce Th2-type cytokines, thus supporting the Th2-type dominance at the maternal-fetal interface (6, 17). Similarly, human cytotrophoblasts were shown to produce bioactive IL-10 that is able to suppress IFN production in an allogenic mixed lymphocyte reaction (18, 19, 20). Moreover, it has been demonstrated that human T cell clones generated from decidua produce Th2-type cytokines and that this secretion is reduced in decidual T cells from women suffering from unexplained recurrent spontaneous abortions (21, 22).
Despite these findings, the production of IL-10 and its regulation in the human uterus need to be further elucidated. To this aim, in the present study we examined nonlymphoid cells of the uterus for spontaneous and stimulated IL-10 production. The expression of IL-10 receptor (IL-10R) was also evaluated. The results obtained show that both cycling and pregnant endometria express the genes for IL-10 and its receptor. However, the production of the cytokine and the expression of IL-10R are up-regulated in early decidua compared with those in menstrual cycle-dependent endometrium. Moreover, the data support the direct involvement of short-term treatment with IL-1ß, but not gonadal steroid hormones, in the regulation of endometrial/decidual IL-10 production. Finally, the lack of an autocrine role of IL-10 as a regulator of specific endometrial/decidual functions suggests that the cytokine might act more in a paracrine than in an autocrine manner.
Materials and Methods
Reagents
LPS from Escherichia coli O55:B5, 17ß-estradiol, and progesterone were purchased from Sigma (St. Louis, MO). Human recombinant IL-1ß was obtained from Amersham International (Little Chalfont, UK). Culture medium consisted of Ham’s F-10 (Euroclone, Wetherby, UK) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, and 10% heat-inactivated FCS (all from Euroclone). Collagenase A was purchased from Roche Molecular Biochemicals (Milan, Italy), and hyaluronidase was obtained from Sigma. Anti-CD45-fluorescein isothiocyanate (FITC)/CD14-phycoerythrin was purchased from BD Biosciences (Mountain View, CA). Primers for IL-10, IL-10R, and hypoxanthine phosphoribosyltransferase 1 (HPRT) were obtained from Amersham Pharmacia Biotech (Milan, Italy).
Sample collection
Human endometrial tissues were obtained from reproductive-age women undergoing laparoscopy for benign ovarian cysts. The criteria for inclusion were 1) the day of the last menstrual period was certain; 2) the patients were normally cycling and did not receive hormones for at least 3 months before surgery; and 3) there was no evidence of endometritis, endometriosis, or previous autoimmune or neoplastic disorders. Based on the date of the last menstrual period and the histological examination of the samples, 15 women were in the proliferative phase, and 15 were in the secretory phase of the cycle. All tissues of uterine endometrium were obtained at the time of laparoscopy using an endometrial biopsy curette.
Decidual tissues were obtained from healthy women undergoing elective termination of normal pregnancies between 8–13 wk gestation. The operative method used was cervical dilatation, followed by vacuum extraction of the products of conception. Special care was taken to avoid contamination by trophoblastic cells. Specimens of endometrium and decidua were rinsed several times in PBS and then processed immediately.
Approval for this research project was granted by the local human institutional investigation committee. All patients were informed in detail about the aims and procedures of the study and subsequently gave their written consent to the sample collection.
Cell culture
Establishment of stromal and epithelial cell monolayers from normal endometrial tissue has been described in detail in previous studies (23, 24). Diffuse and strong cytoplasmic immunostaining for vimentin was demonstrated in nearly all (90%) cultured endometrial stromal cells. Similar results were observed for cytokeratins on cultured endometrial epithelial cells. Briefly, tissue was gently minced into small pieces (1–2 mm3) and incubated for 2 h at 37 C in a shaking water bath in 10 ml Ham’s F-10 containing 0.2% collagenase. At the end of the incubation, single stromal cells were separated from large clumps of epithelium by a 10-min period of differential sedimentation at unity gravity. The top 8 ml medium, containing predominantly stromal cells, were then slowly removed, and the cells were collected by centrifugation. The stromal-enriched fraction was washed twice in culture medium and allowed to adhere selectively to tissue culture dishes for 15 min. Thereafter, any nonattached epithelial cells still present were removed, and a purified stromal preparation was obtained on the surface of the culture dishes. For epithelial isolation, the bottom 2 ml sedimentation medium were collected, washed, and layered over 10 ml fresh medium for two additional 5-min sedimentation periods. Dissociation of glands in single cells or very small clumps was achieved by digesting the pellet with 4 ml of a 0.05% trypsin-0.02% EDTA solution for 3–5 min. Final purification of epithelial cells was obtained by selective plating of any remaining stromal cells onto plastic substrate.
Decidual tissue was minced thoroughly between two scalpels and digested for 1 h at 37 C with gentle agitation in Ham’s F-10 with 0.1% collagenase and 0.2% hyaluronidase. Decidual cells were washed and separated from dead cells and red cells by Ficoll-Hypaque density gradient. The cells at the interface were removed, plated, and left at 37 C overnight, then washed several times to remove nonadherent cells and debris.
Quantification of IL-10 in conditioned medium (CM)
Endometrial or decidual cells were plated at similar density and cultured in Ham’s F-10 with 10% FCS and antibiotics in a humidified atmosphere of 95% air and 5% CO2 at 37 C. After a minimum of 8 d, during which culture medium was changed every other day, subconfluent cells either were left unstimulated or were cultured with the following reagents: LPS (1–1000 ng/ml), IL-1ß (50 and 500 pg/ml), estradiol (3–300 ng/ml), and progesterone (3–300 ng/ml). After 48-h incubation at 37 C with 5% CO2, cell-free CM was harvested and kept (-20 C) until determination of IL-10 production by ELISA. Cells were counted, and flow cytometric analysis was performed to determine the possible contamination of CD45/CD14-positive cells in these cultures. Cultures in which there were more than 2% CD45-positive cells were not included in the study.
IL-10 was measured using an ELISA employing the multiple Ab sandwich principle (Quantikine, R\|[amp ]\|D Systems, Inc., Minneapolis, MN). Plates were read by a microplate reader, and absorbancy was transformed to cytokine concentration (picograms per milliliter) using a standard curve. The minimum detectable dose of IL-10 was 3.9 pg/ml. The inter- and intraassay coefficients of variation were 7% and 4%, respectively.
Extraction of mRNA, RT-PCR, and sequence analysis for IL-10
The RT-PCR specific for IL-10 transcript was performed as previously described (25). Total RNA was isolated by acid guanidium isothiocyanate/phenol chloroform extraction. RNA (1 µg) was transcribed into cDNA using 1 mM of each dNTP, 1 U RNasin, 100 pmol random hexamer primers, and 200 U reverse transcriptase (all from Perkin-Elmer Corp., Milan, Italy) in a total volume of 20 µl. The reaction mixture was run at 42 C for 1 h, followed by a 5-min incubation at 95 C, and then quick-chilled on ice. The oligonucleotide primers used for the PCR reaction had the following sequences: human IL-10 forward, 5'-CTGTGAAAACAAGAGCAAGGC-3'; human IL-10 reverse, 5'-GAAGCTTCTGTTGGCTCCC-3'. IL-10 primers amplified a 500-bp fragment. The PCR reactions were performed on the entire cDNA product according to the instructions provided with the GeneAmp Amplification Reagent Kit (Perkin-Elmer Corp.). Samples were initially denatured at 94 C for 3 min, then heated at 94 C (20 sec), cooled at 61 C (20 sec), and heated at 72 C (20 sec) for 35 cycles, with a final extension at 72 C for 5 min. In each experiment a negative control was prepared using all reagents and substituting 1 µl water for the reverse transcriptase. The integrity of RNA and the absence of genomic contamination were assessed by amplification of the HPRT gene with intron-spanning primers (forward, 5'-GCTTGCTGGTGAAAAGGACC-3'; reverse, 5'-GTCAAGGGCATATCCTACAAC-3') according to the following PCR protocol: 94 C (20 sec), cooled at 52 C (20 sec), and heated at 72 C (20 sec) for 34 cycles. The small size of the intron (170 bp) ensured that both cDNA and genomic DNA were readily amplified, giving rise to 97- and 267-bp fragments, respectively. PCR products were visualized on a 4% agarose gel stained with ethidium bromide.
The nucleotide sequence of the PCR products was confirmed by sequence analysis. Sequencing reactions were prepared using the BigDye terminator chemistry (PE Applied Biosystems, Foster City, CA) and were analyzed on the ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems).
Semiquantitative RT-PCR for IL-10R
The RT-PCR specific for IL-10R transcripts was performed as previously described (26). Primer sequences specific for IL-10R were: forward, 5'-CCATCTTGCTGACAACTTCC-3'; and reverse, 5'-GTGTCTGATACTGTCTTGGC-3'. The specificities of the products generated by the indicated primers were verified by sequence analysis. A titration series of RT-PCR cycle numbers was performed to verify the linear phase of the amplification of both IL-10R and HPRT, used as an internal control. Twenty-eight cycles were employed to study both IL-10R and HPRT gene expression. PCR products were separated on agarose gels stained with ethidium bromide, and the bands were densitometrically scanned. Data were standardized against signals from parallel reactions for the HPRT gene.
Flow cytometric analysis for IL-10-binding sites
All necessary components were contained in a human IL-10 Fluorokine kit (R\|[amp ]\|D Systems, Inc.) (26). Cells were detached by trypsinization. At 1 x 106/ml, cells were incubated with 10 µl biotinylated recombinant human IL-10 in siliconized tubes for 60 min at 4 C. In the negative control, IL-10 was replaced by biotinylated soybean trypsin inhibitor. To prove the specificity of the binding, 10 µl blocking Ab were preincubated together with the biotinylated IL-10 in a separate tube before addition to serum-blocked cells. After incubation and without washing, 10 µl avidin-FITC reagent were added to each tube and incubated for an additional 30 min. Then the cells were washed with 2 ml of the 1x RDF1 buffer contained in the kit. All samples were analyzed on a flow cytometer (FACStar, BD Biosciences). Application of a gate on FL1 (green fluorescence) included IL-10/avidin/FITC-labeled cells.
Functional studies
Proliferation assay. For cell proliferation studies, epithelial, stromal, and decidual cells were plated in 35-mm tissue culture plates at a density varying between 0.5 x 105 and 1 x 105/well. After a minimum of 8 d, during which culture medium was changed every other day, the seeding medium was replaced by 1 ml/well fresh medium containing 0.1% BSA and different doses of IL-10. Controls received only the vehicle. The medium with and without IL-10 was changed every day. After 10 d of treatment, cells were harvested by a 10-min incubation in 0.05% trypsin-0.02% EDTA solution and counted using a cell counter (Seac, Firenze, Italy). The experiments were always performed in duplicate.
TNF secretion.
Decidual cells cultured at 5 x 105 cells/ml were treated with and without different concentrations of IL-10. The levels of TNF in medium conditioned by 48-h decidual cell cultures were measured using an ELISA kit (Bender MedSystems, Vienna, Austria). Assays were performed according to the manufacturer’s instructions; the lower limit of detection was 5 pg/ml. The inter- and intraassay coefficients of variation were 7.4% and 6.9%, respectively. All samples were assayed in duplicate. Results are expressed as picograms per milliliter.
Statistical analysis
Data are expressed as the mean ±SEM. Differences between groups were compared by one-way ANOVA. The Fisher least significant difference test was used as a posttest to determine significant differences between groups. P < 0.05 was considered statistically significant.
Results
Analysis of IL-10 mRNA expression and IL-10 secretion
Endometrial cells derived from 30 different tissues and decidual cells obtained from 23 different samples were tested for IL-10 mRNA expression by RT-PCR and for protein secretion by ELISA. During cell isolation and culture, great effort was directed toward the complete elimination of immune cells, themselves potential cytokine-producing contaminants. Phenotypic analysis was performed on each culture examined, and only cell populations 98% free of CD45- and CD14-positive cells were included in the study. RT-PCR products were consistently detected in all samples of decidual and endometrial cultures analyzed regardless of the phase of the menstrual cycle. Figure 1
(upper panel) shows representative examples of DNA products from RT-PCR reactions. Estimated and actual sizes of the PCR products were 500 bp. The identity of the amplified products with the primer-defined IL-10 DNA sequence was confirmed by sequence analysis (data not shown). Genomic DNA contamination of all RNA samples was controlled by RT-PCR of the constitutively expressed HPRT gene. All HPRT amplifications revealed the correct 97-bp DNA fragment, but no 267-bp products that would have indicated genomic DNA contamination (Fig. 1, lower panel). Of the 23 decidual cultures examined, 20 (86.9%) were also found to secrete detectable levels of IL-10. In contrast, the cytokine was detected in a limited percentage (26.6%) of CM from endometrial stromal cells obtained in both proliferative and secretory phases of the cycle. None of the epithelial cultures was found to secrete detectable levels of IL-10 (Fig. 2, upper panel). IL-10 concentrations in cell cultures in which IL-10 was detectable are shown in Fig. 2 (lower panel).
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Studies of the modulation of IL-10 secretion were limited to decidual and endometrial stromal cells, because the epithelium did not release significant amount of the cytokine. Figure 3 shows IL-10 production by endometrial stromal (A; n = 7) and decidual (B; n = 9) cell cultures after incubation with various concentrations of LPS. LPS had a major and dose-dependent effect on IL-10 production by both types of cells. More importantly, IL-1ß alone, at concentrations of 50 and 500 pg/ml, increased IL-10 secretion by 31% and 51%, respectively, in cycling endometrium and by 7% and 24%, respectively, in decidua (Fig. 3). In contrast, a 48-h incubation with estradiol and progesterone at concentrations ranging from 3–300 ng/ml, given alone or in association with LPS, had no effect on endometrial IL-10 secretion (data not shown).
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IL-10R gene expression was studied by estimating IL-10R mRNA levels using semiquantitative RT-PCR in endometrial stromal (n = 10), endometrial epithelial (n = 10), and decidual (n = 10) cell cultures. RT-PCR products were generated by applying increasing cycle numbers to cDNA of decidual cells. The resultant single bands of the expected sizes showed intensities increasing in dependence of the applied cycle number. RT-PCR products were consistently detected in all samples of decidual and endometrial cultures analyzed (data not shown). Results from the semiquantitative RT-PCR experiments indicated that levels of IL-10R mRNA were significantly increased in early pregnant compared with cycling endometrium. Indeed, target/standard ratios were similar in stromal and epithelial cells independent of the phase of the cycle, but were significantly increased in decidual cells (P < 0.05; Fig. 4).
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shows the resulting histograms of the flow cytometric studies on decidual cells. The percentage of labeled cells varied between 12–70%. Unspecific binding was clearly ruled out by negative control experiments (IL-10 neutralized by anti-IL-10 Ab). Thus, we postulated a specific binding activity for IL-10 on human decidual cells.
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Cell proliferation studies.
In five separate experiments, decidual cells, endometrial stroma, and endometrial epithelium were cultured in presence or absence of various doses of IL-10, and cell proliferation was determined by cell counting after 10 d of culture. The proliferation of these cell types was not affected by the presence of IL-10 (Table 1).
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secretion.
Nineteen samples of decidual cells were treated with different amounts of IL-10, and levels of TNF were evaluated in supernatant from 48 h of culture. Nonstimulated decidual cells (5 x 105) produced 29.56 ± 6.2.6 pg/ml TNF, and IL-10 at concentrations from 5–25 ng/ml did not affect secretion of the cytokine (Table 2).
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IL-10 is thought to be a propitious cytokine to the success of pregnancy. In agreement with this concept, reduced IL-10 production has been reported in pathological pregnancies, distinguishing them from normal ones (13, 16, 27, 28, 29, 30). IL-10 is suggested to be involved in the maintenance of pregnancy by corpus luteum maturation (31), direct inhibition of the synthesis of Th1-type cytokines, which are potentially deleterious for the conceptus, and suppression of the significant proportion of NK-like cells and other inflammatory cells at the uteroplacental interface (8). Moreover, uterine IL-10 has been demonstrated to have a dichotomous effect on human leukocyte antigen expression on trophoblast cells, inducing human leukocyte antigen G expression while down-regulating classical class I and class II antigens (32).
Placental cytotrophoblasts and decidual T cells have been shown to be sources of IL-10 in the human uterus (20, 21). Moreover, it has been reported that decidual cells derived from term placentas are able to release significant quantities of IL-10 after stimulation with LPS and IL-1ß (33, 34). Extending these findings, the results of this study clearly indicate that IL-10 mRNA is already expressed at the endometrial level, that IL-10 secretion is up-regulated in early pregnancy-decidua, and, more importantly, that cytokine synthesis can be stimulated by IL-1ß, but not by short-term incubation with sex steroids. The idea of evaluating the potential role of sex steroids on endometrial IL-10 secretion derived from recent evidence indicating that factors other than cytokines have important regulatory influences on IL-10 production. These factors include the inflammatory mediators, prostaglandin E2 and nitric oxide, and the two major stress hormones, glucocorticoids and catecholamines (35, 36). Steroid receptors as ligand-activated nuclear transcription factors might bind to hormone-responsive elements in the 5'-flanking region of the IL-10 promoter, or steroid receptor-mediated signals may converge upon the transcriptionally active complex of proteins, including members of the activating protein-1 (AP-1) and nuclear factor-
B families (36). Our results do not actually support a direct role of steroid hormones in regulating IL-10 secretion by endometrial cells, although other cell types have been shown to respond differently. Indeed, although similar results were observed for the production of IL-10 by antigen-specific T cell lines and trophoblast-activated peripheral blood mononuclear cells stimulated with progesterone (21, 37), Correale et al. (36) found that estradiol is able to enhance the secretion of antigen- or anti-CD3-stimulated IL-10 by CD4+ T cell clones.
In contrast to sex steroids and in line with data reported by Dudley et al. (33), IL-1ß, a potent primary inflammatory cytokine, increased IL-10 secretion by cycling and early pregnancy-endometrial cells. This effect was not totally unexpected, because IL-10 promoter contains an AP-1 recognition site and IL-1 can induce AP-1, although in monocytes this stimulation could not be observed in the absence of LPS (38). Importantly, the IL-1 system is one of the major pathways involved in the events regulating maternal-fetal interaction during embryonic implantation. Blockade of endometrial IL-1R type I by its natural receptor antagonist prevents implantation in mice (39). In human endometrium, IL-1ß mRNA appears in the midluteal phase, and in vitro decidualization of endometrial stromal cells by steroid stimulation for 9 d results in the expression of the cytokine (39, 40). Therefore, based on these findings and on the results presented herein, IL-1 is likely to be one of the factors directly implicated in the increase in IL-10 secretion by decidual cells. The decidual up-regulation of both cytokines suggests their concerted involvement in the regulation of early phases of pregnancy exerted by the decidua through control of trophoblast invasion, nutrition of the blastocyst, endocrine secretion, and protection of the embryo from maternal immune rejection (41).
Interestingly, we present here the first clear indication of the expression, at both mRNA and protein levels, of IL-10R on human decidual cells. Moreover, similarly to what was observed for IL-10, IL-10R expression is up-regulated in pregnant compared with cycling endometrium. Thus, in search of a functional significance for the presence of IL-10R on endometrial cells, especially during early pregnancy, we looked for biological responses of cultured cells to IL-10 treatment. Previous studies have demonstrated the ability of IL-10 to influence the growth rate of specific nonhemopoietic cell types, with both inhibitory and stimulatory effects. Michel et al. (26) reported a moderate and dose-dependent inhibition of IL-10 on keratinocyte proliferation. In contrast, IL-10 has been shown to enhance the growth of melanoma cells, acting as an autocrine growth factor (42). Other cell types, such as microglia, which express IL-10R, do not respond to IL-10 by a modification of mitogenic events (43). In this context we evaluated the potential action of the cytokine on endometrial proliferation, but we could not demonstrate by direct cellular count any effect on the endometrial growth rate.
IL-10 is also greatly involved in controlling the overactivity of immune reactions, including the overproduction of cytokines. Typically, IL-10 is able to inhibit the production of inflammatory mediators, including TNF, IL-1, and IL-6 (44). Thus, given the well known detrimental effect of TNF on gestation (7), in the present study we evaluated whether IL-10 was able to reduce the production of TNF by decidual cells. From our data, the cytokine itself does not seem to be able to affect this function.
The IL-10R belongs to the family of IFN and IFN receptors (45, 46). Members of this cytokine receptor family can phosphorylate signal transducer and activating transcription factors. Interestingly, it has been demonstrated that the expression of the IL-10R ligand binding chain on certain cells is not sufficient to confer IL-10 responsiveness (47). Specific murine fibroblast lines expressing IL-10R are unresponsive to IL-10, thus indicating that they lack at least one critical component for generation of a functional IL-10 signaling pathway involving signal transducer and activating transcription factors. This possibility may also be true for endometrial cells. Alternatively, similarly to what was observed for microglia, in which IL-10 alone does not affect the activities of IL-1, IL-6, or TNF, but suppresses the effects of LPS on these cytokine activities, IL-10 might actually act on stimulated decidual cells (43). It should be noted, however, that IL-10, up-regulated in different cell types in early pregnancy, most likely exerts its paracrine crucial action on immune cells resident within the decidua to suppress a potential harmful maternal response in vivo. Indeed, IL-10 secreted by cytotrophoblasts has been shown to suppress IFN production in an allogenic mixed lymphocyte reaction, thus supporting its role in the inhibition of lymphoid cell functions (19). Nevertheless, the regulation and the functional consequences of increased IL-10R expression in early pregnant endometrium remain to be further investigated.
In conclusion, the results of this study support the following observations: 1) the genes for IL-10 and IL-10R are expressed in human endometrial cells, and their expression is up-regulated in early pregnant vs. cycling endometrium; 2) IL-1ß is able to modulate endometrial/decidual IL-10 production, which is unaffected by a direct short-term treatment with gonadal steroid hormones; and 3) IL-10 alone does not have any direct effect on TNF production and proliferation of decidual cells. In light of this evidence, human endometrium, especially during pregnancy, may be included among those cell populations, such as trophoblasts and T cells, responsible for IL-10 predominance at the uterine level.
Acknowledgments
Footnotes
Abbreviations: Ab, Antibody; AP-1, activating protein-1; CM, conditioned medium; FITC, fluorescein isothiocyanate; HPRT, hypoxanthine phosphoribosyltransferase; IFN, interferon-; IL-10R, IL-10 receptor; LPS, lipopolysaccharide; NK, natural killer; Th1, Th2, T helper 1, Th helper 2.
Received March 20, 2002.
Accepted August 28, 2002.
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