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Original Studies |
Department of Gynecology and Obstetrics (H.-Y.H., Y.W., J.S.K., F.R., M.L.P.), Stanford University School of Medicine, Stanford, California 94305; and Department of Obstetrics and Gynecology (H.-Y.H., Y.-K.S.), Lin-Kou Medical Center, Chang Gung Memorial Hospital and University School of Medicine, Taipei, Taiwan
Address all correspondence and requests for reprints to: Hong-Yuan Huang, Department of Obstetrics and Gynecology, Lin-Kou Medical Center, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan. E-mail: hykh{at}ms18.hinet.net
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, the interleukin (IL)-1 system has also been implicated as a
major factor in these events. IL-1 is a family of polypeptides
comprised of two agonists (IL-1
and IL-1ß) and an inhibitor [IL-1
receptor antagonist (IL-1ra)] (6). Two receptors (IL-1R)
have been identified and characterized as type I (IL-1R tI; Ref.
7) and type II (IL-1R tII; Ref. 8). Both IL-1
agonist and antagonist are recognized by IL-1R tI, and both trigger
signal responses in target cells (9, 10). IL-1R t II
(8) is also found on many cells but primarily on
neutrophils, monocytes, and B lymphocytes. Recent knowledge of IL-1
expression in both human endometrium and in the embryo has led to a
more detailed outline of implantation events. The presence of the IL-1
system has been documented in human endometrium (11, 12, 13, 14, 15, 16).
IL-1-mediated inhibition of endometrial stromal cell differentiation
has also been described (17, 18). The results obtained
from immunohistochemical studies have localized the complete IL-1
system in human oocytes and embryos at all developmental stages
(19, 20). These results suggest that the entire IL-1
system may play an important role in embryo implantation and
decidualization of stromal cells.
Human endometrium has been shown to express more IL-1ra in the follicular phase, compared with luteal phase (21). In addition, repeated injections of the IL-1ra into pregnant mice, beginning 2 days before the onset of implantation, resulted in the apparent failure of blastocyst to implant, suggesting an inhibitory effect of IL-1ra on embryo implantation (15). The mechanism by which this occurs remains unclear, although it has been suggested that IL-1ra prevents embryo implantation by a direct effect through microvilli in mouse endometrium (22). Interestingly, repeated injections of recombinant IL-1ra in pregnant wild-type or IL-1R tI null female mice did not inhibit embryo implantation (23).
In the present study, we have examined the effects of sex steroid hormones (E and P) on human endometrial stromal cell expression of IL-1ß, IL-1ra, and IL-1R tI messenger RNA (mRNA) using RT-PCR. To further test the hypothesis that an appropriate ratio of IL-1ß to IL-1ra might impact the process of embryo implantation, confluent human endometrial stromal cells stimulated with steroid hormones were further treated with increasing concentrations of recombinant IL-1ß to determine the quantitative mRNA and protein expression of IL-1ß and IL-1ra, and specifically to evaluate the ratio of agonist to antagonist expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human luteal phase endometrium (n = 8) was obtained from surgical specimens of normally cycling women undergoing hysterectomy for benign reasons, in accordance with the guidelines of the Declaration of Helsinki after informed consent and with approval by the Stanford University Institutional Review Board. The tissue samples used for this study were histologically normal. Stromal cells were separated from the glandular epithelium after collagenade digestion and were cultured using an established in vitro model as previously described (24, 25). Cells were cultured in 75% DMEM (Gibco BRL, Grand Island, NY) and 25% MCDB-105 (Sigma, St. Louis, MO), containing antibiotics, 5 µg/mL insulin (Sigma), and 10% charcoal-stripped FBS (contains less than 5 pg/mL of E and less than 10 ng/dL of P; Gimmini, Calabasas, CA). Cultures prepared by this method contained less than 0.1% of endometrial epithelial or vascular cell (24).
Hormonal treatment
Stromal cells (1–5 passages) were plated at 2–105/well in 24-well culture plates (Falcon, Becton Dickinson and Co., Lincoln Park, NJ) and cultured in standard medium. After confluence (designated as day 1), cell cultures were treated with serum-free standard medium supplemented with 10 µg/mL human apotransferrin (Sigma), 50 µg/mL ascorbic acid (Sigma), 1 µmol/L P (Sigma), 10 nmol/L E (Sigma), 20 ng/mL epidermal growth factor (Sigma), and 1 mg/mL BSA (Irvine Inc., Santa Ana, CA) for 9 consecutive days to mimic the usual in vivo implantation period (24, 25). Control confluent cells were cultured in the same medium in the absence of E and P. Unless indicated otherwise, standard medium and serum-free medium were renewed every 2–3 days throughout the culture period. Conditioned serum-free standard medium was collected and frozen at -70 C until assayed for endogenous IL-1ß, IL-1ra, and PRL production.
Enzyme-linked immunosorbent assay (ELISA) for IL-1ß, IL-1ra, and PRL levels in conditioned medium
Conditioned media were collected, before addition of cytokine, for measurement of endogenously produced IL-1ß using an ELISA kit (R&D Systems, Minneapolis, MN) with a detection limit of 1 pg/mL and an intraassay precision of 2.3–3.4% and interassay precision of 3.4–7.1% and for measurement of IL-1ra with a detection limit of 14 pg/mL and an intraassay precision of 2.7–8.3% and interassay precision of 4.9–5.9%. As a marker of decidualization, PRL, in conditioned medium derived from stromal cell cultures, was measured by ELISA (Diagnostic Systems Laboratories, Inc. Webster, TX) with a detection limit of 0.14 ng/mL and intraassay and interassay coefficients of variation of 5.5–9.0% and 6.6–10.4%, respectively. All samples were assayed in triplicate.
Dose response study of recombinant human IL-1ß (rhIL-1ß)
Confluent stromal cells, treated with steroid hormones for 9 days, were stimulated with rhIL-1ß (1 x 105 IU/µg, Genzyme Corp., Cambridge, MA) in a dose-dependent study (0–1000 IU/mL) for a further 24 h. Minimums of three experiments of individual cultures were performed.
RNA analysis
Total RNA was extracted from cultured stromal cells using the guanidinium isothiocyanate method (RNAzol, Tel-Test, Friendswood, TX). RNA concentration was quantified by measuring optical density with a Spectronic 601 spectrophotometer (Milton Roy Co., Rochester, NY). RNA was diluted to 1 µg/µL for RT-PCR. All the experiments were performed a minimum of 3 times with similar results.
Preparation of oligonucleotide primers for RT-PCR
Specific sequences of oligonucleotide primers for detecting
stromal cell expression of human IL-1ß (26), IL-1ra
(27), and IL-1R tI (28) were obtained from
the GenBank Database of the National Center for Biotechnology
Information of the NIH. The corresponding primers were synthesized at
the Beckman Coulter, Inc. Center, Stanford University
Medical Center, Stanford, CA. To ensure that the product detected
resulted from amplification of specific complementary DNA (cDNA) in
question, rather than contamination of other cDNAs, all primers were
designed to span the exon and intron regions. ß-actin message was
amplified as a control molecule using primers for the human sequences
obtained from CLONTECH Laboratories, Inc., Palo Alto, CA
(29). Human luteal endometrium from endometrial biopsy
specimens of normal cycling women is known to express all these
transcripts and was used as a positive control to identify cDNA
fragments generated using the various primers. As a negative control, a
defined volume of cultured medium was extracted and subjected to the
same RT-PCR reaction. The summary of oligonucleotide primer sequences
is listed in Table 1
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For RT-PCR, the GenAmp RNA PCR kit (Perkin-Elmer Corp., Foster City, CA) was used. A total of 19 µL RT-Master Mix for each sample were prepared, containing 5 mmol/L MgCl2, 1x PCR-Buffer II, 1 mmol/L of each deoxynucleotide triphosphate, 2.5 µmol/L Oligod(T)16, 20 IU ribonuclease inhibitor, and 100 IU M-MLV (molony murine leukemia virus) reverse transcriptase (Perkin-Elmer Corp.). The reactions were started with 1 µg total RNA extracted from stromal cells in a total vol of 20 µL RT-Master Mix and filled into a 0.5-mL thin-wall PCR-tube (Applied Scientific, South San Francisco, CA). RT-Master Mix in PCR-tubes was covered with 50 µL light white mineral oil (Sigma). The RT reaction was carried out in the DNA Thermal Cycler 480 (Perkin-Elmer Corp. GeneAmp, PCR Instrument System, Branchburg, NJ) using a program with one 15-min RT cycle at 42 C, followed by 5 min at 99 C, then quenched at 4 C. Products were stored at -20 C until the subsequent PCR.
PCR
Aliquots of the RT products were subjected to PCR in the PCR Master Mix containing 2 mmol/L MgCl2 Solution, 1x PCR-Buffer II, 2.5 IU AmpliTaq DNA polymerase (Perkin-Elmer Corp.) and corresponding paired primers with the concentration of 0.2 µmol/L, to a total vol of 100 µL. PCR was performed simultaneously from a single Master Mix in the different tubes with each primer. PCR cycles were composed of 1 cycle of 95 C for 5 min to denature all proteins, 30 cycles of 45 sec at 94 C, 45 sec at 55 C, and 60 sec at 72 C. The reaction was terminated at 72 C for 5 min and quenched at 4 C.
Agarose gel electrophoresis
Two percent agarose gel (Life Technologies, Inc., Rockville, MD) electrophoresis was carried out in an H5 electrophoresis chamber. Gels were stained with ethidiumbromide (Sigma). Aliquots (25 µL) of each PCR product and dye buffer were analyzed in parallel with a DNA ladder (Life Technologies, Inc.) as a standard. After completion of electrophoresis, the gel blot was analyzed, and photocopies of the blot were printed by ultraviolet densitometry (Bio-Rad Laboratories, Inc., Hercules, CA).
Quantitative competitive PCR (QC-PCR)
Using an internal standard cDNA for QC-PCR, as previously
described (25, 30, 31), quantitative mRNA expression of
IL-1ß, IL-1ra, and IL-1R tI in cultured stromal cells was determined.
A competitive cDNA fragment was constructed by deletion of a 273-bp
fragment from the IL-1ß target cDNA to be detected using an
established methodology, as previously described (25). The
deleted cDNA fragment was synthesized from 1 µg human endometrial RNA
amplified with the 5'-end original primer and 3'-end competitive primer
(listed in Table 1
), then purified from 2% agarose gel with an agarose
gel DNA extraction kit (Roche Molecular Biochemicals,
Mannheim, Germany). Competitive cDNA fragments for IL-1ra and
IL-1R tI were constructed by deletion of 274-bp and 94-bp fragments,
respectively, from the corresponding target cDNA using similar
methodology. To establish the equivalence of each target cDNA to
internal standard cDNA used in QC-PCR, a serial dilution of competitive
cDNA for IL-1ß added to each PCR sample was coamplified with target
cDNA. After completion of RT, a defined amount of competitive cDNA,
determined by equimolar point for IL-1ß (8 fg), IL-1ra (250 fg),
IL-1R tI (4 pg), and the corresponding specific target cDNA,
coamplified in one reaction with the same primers are used. Aliquots
(25 µL from 30 µL) of each PCR product were electrophorsed on 2%
agarose gels and scanned by ultraviolet densitometry. The ability of
increasing concentrations of IL-1ß to modulate IL-1ß, IL-1ra, and
IL-1R tI mRNA expression was measured quantitatively by calculating the
ratio of target cDNA to internal standard cDNA (32).
Solubilization of stromal cells
To further determine the IL-1ß regulation of stromal cell intracellular protein contents of IL-1ß and IL-1ra, confluent stromal cells, treated with steroid hormones for 9 days, were stimulated with rhIL-1ß (Genzyme) in a dose-dependent study (0–1000 IU/mL) for a further 12 h and 24 h, respectively. At the end of cultures, stromal cells were solubilized twice in 0.01% Triton-X 100 in PBS and rotated at 4 C overnight. Supernatants were removed after centrifugation, for measurement of IL-1ß and IL-1ra using ELISA (R&D Systems). For semiquantitation, the volume of samples was adjusted according to the protein concentration as determined by protein assay kit (µg/mL; Bio-Rad Laboratories, Inc.).
Data analysis
Student’s t test and ANOVA were used for statistical analysis between groups. P < 0.05 was accepted as indicating statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of ovarian steroids on PRL production, a known marker of decidualization, by human endometrial stromal cells treated for 9 days with E and P in serum-free medium, was 4.3 ± 0.3 ng/106 cells (the mean ± SD of PRL levels obtained from 22 representative experiments). This low level of PRL, compared with cells in control, nonsteroid-treated cells, documents that endometrial cells were not decidualized. In addition, there was no detectable IL-1ß (<3.9 pg/106 cells/mL) and IL-1ra (<31.2 pg/106 cells/mL) in conditioned media from cultured human endometrial stromal cells. There was also no detectable IL-1ra in conditioned media from stromal cells cultured for 24 h with increasing concentrations of IL-1ß.
IL-1 system mRNA expression in cultured human stromal cells
Figure 1 shows that hormonally
treated endometrial stromal cells produced four different-sized bands,
on RT-PCR, corresponding to ß-actin (838 bp), IL-1ß (548 bp),
IL-1ra (424 bp), and IL-1R tI (284 bp). Cultured stromal cells without
steroid stimulation and follicular phase endometrial biopsy specimens
expressed IL-1R tI only.
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Figure 2 shows IL-1ß stimulation
of IL-1ra mRNA expression in a dose-dependent study. A 151-bp band
corresponding to competitive cDNA as an internal standard and a 424-bp
band corresponding to target cDNA were obtained by PCR coamplification
with a defined amount of competitive cDNA. The ratio was determined by
the density of target cDNA to internal standard cDNA. The
representative blot demonstrates a dose-dependent increase in IL-1ra
mRNA with increasing concentration of IL-1ß (1–1000 U/mL;
P < 0.05). Values are the mean ±
SD of measurements on three representative
blots.
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Figure 3 shows that IL-1ß
regulates stromal cell IL-1ß mRNA expression in a dose-dependent
manner. A 276-bp band corresponding to competitive cDNA as an internal
standard and a 548-bp band corresponding to target cDNA were obtained
by PCR coamplification with a defined amount of competitive cDNA. A
representative experiment revealed a dose-dependent up-regulation of
IL-1ß mRNA expression with increasing concentration of IL-1ß
(1–1000 U/mL; P < 0.001). Values are the mean ±
SD of measurements on three blots. Figure 4 shows that the ratio of IL-1ß to
IL-1ra mRNA in human endometrial stromal cells was increased
significantly in the presence of 1 IU/mL IL-1ß (P <
0.05) but remained constant with increasing concentrations of IL-1ß
(1–1000 IU/mL). Values are the mean ± SD
of measurements on two representative experiments of different
cultures.
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Figure 5 shows the result of IL-1ß
regulation of stromal cell IL-1R tI mRNA expression in a dose-dependent
study. A 191-bp band corresponding to competitive cDNA as an internal
standard and a 284-bp band corresponding to target cDNA were obtained
by PCR coamplification with a defined amount of competitive cDNA. The
ratio of target-to-internal standard cDNA demonstrated a
down-regulation of IL-1R tI mRNA expression with increasing
concentrations of IL-1ß (1–1000 U/mL; P < 0.05).
Values are the mean ± SD of measurements on
three blots.
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To demonstrate the gene product of stromal cell IL-1ß and IL-1ra
transcription, human endometrial stromal cells were treated with
increasing concentrations of IL-1ß for 12 and 24 h. Cells were
solubilized at each timepoint for assay of intracellular IL-1ß and
IL-1ra levels. Figure 6 shows that
intracellular IL-1ß was up-regulated by IL-1ß in human endometrial
stromal cells treated for 12 and 24 h, respectively. In addition,
the intracellular IL-1ra was only up-regulated by IL-1ß after 24
h of culture with IL-1ß, to a maximal level, at 10 IU/mL of IL-1ß.
Similar results were obtained in two separate experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous report, endometrial stromal cell IL-1ra showed greater expression in follicular endometrium, in comparison with luteal endometrium, suggesting an inhibitory effect of IL-1ra in embryo implantation (21). Repeated injections of the IL-1ra into pregnant mice, beginning 2 days before the onset of implantation, resulted in the apparent failure of blastocyst implantation, suggesting that IL-1ra has an inhibitory effect on embryo implantation (15). The mechanism by which this occurs remains unclear, although it has been suggested that the IL-1ra may bind to its receptor expressed in the uterine tissue and interfere with the onset of uterine receptivity. On the other hand, others demonstrated that neither IL-1ra nor the monoclonal antibody 35F5 affected embryo implantation in either wild-type or IL-1 receptor-deficient mice (23). Our results suggest that human endometrial stromal cells express both IL-1ß and IL-1ra, resulting perhaps in an appropriate ratio of agonist-to-receptor antagonist, which favors implantation.
The IL-1 system is a family of polypeptides comprised of IL-1 agonist,
IL-1 ra, and IL-1 receptors. Both IL-1 agonist and receptor antagonist
will bind with IL-1 receptor. Two forms of IL-1ra have been identified:
an intracellular form (27), which is commonly expressed in
epithelial cells; and a secretory form (37). IL-1ra is a
specific inhibitor, which competes with IL-1 and IL-1ß for the
binding site of IL-1R tI and blocks signal transduction by IL-1
(38, 39). Recent knowledge of IL-1 expression in human
endometrium and embryo has led to a more detailed outline of
implantation events. Our earlier reports documented expression of the
IL-1 system in single blastomeres of preimplantation human and mouse
embryos (40, 41). Individual embryos expressed peak IL-1ra
mRNA expression from mouse embryo at the blastocyst stage, either
cultured alone or cocultured with helper cells (42, 43).
IL-1ß and TGF may play crucial roles at the embryo-maternal interface
during trophoblast invasion, by regulating stromal cell expression of
collagenase and their inhibitors, all of which are known to be
important in trophoblast invasion (25). These results
suggest that the entire IL-1 system may be relevant for human
endometrial and embryo physiology. This family of molecules must be
considered to display another paracrine language that may be important
to endometrial and embryo cross-talk during embryo
implantation.
Sex steroids and growth factors have been shown to regulate the stromal cell growth and differentiation in vitro (24, 44). Our results revealed that IL-1ß and IL-1ra were expressed in human endometrial stromal cells after steroid stimulation for 9 days, consistent with such expression in luteal endometrium. These results are also consistent with the central role in the regulation of embryo implantation that decidual cells are believed to play through control of trophoblast invasion (45), nutrition of the blastocyst (46), endocrine secretion (47), and protection of the embryo from maternal immune rejection (48). Furthermore, our results demonstrated that IL-1R tI was expressed in both the control and stimulated groups, suggesting a receptor-ligand relation in human endometrial stromal cells. Stromal cell IL-1R tI mRNA expression is down-regulated by its agonist, which is compatible with the previously published observations that the down-regulation occurs in intestinal epithelial cells (49) and kidney epithelium (31). These results suggest a possible mechanism to limit the effect of IL-1 on human endometrial stromal cell function during the implantation process, by down-regulating the expression of the Type I IL-1 receptor. In addition, our results also demonstrated that there is not any detectable IL-1ß and IL-1ra in conditioned media of cultured stromal cells cultured with or without IL-1ß. The gene product of human endometrial stromal cells is intracellular, and its expression is dependent on IL-1ß.
In conclusion, much evidence has implicated cytokines, especially the IL-1 system, in mediating many aspects of embryo implantation. Our results suggest that the entire IL-1 system, especially IL-1 agonist and receptor antagonist, may be relevant in mediating embryonic implantation. An appropriate ratio of IL-1 agonist to receptor antagonist may be critical to initiate and maintain successful implantation at the local embryo-epithelial interface.
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Acknowledgments |
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Footnotes |
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Received March 15, 2000.
Revised September 22, 2000.
Accepted November 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin-1 ß
levels are identical. Nature. 324:266–268.[CrossRef][Medline]
, and transforming
growth factor ß expression in endometrium, placenta, and
placental membrane. Am J Obstet Gynecol. 163:1430–1437.[Medline]
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