In Vivo Infusion of Interleukin-1 and Chorionic Gonadotropin Induces Endometrial Changes that Mimic Early Pregnancy Events in the Baboon
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《内分泌学杂志》
Department of Obstetrics and Gynecology, University of Illinois, Chicago, Illinois 60212-7313
Abstract
Both human chorionic gonadotropin (hCG) and IL-1 induce changes in the endometrium that are associated with the establishment of pregnancy. We investigated the synergistic effect of these two embryonic signals on endometrial function using a baboon model of simulated pregnancy. Recombinant hCG (30 IU/d) was infused between d 6 and 10 post ovulation (PO) to mimic blastocyst transit. On the expected day of implantation (d 10 PO), IL-1 (12 ng/d) or IL-1 receptor antagonist (IL-1Ra; 12 ng/d) was infused for an additional 5 d. Endometria were harvested on d 15 PO. Both hCG and hCG plus IL-1 induced marked differences in the distribution of -smooth muscle actin, proliferation marker Ki67, decidualization marker IGF-binding protein-1, and cyclooxygenase-1. The most marked effect of IL-1 was the induction of IGF-binding protein-1 protein in stromal cells close to the apical surface, whereas cyclooxygenase-1 was down-regulated in the glandular epithelium. Protein arrays of uterine flushings showed significant suppression of death receptors, Fas and TNF receptor 1, in the hCG- with or without IL-1-treated groups, suggesting an inhibition of apoptosis. Additionally, cytotoxic T lymphocyte antigen-4, matrix metalloproteinase-3, and IL-4 were suppressed in treated animals compared with controls. However, no differences were observed in cytokine profile between hCG-treated and hCG- plus IL-1-treated baboons. This study confirms that in preparation for pregnancy, the primate endometrium undergoes both morphological and functional changes, which are modulated by hCG and IL-1, that lead to the inhibition of apoptosis and the development of an immunotolerant environment. These changes suggest that infusion of IL-1 at the time of implantation into the nonpregnant baboon treated with hCG synergizes with hCG and mimics the early endometrial events associated with the presence of an embryo.
Introduction
THE INTERACTION BETWEEN maternal endometrium and embryo during implantation is a complex process, the details of which are still not fully understood. Implantation involves inflammatory responses in the human endometrium (for a review, see Ref. 1). Inflammatory mediators, such as prostaglandins (PGs), chemokines, and cytokines, have been associated with these events (2). Although the precise mechanisms regulating implantation are still unknown, increasing evidence suggests an interaction between these mediators.
Human chorionic gonadotropin (hCG) is one of the major hormonal products of first trimester human placental trophoblast cells. Besides its well-known luteotropic function, it appears to play an important role in the preparation of the endometrium for blastocyst implantation (for a review, see Ref. 3). Injection of hCG reduces apoptosis in human endometrium (4). Infusion of hCG into nonpregnant baboons functionally alters the major cell types present in baboon uterine endometrium (5, 6).
IL-1 is a proinflammatory cytokine expressed in the endometrium throughout the menstrual cycle, particularly around the time of implantation (7). It is postulated to mediate the dialogue between the blastocyst and the endometrium during implantation (8). IL-1 induces cyclooxygenase-2 (COX-2) and PG production in human and baboon endometrial stromal cells and an epithelial cell line (9, 10, 11, 12). The role of IL-1 during decidualization was recently reviewed (13).
We hypothesized that the addition of IL-1 may potentiate changes in endometrium observed after hCG infusion in the nonpregnant baboon. In this study we report endometrial changes observed in animals treated with both hCG and IL-1 and demonstrate that the addition of IL-1 to the hCG paradigm mimics changes observed in early pregnancy when an embryo is present.
Materials and Methods
Materials
Recombinant hCG was obtained from Dr. A. F. Parlow (National Hormone and Pituitary Program, Harbor-University of California-Los Angeles Medical Center, Torrance, CA), and from Serono Laboratories (Rockland, MA). Recombinant human IL-1 and IL-1 receptor antagonist (IL-1Ra) were obtained from R&D Systems, Inc. (Minneapolis, MN). Mouse monoclonal antihuman smooth muscle actin antibody (clone 1A4, catalog no. M0851) was purchased from DakoCytomation (Glastrup, Denmark). Ki67 antibody was purchased from BD Biosciences (San Jose, CA). Mouse monoclonal antibody to IGF-binding protein-1 (IGFBP-1; B2H10) was provided by Dr. Stephen C. Bell (University of Leicester, Leicester, UK). COX-1 and COX-2 antibodies were provided by Dr. S. K. Dey (Vanderbilt University Medical Center, Nashville, TN).
Tissue collection
Uterine tissue was obtained from adult female baboons (Papio anubis). All experimental procedures were approved by the animal care committee of University of Illinois (Chicago, IL).
Uteri from pregnant baboons (d 15–22 of pregnancy) were obtained by endometriectomy or hysterectomy after the stage of pregnancy was confirmed by ultrasound and by circulating levels of CG, estrogen, and progesterone, as previously described (14, 15). After visualization of the site of attachment, the uterus was divided into three regions: region I, tissue directly under the implantation site; region II, tissue immediately adjacent to the implantation site (data in this paper incorporate regions I and II as the implantation site); and region III, tissue opposite the implantation site (designated the nonimplantation site).
For simulated pregnancy studies, uterine tissue was obtained from four different groups: 1) no treatment (control animals), 2) baboons treated with hCG, 3) baboons treated with hCG and IL-1, and 4) baboons treated with hCG and IL-1Ra. Endometrial tissues (n = 3 from each group) were obtained on d 14–15 post ovulation (PO). Ovulation was detected in cycling female baboons by measuring peripheral serum levels of estradiol (16), beginning 7 d after the first day of menses (Fig. 1). The day of the estradiol surge was designated d –1; d 0 was the day of the ovulatory LH surge, and d 1 was the day of ovulation. At the time of corpus luteum rescue in the baboon (d 5 PO), the oviductal cannula was attached to an Alzet osmotic minipump (ALZA Corp., Mountain View, CA), and recombinant hCG was infused at a rate of 1.25 IU/h for 5 d, as previously described (5). On the presumed day of implantation (d 10 PO), IL-1 infusion was begun to mimic the embryonic stimulus. IL-1 (or IL-1Ra) at a dose 0.5 ng/h was administered through the pump together with hCG for an additional 5 d. The rationale for the dose used in this study is based on previous in vitro studies which reported that the human cytotrophoblast secretes approximately 1.2 ng IL-1/d (17). The concentration of IL-1 in culture fluid after 24 h of human embryo culture was estimated to be approximately 66 pg/ml in viable pregnancy cycles (18). The experiment was terminated on d 15 PO (which corresponds to d 5 after implantation in pregnant baboons). At the time of surgery, uterine flushings were obtained by perfusing the uterine lumen with 5 ml sterile Ca2+- and Mg2+-free Hanks’ buffered salt solution before harvesting endometrial tissue. Perfusion of the uterine lumen was accomplished by inserting two 10-ml syringes attached to 21-gauge needles into the uterine cavity. The cervix and fallopian tubes were manually compressed, and infusion of fluid from the syringe inserted at the upper fundus of the uterus was collected in the empty syringe inserted into the midportion of the uterine lumen. Care was taken to ensure that the uterine flushings were not contaminated with blood.
Histology and immunohistochemistry
Uterine tissues were immersion-fixed in Bouin’s solution or 10% buffered formalin for 24 h at room temperature, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin (19). For histological analyses, tissue sections (5 μm) were stained with Gomori’s Trichrome stain. For immunohistochemistry, a monoclonal antibody to -smooth muscle actin (-SMA; 1:250; DakoCytomation) (20), IGFBP-1 (1:2000) (19), Ki67 (1:250; BD Biosciences, San Jose, CA), and rabbit polyclonal antibodies to COX-1 (1:750) and COX-2 (1:50) were used. The immunolocalization of proteins was performed in fixed, paraffin-embedded sections using the Vectastain Elite ABC Detection Kit (Vector Laboratories, Inc., Burlingame, CA) with 3,3'-diaminobenzidine as the chromagen. Negative controls consisted of preimmune serum at the same concentration as the immune serum. The stained sections were evaluated on a Nikon microscope ECLIPSE E400 (Nikon Corp., Tokyo, Japan). Images were captured using a digital Spot Camera and the Image-Pro Plus software package (Media Cybernetics, San Diego, CA).
RT-PCR detection of IL-1
RNA from the implantation site of a pregnant (d 18 of pregnancy) baboon and from cytotrophoblasts isolated from term placenta (using the methods described in Refs. 21 and 22 , with cell purity as described in Ref. 23) was extracted using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) according to the protocol provided by the manufacturer. RT was performed in a final volume of 20 μl with 1 μg total cellular RNA and 50 U Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Inc., Grand Island, NY). Primers specific for IL-1 (5'-TGC TGC AGA GGC AGG GAG CCC-3' and 3'-AGG GAT CCT CTT CCC ATT CCA-5') were designed to span through the fourth intron in the human IL-1 gene (data from National Center for Biotechnology Information nucleotide database: gene accession no. M15840 and mRNA sequence NM_000576). After 32 amplification cycles, the PCR products were analyzed by electrophoresis on 2.5% agarose gels. The PCR product for IL-1 mRNA (246 bp) was verified by DNA sequencing and was found to be identical with the GenBank sequence for IL-1. The baboon sample also contained another nonspecific PCR product (205 bp) not related to the IL-1 mRNA. Parallel semiquantitative analysis of samples was also conducted to detect both IGFBP-1 mRNA and the internal standard H3.3 (24).
Detection of prolactin and IGFBP-1 in uterine flushings
Prolactin and IGFBP-1 in uterine flushings were measured using ELISA kits (Diagnostic Systems Laboratories, Inc., Webster, TX) according to the manufacturer’s protocol. The detection limit for prolactin is 0.14 ng/ml, and the assay does not cross-react with LH, FSH, GH, TSH, ACTH, hCG, or -hCG. The intraassay coefficient of variation is 7.9–8.2%, and the interassay coefficient of variation is 9.4–10.4%. For IGFBP-1, the minimum detection limit is 0.25 ng/ml, and the assay is highly specific for this molecule. The intraassay coefficient of variation is 1.7–4.6%, and interassay coefficient of variation is 6.2–7.6%.
Cytokine array in uterine flushings
The protein concentration in uterine flushes was estimated by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Cytokines in uterine flushings (5 μg protein) were analyzed with TranSignal Human Cytokine Antibody Array 3.0 membranes (catalogue no. MA6160, Panomics, Inc., Redwood City, CA). The array allows for simultaneous detection of 36 cytokines and provides positive and negative controls. Briefly, the sample was incubated for 1.5 h with membrane-immobilized capture antibodies specific to a particular cytokine protein. Unbound proteins were washed away. The second biotin-conjugated antibody was allowed to bind for 1.5 h to a second epitope on the protein. The 1-h incubation with streptavidin-horseradish peroxidase allowed visualization of proteins after detection of chemiluminescent signal on Kodak film (Eastman Kodak, Rochester, NY). The bands were scanned and quantified by densitometry on a densitometer (version 4.0, Molecular Dynamics, Inc., Sunnyvale, CA). The surrounding background and negative controls were subtracted. Data were normalized to the average of positive controls on the individual membranes.
Statistical analyses
All statistical analyses were performed using SPSS 12.0 (SPSS, Inc., Chicago, IL). The means of the cytokine array data were analyzed by one-way ANOVA at a significance level of P < 0.05. When the overall ANOVA was significant, post hoc tests were performed using Tukey’s correction for multiple comparisons.
Results
Presence of IL-1 at the implantation site in pregnant baboon
The expression of IL-1 at the implantation site was confirmed by mRNA expression on d 18 of pregnancy (Fig. 2A). The mRNA for IL-1 was also detected in human cytotrophoblasts, suggesting that the conceptus is a possible source of IL-1 at the implantation site. Parallel semiquantitative RT-PCR analysis of samples confirmed the expected presence of IGFBP-1 mRNA (a marker of decidualization) at the implantation site, but not in the cytotrophoblasts (Fig. 2B).
Immunolocalization of -SMA, COX-2, and IGFBP-1 in early pregnant baboon
At the implantation site of the baboon, the decidualization process in vivo is characterized by sequential changes early in pregnancy. By d 15 of pregnancy, -SMA begins to be down-regulated at the implantation site (Fig. 3A). Coincident with this decrease is an increase in COX-2 protein in stromal cells directly below the implantation site (25) (illustrated in Fig. 3B); subsequently, IGFBP-1 appears in stromal fibroblasts (Fig. 3C) undergoing decidualization in close proximity to the invading cytotrophoblasts.
Morphology and immunolocalization of -SMA, Ki67, and IGFBP-1 in animals treated with hCG in the presence of IL-1
In intact baboons, the infusion of hCG at the time of implantation results in morphological changes distinct from those seen at a comparable time during a normal menstrual cycle (5). In this study we investigated the effects of IL-1 infusion into hCG-treated animals.
The tissue sections isolated from the four treatment groups [no treatment (group 1), hCG-treated (group 2), hCG and IL-1 treated (group 3), and hCG and IL-1Ra treated (group 4)] were first histologically evaluated after staining with Gomori’s Trichrome stain (Fig. 4, A–D). In control (Fig. 4A) and hCG- and IL-1Ra-treated animals (Fig. 4D), there were no differences in the appearance of the luminal epithelium and the underlying stromal cells. The hCG and hCG plus IL-1 treatments resulted in hypertrophy of the surface epithelium and the formation of distinct epithelial plaques in the luminal epithelium (Fig. 4, B and C). In tissues from hCG-treated and hCG- plus IL-1Ra-treated baboons, blue staining representing collagen deposits was evident (Fig. 4, B and D). These deposits were only faintly seen in hCG- and IL-1-treated animals (Fig. 4C).
Epithelial plaques did not stain for the cytoskeletal protein -SMA (Fig. 4, F and G). Cytokeratin staining confirmed the epithelial origin of the plaque cells (data not shown). Staining for the proliferation marker Ki67 demonstrated that the epithelial plaques together with the stromal fibroblasts below them were actively proliferating in response to hCG and IL-1 stimulation (Fig. 4K). In hCG-treated animals, the most active proliferation was observed in the epithelial plaques (Fig. 4J). In control and hCG- plus IL-1Ra-treated groups, there were only a few randomly scattered Ki67-positive cells (Fig. 4, I and L). The stromal fibroblasts directly below the epithelial plaques also stained positively for -SMA in animals treated with hCG and IL-1 (Fig. 4G). Staining for -SMA was absent in the subepithelial stromal cells directly below the uterine lumen in the hCG- and IL-1-treated animals (Fig. 4G). The studies in the simulated pregnant baboon model (16) suggested that hCG together with estrogen and progesterone are insufficient to induce IGFBP-1 expression in decidualized stromal cells. However, glandular epithelial synthesis of IGFBP-1 is maintained, perhaps as a direct effect of hCG on endometrial secretory activity (16). In the animals treated with hCG in the presence of IL-1, the stromal cells close to the apical surface stained intensively for IGFBP-1 (Fig. 4O), and staining was also detected in stromal cells underneath the epithelial plaques (Fig. 4O). In addition, the cells close to the apical surface did not stain for either Ki67 (Fig. 4K) or -SMA (Fig. 4G), which is identical with our previous observations at the implantation site in early pregnancy (as illustrated in Fig. 3A).
Localization of COX-1 and COX-2 protein in the endometrium of baboons treated with hCG and IL-1
COX is the rate-limiting enzyme in the conversion of arachidonic acid to PGs. Two COX isozymes have been identified, COX-1 and COX-2. In control animals on d 14 PO (Fig. 5A) and in animals treated with hCG and IL-1Ra (Fig. 5D), intense COX-1 staining was observed on luminal epithelium and in the epithelial glands in controls (Fig. 5, A, E, and I). Similar to our previous report for COX-1 mRNA expression in early pregnant endometrial tissues (d 17–19 of pregnancy) (25), the infusion of hCG (Fig. 5B) and hCG plus IL-1 (Fig. 5C) resulted in the loss of COX-1 in epithelial cells at the apical surface. In the deeper epithelial glands of hCG- and IL-1-treated animals (Fig. 5, G and K), the presence of COX-1 protein was significantly decreased compared with that after hCG treatment (Fig. 5, F and J).
COX-2 expression was different from COX-1 expression. In control animals on d 14 PO, weak positive staining was observed in luminal epithelium, whereas glandular epithelium remained negative. We expected to observe staining in stromal cells undergoing decidualization, but there was no significant positive staining evident in animals treated with hCG and IL-1 on d 15 PO (data not shown), in contrast to what was observed in pregnant baboons when a conceptus was present (Fig. 3B) (25).
Immunolocalization of COX-1 in endometrium of early pregnant baboons
There were two distinctly different patterns in endometrium of pregnant baboons (d 22 of pregnancy): the area close to the implantation site (Fig. 6, B and D) and the area opposite the implantation site (nonimplantation site; Fig. 6, A and C).
The luminal epithelium of the endometrium from the nonimplantation site stained for COX-1 (Fig. 6A), similar to that seen with the nonpregnant control (Fig. 5A) and hCG- and IL-1Ra-treated animals (Fig. 5D). The implantation site had a remodeled apical area that stained weakly for COX-1 (Fig. 6B). The weaker staining for COX-1 in the epithelial glands in the midfunctionalis region in the endometrium of pregnant animals (Fig. 6, C and D) is similar to that observed in the CG- and IL-1-treated animals (Fig. 5, G and K).
Prolactin and IGFBP-1 detection in uterine flushings
In humans, IGFBP-1 begins to be secreted by stromal cells surrounding the spiral arteries during the late secretory phase of the menstrual cycle, whereas in baboons, IGFBP-1 is secreted from the glands in response to progesterone (19, 26). The production of decidual prolactin begins by d 22 of the menstrual cycle in humans and continues to increase throughout gestation (27). In baboons with the onset of pregnancy, prolactin expression increases steadily and is evident primarily in the decidual tissue (28).
Because we observed both morphological and immunohistological changes in hCG- and IL-1-treated animals consistent with the decidualization process in stromal cells (Fig. 4O), we assayed uterine flushings from all four treatment groups for prolactin and IGFBP-1. Because of the limited number of flushings (n = 3/experimental group), the interanimal variability was high, and no statistically significant differences between the experimental groups were evident. However, there was a trend toward an increase in the mean values of both prolactin and IGFBP-1 in hCG-treated (13.9 ± 14.6 ng/mg protein or 10.8 ± 8.0 ng/μg protein, respectively) and of prolactin and IGFBP-1 in hCG- plus IL-1-treated (16.5 ± 12.2 ng/mg protein or 9.2 ± 4.4 ng/μg protein, respectively) animals. This is compared with prolactin and IGFBP-1 in controls (1.6 ± 2.7 ng/mg protein or 6.3 ± 5.0 ng/μg protein, respectively) and with prolactin and IGFBP-1 in hCG- plus IL-1Ra-treated animals (10.6 ± 4.1 ng/mg protein or 5.0 ± 3.9 ng/μg protein, respectively). Based on previous studies (19, 26, 28), the increased levels in the hCG-treated and hCG- plus IL-1-treated groups probably reflect a contribution primarily from the glandular epithelium and, to a lesser extent, from the stromal cells.
Analysis of cytokines in uterine flushings
There is evidence that cytokines produced by the maternal endometrium and the developing embryo play a crucial role during implantation (see review in Ref. 29). Cytokines are often considered in the context of their T helper type 1 (Th1)/Th2 classification (see review in Ref. 30).
The uterine flushings were analyzed for the presence of cytokines by a protein array. Of the 36 cytokines spotted on the array, the majority were detectable in uterine flushings. Cytokine values showed a high interanimal variation within the same treatment group. Overall, however, there was a trend toward a decrease in cytokine levels in treated animals compared with controls (no treatment).
In uterine flushings of treated animals, we observed a decrease in Th1 cytokines, IL-2 (Fig. 7A), TNF- (Fig. 7B), and interferon- (Fig. 7C), compared with controls. IL-12 exists as two isoforms (p40 and p70), and only the heterodimer is bioactive. This array only measured the IL-12 (p40) form, and levels were similar in all four treatment groups (Fig. 7A). Therefore, if there is indeed a down-regulation of IL-12, as is predicted to be favorable for pregnancy (30), it could be associated with regulation of the IL-12 (p70) form.
In contrast with the classical Th1/Th2 concept, there was no increase in Th2 cytokine levels in treated animals compared with controls. However, there was a significant decrease in levels of IL-4 (Fig. 7A). The assay also detected the Th2 cytokines IL-3 (Fig. 7C), IL-5, IL-6, IL-6 receptor, and IL-10 (data not shown), but overall they were present at low concentrations.
We detected a borderline decrease in Fas (Fig. 7B) in uterine flushings from animals treated with hCG (P = 0.093) and a significant decrease in Fas in the presence of hCG and IL-1 or IL-1Ra (P = 0.025) compared with the controls. In all treated animals, there was less TNF- and TNF receptor-1 and -2 (TNFR-1 and TNFR-2; Fig. 7A) detectable, although only the decrease in TNFR-1 was statistically significant (P < 0.05).
The other molecules active at the maternal-fetal interface, such as growth factors (vascular endothelial growth factor, epidermal growth factor, granulocyte-macrophage colony-stimulating factor, and TGF-), leptin, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein-1, and macrophage inflammatory protein-4, were detected in uterine flushings, but the differences between control and treated animals did not approach statistical significance (data not shown). Similarly, no statistical differences were observed for interferon--inducible protein-10 (CXCL10) and eotaxin (data not shown). In contrast, matrix metalloproteinase-3 (Fig. 7A) and cytotoxic T lymphocyte-4 (CTLA-4; Fig. 7C) were significantly decreased in treated animals compared with controls.
Discussion
Successful embryo implantation requires precise synchrony between the blastocyst and the uterine environment, where a complex series of interactions occurs. The aims of this study were to investigate the possible in vivo relationship between two embryonic factors, hCG and IL-1, in preparing the endometrium for the establishment of pregnancy. hCG, which is required to maintain pregnancy in the primate, exerts its effects through LH/hCG receptors by stimulating progesterone secretion from the corpus luteum during the first trimester. hCG signals to the endometrium and modulates the uterine environment before implantation (5). Other conceptus-mediated factors are required for the differentiation of stromal cells to the decidual phenotype, because decidualization occurs at the implantation site of baboons (31), but not in hCG-induced pseudopregnancy (16). Our in vitro studies have also previously demonstrated that soluble molecules from cytotrophoblasts were responsible for the induction of decidualization in baboon stromal fibroblasts in a coculture model (23). In the present study we confirmed that IL-1 mRNA is present in cytotrophoblasts and at the implantation site of the baboon. The immunohistochemical evidence confirms that IL-1 enhances the previously reported cellular transformation of the endometrium induced by hCG (5), and the changes are similar to those observed in early pregnancy.
Our data suggest two distinct patterns associated with the IL-1 effect. First, the presence of IL-1 specifically contributes to decidualization (as evidenced by IGFBP-1 expression) and COX-1 protein regulation, which are functional changes associated with the presence of the conceptus during early pregnancy (25, 31). The effect of IL-1 on in vivo decidualization is in agreement with our previous in vitro studies in both baboon and human stromal fibroblasts (11). Based on our previous studies (11, 25, 32), we would also propose that a synergism among hCG, IL-1, and progesterone is critical for the changes to be induced. In addition, IL-1 decreases COX-1 protein in epithelial cells of baboons, which is comparable to observations during early pregnancy. Secondly, IL-1 in our model does not influence changes in cytokine levels detected in uterine flushings; hCG alone is capable of regulating their secretion.
Over the past decade, the importance of COX-1 and -2 in reproduction has been well documented (see review in Ref. 33). Up-regulation of COX-2 gene expression in response to hCG has been demonstrated in both human endometrial stromal and epithelial cells (34, 35). We and others have demonstrated that IL-1 can elevate both PGE2 and PGF2 levels by increasing COX-2 in human and baboon stromal cells in vitro (9, 10, 11, 12), and that the inhibition of COX-2 causes a partial decrease in the in vitro decidualization response (11). During early pregnancy in the baboon, COX-2 was strongly expressed in the stromal cells at the site of implantation (25). In contrast, our current study did not detect an up-regulation of COX-2 in baboon endometrium in animals treated with hCG and IL-1 on d 15 PO. We conclude that hCG and IL-1, when infused in vivo, were not sufficient to induce COX-2 expression, and that additional factors from the conceptus may be necessary for its induction at the implantation site during pregnancy.
COX-1 is constitutively expressed in most cell types and has been considered to be a housekeeping gene, although our studies show that its expression is hormonally regulated in baboon uterus (25). COX-1 mRNA expression in epithelial cells of the endometrium was up-regulated during the secretory phase and was down-regulated with the establishment of pregnancy (25). In this study, COX-1 protein was localized at the luminal epithelium and in epithelial glands of control animals on d 14 PO and was markedly decreased in the epithelial cells of hCG- and IL-1-treated animals, similar to what was observed in early pregnancy. Similarly, the expression of COX-1 and COX-2 protein was reported to be decreased in early pregnancy in the rat uterus (36). In mice, it was recently demonstrated that a compensatory mechanism via COX-1 up-regulation substantially rescues COX-2-deficient female infertility in a genetic background-dependent manner (37).
In the rat, the biosynthesis and catabolism of PGF2 are regulated by progesterone (36). The absence of COX-1 expression in antiprogestin-treated animals strongly implicates a role for progesterone in COX-1 regulation (25), but the absence of COX-1 down-regulation in hCG-treated animals also suggests a role for IL-1 in COX-1 regulation. Although the role of PGs during implantation is recognized, nothing is known about the regulation of COX-1 during the establishment of pregnancy. In synovial cells, IL-1-induced COX-1 and COX-2 up-regulation was inhibited by the glucocorticoid receptor inhibitor dexamethasone (38). In glial cells, regulation of COX-1 expression was associated with histone acetylation (39), because the histone deacetylase inhibitors stimulated COX-1 promoter activity. Modification of chromatin structure is part of the response to IL-1 in myometrial cells (40). IL-1 treatment increases polymerase II cross-linking to COX-2 promoter and increases histone H4 acetylation at specific sites (40). Additional studies are required to address the regulatory mechanism of COX-1 regulation during early pregnancy and the possible involvement of IL-1 in this process.
IL-1 is a family of polypeptides comprised of two agonists, IL-1 and IL-1, and a naturally present inhibitor, IL-1Ra. One surprising observation in our study was the ability of hCG and IL-1Ra to suppress the effects of hCG on the morphological transformation of endometrium (such as the formation of epithelial plaques). In mice, ip injection of IL-1Ra prevents implantation (41). It has been suggested that this is due to the down-regulation of integrins 4, V, and 3 on epithelial cells (42, 43). There is very little evidence relating to the interactions between IL-1Ra and hCG described in the literature to date. We can only hypothesize that additional IL-1Ra binding to IL-1RI and prevention of IL-1 binding and receptor activation in the endometrium at the local level could interfere with specific hCG-induced responses. The balance between IL-1 and IL-1Ra in tissues plays an important role in the susceptibility to and the severity of many diseases (44). The importance of IL-1 regulation during the establishment of human pregnancy has also been suggested based on the observation that the decreased expression of IL-1 and IL-6 in the midsecretory phase is associated with habitual abortion (45).
Classical Th1/Th2 dogma suggests a decrease in Th1 (IL-2, interferon-, TNF-, and IL-12) cytokines and an increase in Th2 (IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13) cytokines during pregnancy. In terms of the Th1/Th2 paradigm, the expected decline in Th1 cytokines was detected in uterine flushings of treated animals compared with controls. However, the expected increase in Th2 cytokines was not evident. Chaouat et al. (30) reviewed in detail the limits of the Th1/Th2 paradigm and concluded that the Th1/Th2 paradigm in pregnancy appears to be obsolete and an oversimplification. Rather, step-specific events have to be considered, and a key role is seen in local tissue remodeling, in which immune cytokines may play an important role. It is also possible that the placenta and/or endometrium are capable of regulating the cytokine milieu at the site of implantation without significantly altering the Th1/Th2 paradigm due to the large number of cytokines at the maternal-fetal interface.
The sloughing off of the endometrium at the start of menstruation occurs by programmed cell death (apoptosis). In a recent study, a bolus injection of hCG to nonpregnant woman decreased apoptosis in the endometrium on d 26 of the menstrual cycle compared with control tissues obtained at the same time in the menstrual cycle (4). The results suggested that both exogenous progesterone and hCG can influence the human endometrium during the late secretory phase and can prevent or delay the start of endometrial apoptosis (4). It is of interest in the current study that treatment with hCG and IL-1 resulted in a significant suppression of Fas and TNFR-1, which are the best-characterized death receptors (see review in Ref. 46). The major function of the receptor Fas (Apo-1, CD95) and Fas ligand interaction is the induction of apoptosis in cells carrying Fas. TNFR-1 activation initiates downstream events leading to apoptosis through caspase-8 or activation of nuclear factor-B transcription factor and activation of c-Jun NH2 kinase (47). The increased presence of Fas and TNFR-1 in uterine flushes of control animals confirms the premenstrual start of endometrial apoptosis and the shedding of Fas and TNFR-1 from the surface of uterine cells. Similarly, soluble Fas receptor was increased in the plasma of patients with congestive heart failure during presumed activation of myocardial apoptotic cell death (48). Also, TNFR-1 is shed into the serum during liver damage in chronic hepatitis patients (49).
Down-regulation of TNFR-1, IL-4, matrix metalloproteinase-3, and CTLA-4 in uterine flushes of treated animals seems to be primarily regulated by hCG, because the addition of IL-1 or its antagonist, IL-1Ra, did not result in a different outcome. It has been hypothesized that hCG may be the placental link for the development of local immune tolerance during and after implantation by induction of T cell apoptosis (50).
CTLA-4 is a cell surface glycoprotein cosignaling molecule that may modulate peripheral self-tolerance at the maternal-fetal interface (51). Cosignaling molecules are controllers of T cell responses to antigens. By selectively turning on and/or off costimulatory pathways, the T cell receptor signal could be transiently regulated in positive and negative directions. Therefore, a precise balance between these pathways often determines the outcome of the T cell response (see review in Ref. 52). It has been suggested that abnormal expression of CTLA-4 in fetal tissues at the maternal-fetal interface may be a potentially common etiological factor in human miscarriage (51). A CD4+CD25+ regulatory T cell population isolated from the human decidua, which expresses intracellular CTLA-4, has been reported (53). It has been proposed that these cells are most likely to be important in the regulation of local maternal tolerance toward the fetus. Therefore, it is possible that the CTLA-4 decrease detected in uterine flushings of treated animals may contribute to the alteration of cytokine profiles and the modulation of T cell activation in the uterus during the early implantation period.
However, we are aware that our simulated pregnancy model does not completely reflect the complex interactions that occur between the implanting embryo and the maternal endometrium (54), but what is relevant with our model is that the function of specific embryonic molecules could be selectively delineated. Furthermore, it is evident from our studies that the interaction between hCG and IL-1 does recapitulate many of the changes observed in early pregnancy and provides evidence for some of the mechanisms by which the endometrium modulates both the inhibition of apoptosis and the immune environment that are associated with the establishment of pregnancy.
Acknowledgments
We thank Laura Studee, M.P.H., for help with statistical analysis.
Footnotes
This work was supported by National Institutes of Health Grants HD-36759 and HD-42280 (to A.T.F.) and HD-44713 (to Z.S.).
Abbreviations: COX, Cyclooxygenase; CTLA-4, cytotoxic T lymphocyte antigen-4; hCG, human chorionic gonadotropin; IGFBP-1, IGF-binding protein-1; IL-1Ra, IL-1 receptor antagonist; PG, prostaglandin; PO, post ovulation; -SMA, -smooth muscle actin; Th1, T helper type 1; TNFR-1, TNF receptor type 1.
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Abstract
Both human chorionic gonadotropin (hCG) and IL-1 induce changes in the endometrium that are associated with the establishment of pregnancy. We investigated the synergistic effect of these two embryonic signals on endometrial function using a baboon model of simulated pregnancy. Recombinant hCG (30 IU/d) was infused between d 6 and 10 post ovulation (PO) to mimic blastocyst transit. On the expected day of implantation (d 10 PO), IL-1 (12 ng/d) or IL-1 receptor antagonist (IL-1Ra; 12 ng/d) was infused for an additional 5 d. Endometria were harvested on d 15 PO. Both hCG and hCG plus IL-1 induced marked differences in the distribution of -smooth muscle actin, proliferation marker Ki67, decidualization marker IGF-binding protein-1, and cyclooxygenase-1. The most marked effect of IL-1 was the induction of IGF-binding protein-1 protein in stromal cells close to the apical surface, whereas cyclooxygenase-1 was down-regulated in the glandular epithelium. Protein arrays of uterine flushings showed significant suppression of death receptors, Fas and TNF receptor 1, in the hCG- with or without IL-1-treated groups, suggesting an inhibition of apoptosis. Additionally, cytotoxic T lymphocyte antigen-4, matrix metalloproteinase-3, and IL-4 were suppressed in treated animals compared with controls. However, no differences were observed in cytokine profile between hCG-treated and hCG- plus IL-1-treated baboons. This study confirms that in preparation for pregnancy, the primate endometrium undergoes both morphological and functional changes, which are modulated by hCG and IL-1, that lead to the inhibition of apoptosis and the development of an immunotolerant environment. These changes suggest that infusion of IL-1 at the time of implantation into the nonpregnant baboon treated with hCG synergizes with hCG and mimics the early endometrial events associated with the presence of an embryo.
Introduction
THE INTERACTION BETWEEN maternal endometrium and embryo during implantation is a complex process, the details of which are still not fully understood. Implantation involves inflammatory responses in the human endometrium (for a review, see Ref. 1). Inflammatory mediators, such as prostaglandins (PGs), chemokines, and cytokines, have been associated with these events (2). Although the precise mechanisms regulating implantation are still unknown, increasing evidence suggests an interaction between these mediators.
Human chorionic gonadotropin (hCG) is one of the major hormonal products of first trimester human placental trophoblast cells. Besides its well-known luteotropic function, it appears to play an important role in the preparation of the endometrium for blastocyst implantation (for a review, see Ref. 3). Injection of hCG reduces apoptosis in human endometrium (4). Infusion of hCG into nonpregnant baboons functionally alters the major cell types present in baboon uterine endometrium (5, 6).
IL-1 is a proinflammatory cytokine expressed in the endometrium throughout the menstrual cycle, particularly around the time of implantation (7). It is postulated to mediate the dialogue between the blastocyst and the endometrium during implantation (8). IL-1 induces cyclooxygenase-2 (COX-2) and PG production in human and baboon endometrial stromal cells and an epithelial cell line (9, 10, 11, 12). The role of IL-1 during decidualization was recently reviewed (13).
We hypothesized that the addition of IL-1 may potentiate changes in endometrium observed after hCG infusion in the nonpregnant baboon. In this study we report endometrial changes observed in animals treated with both hCG and IL-1 and demonstrate that the addition of IL-1 to the hCG paradigm mimics changes observed in early pregnancy when an embryo is present.
Materials and Methods
Materials
Recombinant hCG was obtained from Dr. A. F. Parlow (National Hormone and Pituitary Program, Harbor-University of California-Los Angeles Medical Center, Torrance, CA), and from Serono Laboratories (Rockland, MA). Recombinant human IL-1 and IL-1 receptor antagonist (IL-1Ra) were obtained from R&D Systems, Inc. (Minneapolis, MN). Mouse monoclonal antihuman smooth muscle actin antibody (clone 1A4, catalog no. M0851) was purchased from DakoCytomation (Glastrup, Denmark). Ki67 antibody was purchased from BD Biosciences (San Jose, CA). Mouse monoclonal antibody to IGF-binding protein-1 (IGFBP-1; B2H10) was provided by Dr. Stephen C. Bell (University of Leicester, Leicester, UK). COX-1 and COX-2 antibodies were provided by Dr. S. K. Dey (Vanderbilt University Medical Center, Nashville, TN).
Tissue collection
Uterine tissue was obtained from adult female baboons (Papio anubis). All experimental procedures were approved by the animal care committee of University of Illinois (Chicago, IL).
Uteri from pregnant baboons (d 15–22 of pregnancy) were obtained by endometriectomy or hysterectomy after the stage of pregnancy was confirmed by ultrasound and by circulating levels of CG, estrogen, and progesterone, as previously described (14, 15). After visualization of the site of attachment, the uterus was divided into three regions: region I, tissue directly under the implantation site; region II, tissue immediately adjacent to the implantation site (data in this paper incorporate regions I and II as the implantation site); and region III, tissue opposite the implantation site (designated the nonimplantation site).
For simulated pregnancy studies, uterine tissue was obtained from four different groups: 1) no treatment (control animals), 2) baboons treated with hCG, 3) baboons treated with hCG and IL-1, and 4) baboons treated with hCG and IL-1Ra. Endometrial tissues (n = 3 from each group) were obtained on d 14–15 post ovulation (PO). Ovulation was detected in cycling female baboons by measuring peripheral serum levels of estradiol (16), beginning 7 d after the first day of menses (Fig. 1). The day of the estradiol surge was designated d –1; d 0 was the day of the ovulatory LH surge, and d 1 was the day of ovulation. At the time of corpus luteum rescue in the baboon (d 5 PO), the oviductal cannula was attached to an Alzet osmotic minipump (ALZA Corp., Mountain View, CA), and recombinant hCG was infused at a rate of 1.25 IU/h for 5 d, as previously described (5). On the presumed day of implantation (d 10 PO), IL-1 infusion was begun to mimic the embryonic stimulus. IL-1 (or IL-1Ra) at a dose 0.5 ng/h was administered through the pump together with hCG for an additional 5 d. The rationale for the dose used in this study is based on previous in vitro studies which reported that the human cytotrophoblast secretes approximately 1.2 ng IL-1/d (17). The concentration of IL-1 in culture fluid after 24 h of human embryo culture was estimated to be approximately 66 pg/ml in viable pregnancy cycles (18). The experiment was terminated on d 15 PO (which corresponds to d 5 after implantation in pregnant baboons). At the time of surgery, uterine flushings were obtained by perfusing the uterine lumen with 5 ml sterile Ca2+- and Mg2+-free Hanks’ buffered salt solution before harvesting endometrial tissue. Perfusion of the uterine lumen was accomplished by inserting two 10-ml syringes attached to 21-gauge needles into the uterine cavity. The cervix and fallopian tubes were manually compressed, and infusion of fluid from the syringe inserted at the upper fundus of the uterus was collected in the empty syringe inserted into the midportion of the uterine lumen. Care was taken to ensure that the uterine flushings were not contaminated with blood.
Histology and immunohistochemistry
Uterine tissues were immersion-fixed in Bouin’s solution or 10% buffered formalin for 24 h at room temperature, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin (19). For histological analyses, tissue sections (5 μm) were stained with Gomori’s Trichrome stain. For immunohistochemistry, a monoclonal antibody to -smooth muscle actin (-SMA; 1:250; DakoCytomation) (20), IGFBP-1 (1:2000) (19), Ki67 (1:250; BD Biosciences, San Jose, CA), and rabbit polyclonal antibodies to COX-1 (1:750) and COX-2 (1:50) were used. The immunolocalization of proteins was performed in fixed, paraffin-embedded sections using the Vectastain Elite ABC Detection Kit (Vector Laboratories, Inc., Burlingame, CA) with 3,3'-diaminobenzidine as the chromagen. Negative controls consisted of preimmune serum at the same concentration as the immune serum. The stained sections were evaluated on a Nikon microscope ECLIPSE E400 (Nikon Corp., Tokyo, Japan). Images were captured using a digital Spot Camera and the Image-Pro Plus software package (Media Cybernetics, San Diego, CA).
RT-PCR detection of IL-1
RNA from the implantation site of a pregnant (d 18 of pregnancy) baboon and from cytotrophoblasts isolated from term placenta (using the methods described in Refs. 21 and 22 , with cell purity as described in Ref. 23) was extracted using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) according to the protocol provided by the manufacturer. RT was performed in a final volume of 20 μl with 1 μg total cellular RNA and 50 U Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Inc., Grand Island, NY). Primers specific for IL-1 (5'-TGC TGC AGA GGC AGG GAG CCC-3' and 3'-AGG GAT CCT CTT CCC ATT CCA-5') were designed to span through the fourth intron in the human IL-1 gene (data from National Center for Biotechnology Information nucleotide database: gene accession no. M15840 and mRNA sequence NM_000576). After 32 amplification cycles, the PCR products were analyzed by electrophoresis on 2.5% agarose gels. The PCR product for IL-1 mRNA (246 bp) was verified by DNA sequencing and was found to be identical with the GenBank sequence for IL-1. The baboon sample also contained another nonspecific PCR product (205 bp) not related to the IL-1 mRNA. Parallel semiquantitative analysis of samples was also conducted to detect both IGFBP-1 mRNA and the internal standard H3.3 (24).
Detection of prolactin and IGFBP-1 in uterine flushings
Prolactin and IGFBP-1 in uterine flushings were measured using ELISA kits (Diagnostic Systems Laboratories, Inc., Webster, TX) according to the manufacturer’s protocol. The detection limit for prolactin is 0.14 ng/ml, and the assay does not cross-react with LH, FSH, GH, TSH, ACTH, hCG, or -hCG. The intraassay coefficient of variation is 7.9–8.2%, and the interassay coefficient of variation is 9.4–10.4%. For IGFBP-1, the minimum detection limit is 0.25 ng/ml, and the assay is highly specific for this molecule. The intraassay coefficient of variation is 1.7–4.6%, and interassay coefficient of variation is 6.2–7.6%.
Cytokine array in uterine flushings
The protein concentration in uterine flushes was estimated by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Cytokines in uterine flushings (5 μg protein) were analyzed with TranSignal Human Cytokine Antibody Array 3.0 membranes (catalogue no. MA6160, Panomics, Inc., Redwood City, CA). The array allows for simultaneous detection of 36 cytokines and provides positive and negative controls. Briefly, the sample was incubated for 1.5 h with membrane-immobilized capture antibodies specific to a particular cytokine protein. Unbound proteins were washed away. The second biotin-conjugated antibody was allowed to bind for 1.5 h to a second epitope on the protein. The 1-h incubation with streptavidin-horseradish peroxidase allowed visualization of proteins after detection of chemiluminescent signal on Kodak film (Eastman Kodak, Rochester, NY). The bands were scanned and quantified by densitometry on a densitometer (version 4.0, Molecular Dynamics, Inc., Sunnyvale, CA). The surrounding background and negative controls were subtracted. Data were normalized to the average of positive controls on the individual membranes.
Statistical analyses
All statistical analyses were performed using SPSS 12.0 (SPSS, Inc., Chicago, IL). The means of the cytokine array data were analyzed by one-way ANOVA at a significance level of P < 0.05. When the overall ANOVA was significant, post hoc tests were performed using Tukey’s correction for multiple comparisons.
Results
Presence of IL-1 at the implantation site in pregnant baboon
The expression of IL-1 at the implantation site was confirmed by mRNA expression on d 18 of pregnancy (Fig. 2A). The mRNA for IL-1 was also detected in human cytotrophoblasts, suggesting that the conceptus is a possible source of IL-1 at the implantation site. Parallel semiquantitative RT-PCR analysis of samples confirmed the expected presence of IGFBP-1 mRNA (a marker of decidualization) at the implantation site, but not in the cytotrophoblasts (Fig. 2B).
Immunolocalization of -SMA, COX-2, and IGFBP-1 in early pregnant baboon
At the implantation site of the baboon, the decidualization process in vivo is characterized by sequential changes early in pregnancy. By d 15 of pregnancy, -SMA begins to be down-regulated at the implantation site (Fig. 3A). Coincident with this decrease is an increase in COX-2 protein in stromal cells directly below the implantation site (25) (illustrated in Fig. 3B); subsequently, IGFBP-1 appears in stromal fibroblasts (Fig. 3C) undergoing decidualization in close proximity to the invading cytotrophoblasts.
Morphology and immunolocalization of -SMA, Ki67, and IGFBP-1 in animals treated with hCG in the presence of IL-1
In intact baboons, the infusion of hCG at the time of implantation results in morphological changes distinct from those seen at a comparable time during a normal menstrual cycle (5). In this study we investigated the effects of IL-1 infusion into hCG-treated animals.
The tissue sections isolated from the four treatment groups [no treatment (group 1), hCG-treated (group 2), hCG and IL-1 treated (group 3), and hCG and IL-1Ra treated (group 4)] were first histologically evaluated after staining with Gomori’s Trichrome stain (Fig. 4, A–D). In control (Fig. 4A) and hCG- and IL-1Ra-treated animals (Fig. 4D), there were no differences in the appearance of the luminal epithelium and the underlying stromal cells. The hCG and hCG plus IL-1 treatments resulted in hypertrophy of the surface epithelium and the formation of distinct epithelial plaques in the luminal epithelium (Fig. 4, B and C). In tissues from hCG-treated and hCG- plus IL-1Ra-treated baboons, blue staining representing collagen deposits was evident (Fig. 4, B and D). These deposits were only faintly seen in hCG- and IL-1-treated animals (Fig. 4C).
Epithelial plaques did not stain for the cytoskeletal protein -SMA (Fig. 4, F and G). Cytokeratin staining confirmed the epithelial origin of the plaque cells (data not shown). Staining for the proliferation marker Ki67 demonstrated that the epithelial plaques together with the stromal fibroblasts below them were actively proliferating in response to hCG and IL-1 stimulation (Fig. 4K). In hCG-treated animals, the most active proliferation was observed in the epithelial plaques (Fig. 4J). In control and hCG- plus IL-1Ra-treated groups, there were only a few randomly scattered Ki67-positive cells (Fig. 4, I and L). The stromal fibroblasts directly below the epithelial plaques also stained positively for -SMA in animals treated with hCG and IL-1 (Fig. 4G). Staining for -SMA was absent in the subepithelial stromal cells directly below the uterine lumen in the hCG- and IL-1-treated animals (Fig. 4G). The studies in the simulated pregnant baboon model (16) suggested that hCG together with estrogen and progesterone are insufficient to induce IGFBP-1 expression in decidualized stromal cells. However, glandular epithelial synthesis of IGFBP-1 is maintained, perhaps as a direct effect of hCG on endometrial secretory activity (16). In the animals treated with hCG in the presence of IL-1, the stromal cells close to the apical surface stained intensively for IGFBP-1 (Fig. 4O), and staining was also detected in stromal cells underneath the epithelial plaques (Fig. 4O). In addition, the cells close to the apical surface did not stain for either Ki67 (Fig. 4K) or -SMA (Fig. 4G), which is identical with our previous observations at the implantation site in early pregnancy (as illustrated in Fig. 3A).
Localization of COX-1 and COX-2 protein in the endometrium of baboons treated with hCG and IL-1
COX is the rate-limiting enzyme in the conversion of arachidonic acid to PGs. Two COX isozymes have been identified, COX-1 and COX-2. In control animals on d 14 PO (Fig. 5A) and in animals treated with hCG and IL-1Ra (Fig. 5D), intense COX-1 staining was observed on luminal epithelium and in the epithelial glands in controls (Fig. 5, A, E, and I). Similar to our previous report for COX-1 mRNA expression in early pregnant endometrial tissues (d 17–19 of pregnancy) (25), the infusion of hCG (Fig. 5B) and hCG plus IL-1 (Fig. 5C) resulted in the loss of COX-1 in epithelial cells at the apical surface. In the deeper epithelial glands of hCG- and IL-1-treated animals (Fig. 5, G and K), the presence of COX-1 protein was significantly decreased compared with that after hCG treatment (Fig. 5, F and J).
COX-2 expression was different from COX-1 expression. In control animals on d 14 PO, weak positive staining was observed in luminal epithelium, whereas glandular epithelium remained negative. We expected to observe staining in stromal cells undergoing decidualization, but there was no significant positive staining evident in animals treated with hCG and IL-1 on d 15 PO (data not shown), in contrast to what was observed in pregnant baboons when a conceptus was present (Fig. 3B) (25).
Immunolocalization of COX-1 in endometrium of early pregnant baboons
There were two distinctly different patterns in endometrium of pregnant baboons (d 22 of pregnancy): the area close to the implantation site (Fig. 6, B and D) and the area opposite the implantation site (nonimplantation site; Fig. 6, A and C).
The luminal epithelium of the endometrium from the nonimplantation site stained for COX-1 (Fig. 6A), similar to that seen with the nonpregnant control (Fig. 5A) and hCG- and IL-1Ra-treated animals (Fig. 5D). The implantation site had a remodeled apical area that stained weakly for COX-1 (Fig. 6B). The weaker staining for COX-1 in the epithelial glands in the midfunctionalis region in the endometrium of pregnant animals (Fig. 6, C and D) is similar to that observed in the CG- and IL-1-treated animals (Fig. 5, G and K).
Prolactin and IGFBP-1 detection in uterine flushings
In humans, IGFBP-1 begins to be secreted by stromal cells surrounding the spiral arteries during the late secretory phase of the menstrual cycle, whereas in baboons, IGFBP-1 is secreted from the glands in response to progesterone (19, 26). The production of decidual prolactin begins by d 22 of the menstrual cycle in humans and continues to increase throughout gestation (27). In baboons with the onset of pregnancy, prolactin expression increases steadily and is evident primarily in the decidual tissue (28).
Because we observed both morphological and immunohistological changes in hCG- and IL-1-treated animals consistent with the decidualization process in stromal cells (Fig. 4O), we assayed uterine flushings from all four treatment groups for prolactin and IGFBP-1. Because of the limited number of flushings (n = 3/experimental group), the interanimal variability was high, and no statistically significant differences between the experimental groups were evident. However, there was a trend toward an increase in the mean values of both prolactin and IGFBP-1 in hCG-treated (13.9 ± 14.6 ng/mg protein or 10.8 ± 8.0 ng/μg protein, respectively) and of prolactin and IGFBP-1 in hCG- plus IL-1-treated (16.5 ± 12.2 ng/mg protein or 9.2 ± 4.4 ng/μg protein, respectively) animals. This is compared with prolactin and IGFBP-1 in controls (1.6 ± 2.7 ng/mg protein or 6.3 ± 5.0 ng/μg protein, respectively) and with prolactin and IGFBP-1 in hCG- plus IL-1Ra-treated animals (10.6 ± 4.1 ng/mg protein or 5.0 ± 3.9 ng/μg protein, respectively). Based on previous studies (19, 26, 28), the increased levels in the hCG-treated and hCG- plus IL-1-treated groups probably reflect a contribution primarily from the glandular epithelium and, to a lesser extent, from the stromal cells.
Analysis of cytokines in uterine flushings
There is evidence that cytokines produced by the maternal endometrium and the developing embryo play a crucial role during implantation (see review in Ref. 29). Cytokines are often considered in the context of their T helper type 1 (Th1)/Th2 classification (see review in Ref. 30).
The uterine flushings were analyzed for the presence of cytokines by a protein array. Of the 36 cytokines spotted on the array, the majority were detectable in uterine flushings. Cytokine values showed a high interanimal variation within the same treatment group. Overall, however, there was a trend toward a decrease in cytokine levels in treated animals compared with controls (no treatment).
In uterine flushings of treated animals, we observed a decrease in Th1 cytokines, IL-2 (Fig. 7A), TNF- (Fig. 7B), and interferon- (Fig. 7C), compared with controls. IL-12 exists as two isoforms (p40 and p70), and only the heterodimer is bioactive. This array only measured the IL-12 (p40) form, and levels were similar in all four treatment groups (Fig. 7A). Therefore, if there is indeed a down-regulation of IL-12, as is predicted to be favorable for pregnancy (30), it could be associated with regulation of the IL-12 (p70) form.
In contrast with the classical Th1/Th2 concept, there was no increase in Th2 cytokine levels in treated animals compared with controls. However, there was a significant decrease in levels of IL-4 (Fig. 7A). The assay also detected the Th2 cytokines IL-3 (Fig. 7C), IL-5, IL-6, IL-6 receptor, and IL-10 (data not shown), but overall they were present at low concentrations.
We detected a borderline decrease in Fas (Fig. 7B) in uterine flushings from animals treated with hCG (P = 0.093) and a significant decrease in Fas in the presence of hCG and IL-1 or IL-1Ra (P = 0.025) compared with the controls. In all treated animals, there was less TNF- and TNF receptor-1 and -2 (TNFR-1 and TNFR-2; Fig. 7A) detectable, although only the decrease in TNFR-1 was statistically significant (P < 0.05).
The other molecules active at the maternal-fetal interface, such as growth factors (vascular endothelial growth factor, epidermal growth factor, granulocyte-macrophage colony-stimulating factor, and TGF-), leptin, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein-1, and macrophage inflammatory protein-4, were detected in uterine flushings, but the differences between control and treated animals did not approach statistical significance (data not shown). Similarly, no statistical differences were observed for interferon--inducible protein-10 (CXCL10) and eotaxin (data not shown). In contrast, matrix metalloproteinase-3 (Fig. 7A) and cytotoxic T lymphocyte-4 (CTLA-4; Fig. 7C) were significantly decreased in treated animals compared with controls.
Discussion
Successful embryo implantation requires precise synchrony between the blastocyst and the uterine environment, where a complex series of interactions occurs. The aims of this study were to investigate the possible in vivo relationship between two embryonic factors, hCG and IL-1, in preparing the endometrium for the establishment of pregnancy. hCG, which is required to maintain pregnancy in the primate, exerts its effects through LH/hCG receptors by stimulating progesterone secretion from the corpus luteum during the first trimester. hCG signals to the endometrium and modulates the uterine environment before implantation (5). Other conceptus-mediated factors are required for the differentiation of stromal cells to the decidual phenotype, because decidualization occurs at the implantation site of baboons (31), but not in hCG-induced pseudopregnancy (16). Our in vitro studies have also previously demonstrated that soluble molecules from cytotrophoblasts were responsible for the induction of decidualization in baboon stromal fibroblasts in a coculture model (23). In the present study we confirmed that IL-1 mRNA is present in cytotrophoblasts and at the implantation site of the baboon. The immunohistochemical evidence confirms that IL-1 enhances the previously reported cellular transformation of the endometrium induced by hCG (5), and the changes are similar to those observed in early pregnancy.
Our data suggest two distinct patterns associated with the IL-1 effect. First, the presence of IL-1 specifically contributes to decidualization (as evidenced by IGFBP-1 expression) and COX-1 protein regulation, which are functional changes associated with the presence of the conceptus during early pregnancy (25, 31). The effect of IL-1 on in vivo decidualization is in agreement with our previous in vitro studies in both baboon and human stromal fibroblasts (11). Based on our previous studies (11, 25, 32), we would also propose that a synergism among hCG, IL-1, and progesterone is critical for the changes to be induced. In addition, IL-1 decreases COX-1 protein in epithelial cells of baboons, which is comparable to observations during early pregnancy. Secondly, IL-1 in our model does not influence changes in cytokine levels detected in uterine flushings; hCG alone is capable of regulating their secretion.
Over the past decade, the importance of COX-1 and -2 in reproduction has been well documented (see review in Ref. 33). Up-regulation of COX-2 gene expression in response to hCG has been demonstrated in both human endometrial stromal and epithelial cells (34, 35). We and others have demonstrated that IL-1 can elevate both PGE2 and PGF2 levels by increasing COX-2 in human and baboon stromal cells in vitro (9, 10, 11, 12), and that the inhibition of COX-2 causes a partial decrease in the in vitro decidualization response (11). During early pregnancy in the baboon, COX-2 was strongly expressed in the stromal cells at the site of implantation (25). In contrast, our current study did not detect an up-regulation of COX-2 in baboon endometrium in animals treated with hCG and IL-1 on d 15 PO. We conclude that hCG and IL-1, when infused in vivo, were not sufficient to induce COX-2 expression, and that additional factors from the conceptus may be necessary for its induction at the implantation site during pregnancy.
COX-1 is constitutively expressed in most cell types and has been considered to be a housekeeping gene, although our studies show that its expression is hormonally regulated in baboon uterus (25). COX-1 mRNA expression in epithelial cells of the endometrium was up-regulated during the secretory phase and was down-regulated with the establishment of pregnancy (25). In this study, COX-1 protein was localized at the luminal epithelium and in epithelial glands of control animals on d 14 PO and was markedly decreased in the epithelial cells of hCG- and IL-1-treated animals, similar to what was observed in early pregnancy. Similarly, the expression of COX-1 and COX-2 protein was reported to be decreased in early pregnancy in the rat uterus (36). In mice, it was recently demonstrated that a compensatory mechanism via COX-1 up-regulation substantially rescues COX-2-deficient female infertility in a genetic background-dependent manner (37).
In the rat, the biosynthesis and catabolism of PGF2 are regulated by progesterone (36). The absence of COX-1 expression in antiprogestin-treated animals strongly implicates a role for progesterone in COX-1 regulation (25), but the absence of COX-1 down-regulation in hCG-treated animals also suggests a role for IL-1 in COX-1 regulation. Although the role of PGs during implantation is recognized, nothing is known about the regulation of COX-1 during the establishment of pregnancy. In synovial cells, IL-1-induced COX-1 and COX-2 up-regulation was inhibited by the glucocorticoid receptor inhibitor dexamethasone (38). In glial cells, regulation of COX-1 expression was associated with histone acetylation (39), because the histone deacetylase inhibitors stimulated COX-1 promoter activity. Modification of chromatin structure is part of the response to IL-1 in myometrial cells (40). IL-1 treatment increases polymerase II cross-linking to COX-2 promoter and increases histone H4 acetylation at specific sites (40). Additional studies are required to address the regulatory mechanism of COX-1 regulation during early pregnancy and the possible involvement of IL-1 in this process.
IL-1 is a family of polypeptides comprised of two agonists, IL-1 and IL-1, and a naturally present inhibitor, IL-1Ra. One surprising observation in our study was the ability of hCG and IL-1Ra to suppress the effects of hCG on the morphological transformation of endometrium (such as the formation of epithelial plaques). In mice, ip injection of IL-1Ra prevents implantation (41). It has been suggested that this is due to the down-regulation of integrins 4, V, and 3 on epithelial cells (42, 43). There is very little evidence relating to the interactions between IL-1Ra and hCG described in the literature to date. We can only hypothesize that additional IL-1Ra binding to IL-1RI and prevention of IL-1 binding and receptor activation in the endometrium at the local level could interfere with specific hCG-induced responses. The balance between IL-1 and IL-1Ra in tissues plays an important role in the susceptibility to and the severity of many diseases (44). The importance of IL-1 regulation during the establishment of human pregnancy has also been suggested based on the observation that the decreased expression of IL-1 and IL-6 in the midsecretory phase is associated with habitual abortion (45).
Classical Th1/Th2 dogma suggests a decrease in Th1 (IL-2, interferon-, TNF-, and IL-12) cytokines and an increase in Th2 (IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13) cytokines during pregnancy. In terms of the Th1/Th2 paradigm, the expected decline in Th1 cytokines was detected in uterine flushings of treated animals compared with controls. However, the expected increase in Th2 cytokines was not evident. Chaouat et al. (30) reviewed in detail the limits of the Th1/Th2 paradigm and concluded that the Th1/Th2 paradigm in pregnancy appears to be obsolete and an oversimplification. Rather, step-specific events have to be considered, and a key role is seen in local tissue remodeling, in which immune cytokines may play an important role. It is also possible that the placenta and/or endometrium are capable of regulating the cytokine milieu at the site of implantation without significantly altering the Th1/Th2 paradigm due to the large number of cytokines at the maternal-fetal interface.
The sloughing off of the endometrium at the start of menstruation occurs by programmed cell death (apoptosis). In a recent study, a bolus injection of hCG to nonpregnant woman decreased apoptosis in the endometrium on d 26 of the menstrual cycle compared with control tissues obtained at the same time in the menstrual cycle (4). The results suggested that both exogenous progesterone and hCG can influence the human endometrium during the late secretory phase and can prevent or delay the start of endometrial apoptosis (4). It is of interest in the current study that treatment with hCG and IL-1 resulted in a significant suppression of Fas and TNFR-1, which are the best-characterized death receptors (see review in Ref. 46). The major function of the receptor Fas (Apo-1, CD95) and Fas ligand interaction is the induction of apoptosis in cells carrying Fas. TNFR-1 activation initiates downstream events leading to apoptosis through caspase-8 or activation of nuclear factor-B transcription factor and activation of c-Jun NH2 kinase (47). The increased presence of Fas and TNFR-1 in uterine flushes of control animals confirms the premenstrual start of endometrial apoptosis and the shedding of Fas and TNFR-1 from the surface of uterine cells. Similarly, soluble Fas receptor was increased in the plasma of patients with congestive heart failure during presumed activation of myocardial apoptotic cell death (48). Also, TNFR-1 is shed into the serum during liver damage in chronic hepatitis patients (49).
Down-regulation of TNFR-1, IL-4, matrix metalloproteinase-3, and CTLA-4 in uterine flushes of treated animals seems to be primarily regulated by hCG, because the addition of IL-1 or its antagonist, IL-1Ra, did not result in a different outcome. It has been hypothesized that hCG may be the placental link for the development of local immune tolerance during and after implantation by induction of T cell apoptosis (50).
CTLA-4 is a cell surface glycoprotein cosignaling molecule that may modulate peripheral self-tolerance at the maternal-fetal interface (51). Cosignaling molecules are controllers of T cell responses to antigens. By selectively turning on and/or off costimulatory pathways, the T cell receptor signal could be transiently regulated in positive and negative directions. Therefore, a precise balance between these pathways often determines the outcome of the T cell response (see review in Ref. 52). It has been suggested that abnormal expression of CTLA-4 in fetal tissues at the maternal-fetal interface may be a potentially common etiological factor in human miscarriage (51). A CD4+CD25+ regulatory T cell population isolated from the human decidua, which expresses intracellular CTLA-4, has been reported (53). It has been proposed that these cells are most likely to be important in the regulation of local maternal tolerance toward the fetus. Therefore, it is possible that the CTLA-4 decrease detected in uterine flushings of treated animals may contribute to the alteration of cytokine profiles and the modulation of T cell activation in the uterus during the early implantation period.
However, we are aware that our simulated pregnancy model does not completely reflect the complex interactions that occur between the implanting embryo and the maternal endometrium (54), but what is relevant with our model is that the function of specific embryonic molecules could be selectively delineated. Furthermore, it is evident from our studies that the interaction between hCG and IL-1 does recapitulate many of the changes observed in early pregnancy and provides evidence for some of the mechanisms by which the endometrium modulates both the inhibition of apoptosis and the immune environment that are associated with the establishment of pregnancy.
Acknowledgments
We thank Laura Studee, M.P.H., for help with statistical analysis.
Footnotes
This work was supported by National Institutes of Health Grants HD-36759 and HD-42280 (to A.T.F.) and HD-44713 (to Z.S.).
Abbreviations: COX, Cyclooxygenase; CTLA-4, cytotoxic T lymphocyte antigen-4; hCG, human chorionic gonadotropin; IGFBP-1, IGF-binding protein-1; IL-1Ra, IL-1 receptor antagonist; PG, prostaglandin; PO, post ovulation; -SMA, -smooth muscle actin; Th1, T helper type 1; TNFR-1, TNF receptor type 1.
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