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Original Studies |
Division of Endocrinology, Children’s Hospital Medical Center and Perinatal Research Institute, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229
Address all correspondence and requests for reprints to: Dr. Stuart Handwerger, Division of Endocrinology, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. E-mail: stuart.handwerger{at}chmcc.org
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Abstract |
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Introduction |
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(TNF) (1) stimulates PRL release and interleukin-1 (IL-1) (2)
inhibits TRH-stimulated PRL release from rat pituitary cells. IL-2,
which stimulates the growth and differentiation of T and B cells,
natural killer cells, monocytes/macrophages, lymphokine-activated
killer cells, and oligodendrocytes (3, 4, 5, 6), stimulates the release of
PRL, ACTH, and TSH from pituitary cells and inhibits the release of LH
and FSH (7, 8).
In addition to modulating the release of pituitary PRL, cytokines also
affect the synthesis and release of decidual PRL, a protein hormone
expressed by decidualized human endometrial stromal cells that is
identical in sequence to pituitary PRL. IL-1 inhibits the induction of
decidual PRL gene expression during decidualization (9). In contrast,
TNF, which stimulates pituitary PRL release, inhibits decidual PRL
release (10). As human uteroplacental tissues contain a large number of
bone marrow-derived cells (11), (e.g. macrophages, large
granular lymphocytes, and T cells) that synthesize and release IL-1
(12) and TNF (13), and as decidual stromal cells contain receptors
for these cytokines, the effects of IL-1 and TNF on decidual PRL
appear to be due at least in part to a paracrine action.
Although decidual cells have also been shown to contain receptors for IL-2 (14), the effects of IL-2 on the synthesis and release of decidual PRL are unknown. In this study, we have examined the effects of IL-2 on the synthesis and release of decidual PRL and on trans-activation of the decidual PRL (exon 1a) promoter. The findings indicate that IL-2 inhibits the synthesis and release of decidual PRL by inhibiting trans-activation of the decidual PRL promoter, providing further evidence for a role of cytokines in the paracrine regulation of decidual PRL gene expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IL-2 was purchased from Life Technologies (Gaithersburg, MD) and was diluted with RPMI 1640 (Sigma Chemical Co., St. Louis, MO) medium containing 0.1% BSA (Sigma Chemical Co.) before use. Recombinant human insulin was obtained from Sigma Chemical Co., and porcine relaxin was a gift from the National Hormone and Pituitary Program (NIDDK, Bethesda, MD).
Preparation and culture of decidual cells
An enriched fraction of decidual cells was prepared from term human decidual tissue, as previously described (15). The protocol for obtaining decidual tissue was approved by the human investigation committees of Children’s Hospital Medical Center and University of Cincinnati. Briefly, decidual tissue, after being manually separated from chorionic membrane, was digested with 0.35% collagenase and 0.35% hyaluronidase. Decidual cells were isolated by isopynic centrifugation through a 40% Percoll (Pharmacia LKB Biotech, Piscataway, NJ) gradient, then suspended in RPMI 1640 containing 10% FBS (Life Technologies, Grand Island, NY) and antibiotics. The cells were plated at density of 0.5 x 106 cells/well in 24-well culture plates (Costar, Cambridge, MA) coated with collagen (Vitrogen 100, Celtrix Corp., Palo Alto, CA) and incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2 for 48 h.
Experimental conditions
After plating for 48 h, the medium was replaced with serum-free medium. Twenty-four hours later (0 h), the medium in each well was changed to either serum-free medium alone or serum-free medium containing IL-2. The cells were maintained in culture for an additional 120 h with daily medium changes. At the end of each 24 h, the medium was removed and frozen at -20 C until assayed as described below. In selected instances, the cells were removed from the culture plates by trypsinization, suspended in 10 mmol/L HEPES buffer, and lysed by sonication. The suspension was then centrifuged at 10,000 rpm for 10 min, and the supernatant was removed and frozen at -20 C. In one set of experiments, the cells were removed from the culture plates by treatment with collagenase, suspended in medium RPMI, and lysed by sonication. The suspension was then centrifuged at 2000 rpm for 10 min, and the supernatant was removed and frozen at -20 C. In another set of experiments, alkaline phosphatase and lactic dehydrogenase concentrations were determined in the media from control cells and cells that had been exposed for 96 h to IL-2 (50 ng/mL) using automated analysis (16). The viability of the cells after 96 h of exposure to IL-2 (50 ng/mL) was also determined by trypan blue exclusion test.
Determination of total protein and PRL synthesis
In one set of experiments, total protein synthesis and PRL synthesis were determined by measuring the incorporation of [35S]methionine into trichloroacetic acid (TCA)-precipitable proteins and immunoprecipitable PRL as previously described from our laboratory (17). Decidual cells (1.5 x 106 cells) were incubated for 72 h in RPMI medium containing 10% FCS with or without IL-2 (50 ng/mL), with medium changes after each 24-h period. The cells were then incubated for an additional 24 h in medium of the same composition containing 2 µCi/mL [35S]methionine (New England Nuclear Corp., Boston, MA). In the immunoprecipitation experiments, the amount of nonspecific precipitation of [35S]methionine was determined by using nonimmune serum in place of PRL antiserum. Nonspecific counts, which ranged from 5–15% of the total immunoprecipitable counts, were subtracted from total immunoprecipitable counts.
PRL assay
PRL was measured in conditioned medium by homologous RIA, as previously described (18), using materials provided by the National Hormone and Pituitary Program, NIDDK. Conditioned medium was stored at -20 C until assayed. Interassay variation (SE at ED50) was 4% of the mean.
Proliferation assay
The incorporation of [3H]thymidine (TdR) into decidual cell DNA was determined as previously reported (16). Triplicate wells of decidual cells incubated in either control medium or medium containing IL-2 were exposed to 1 µCi TdR. After 24 h of exposure to TdR, the cells were washed once with 500 µL HBSS containing 0.5% BSA and then exposed to 500 µL 5% TCA at 4 C for 3 min. After a final wash with ice-cold 5% TCA, TdR incorporation was determined by solubilizing the remaining DNA and protein in 500 µL NaOH, 2% NaHCO3, and 0.1% SDS and counting in a liquid scintillation counter.
Plasmids
The region from -2977 to +66 bp located 5' of exon 1a of the decidual PRL gene (3-kb fragment) was generated using the PCR technique. Briefly, two DNA fragments (-2927/-1184 and -1489/+66 bp) were amplified from a human placental DNA library using primers encoding the decidual PRL gene, as previously described (19). The two DNA fragments were ligated together, sequenced, and then inserted into pGL3-Basic (pGL3B) luciferase vectors (Promega Corp., Madison, WI). All plasmids used in transfection studies were prepared with a plasmid extraction kit (Qiagen, Santa Clarita, CA), quantitated by 260/280 nm absorbance, and sequenced before use in experiments.
Transfection of in vitro decidualized endometrial stromal cells
To prepare primary cultures of endometrial stromal cells, uterine endometrial tissue was obtained from women with normal menstrual cycles at the time of elective tubal ligation. Informed consent was obtained from patients, and the institutional review boards of Children’s Hospital Medical Center and the University of Cincinnati approved the study. Proliferative or secretory phase endometrium was removed by suction biopsy, and stromal cell cultures were prepared as previously described (9). Primary endometrial stromal cells were cultured in DMEM-2% FBS, 25 U/mL penicillin G, 25 µg/mL streptomycin, and 2.5 µg/mL amphotericin at 37 C. Cells were plated in Falcon six-well plates and treated with 1 µmol/L medroxyprogesterone acetate and 10 nmol/L 17ß-estradiol for 10 days as previously described (9). These steroids were dissolved in ethanol and added to the medium before use. The final concentration of ethanol in the medium never exceeded 0.09% (vol/vol). When the cells were transfected with pGL3B alone or with PGL3B containing the 3-kb PRL promoter, equal amounts of DNA-calcium phosphate precipitate from a single polystyrene tube were added to each of three wells. Cells were incubated for 4 h in the presence of chloroquine (60 ng/mL), rinsed with PBS, and then incubated in growth medium with or without IL-2 for 48 h. At the end of 48 h, the cells were harvested with lysis buffer (Tropix, Inc., Bedford, MA) and centrifuged (12,000 x g for 5 min), and the supernatant was assayed for luciferase using a Berthold 9501 luminometer (Berthold Analytical Instruments, Nashua, NH). All transfection results were normalized to the protein content of the samples.
Statistics
Statistical differences between sample means were calculated by ANOVA, followed by Newman-Keuls test. The results are expressed as the mean ± SE. P < 0.05 was considered to represent a significant statistical difference.
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Results |
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Human decidual cells incubated in medium containing IL-2 (50
ng/mL) for 5 days released significantly less PRL after day 2 than
cells exposed to control medium alone (Fig. 1
). The amounts of PRL released by cells
exposed to IL-2 during the third, fourth, and fifth days of exposure
were 28.1%, 50.2%, and 71.4% less, respectively, than that released
by control cells (P < 0.01 in each instance). The
inhibition was dose dependent, with 50% inhibition of PRL release
occurring at a concentration of approximately 15 ng/mL (Fig. 2).
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, IL-2 (50 ng/mL)-exposed cells
synthesized 65.7% less [35S]methionyl PRL during the
fourth day of exposure to IL-2 than control cells (P =
0.0001). During the same time interval, the total amount of
TCA-precipitable proteins synthesized by the IL-2-exposed cells was not
significantly different from that from control cells.
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Effect of IL-2 on PRL gene expression
The inhibition of PRL synthesis and release was also accompanied
by a decrease in decidual PRL promoter activity. In the experiment
depicted in Fig. 3, decidualized human
endometrial stromal cells were transfected with an expression plasmid
for luciferase coupled to a 3-kb fragment (-2927 to +66) of the
decidual PRL promoter. The transfected cells were then exposed for 2
days to either IL-2 (1 and 10 ng/mL) or control medium alone. The
luciferase activities of decidualized endometrial stromal cells exposed
for 2 days to IL-2 at a concentration of 1 or 10 ng/mL (normalized to
protein content) were 14% and 54% less than that of control cells. In
a control experiment, IL-2 had no effect on luciferase activity when
the cells were transfected with the reporter gene plasmid (pGL3B)
lacking the decidual PRL promoter fragment (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of this study demonstrate that IL-2 also inhibits the synthesis and release of decidual PRL. The inhibition of PRL release by IL-2 is both dose and time dependent. Initial inhibition occurs after 2–3 days of exposure, with a half-maximal effective dose of 15 ng/mL. Inhibition of PRL synthesis in response to IL-2 was demonstrated by a study in which IL-2-exposed decidual cells incorporated significantly less [35S]methionine into PRL than control cells. Over the same time interval, the IL-2-exposed cells synthesized the same amount of total TCA-precipitable [35S]methionyl proteins as control cells, indicating that the inhibition of PRL synthesis is not secondary to a generalized effect of IL-2 on protein synthesis. The inhibition of the synthesis and release of PRL is also not due to an effect of IL-2 on decidual stromal cell proliferation, as IL-2 does not affect the incorporation of [3H]TdR into decidual cell DNA. Furthermore, the inhibitory effect cannot be attributed to IL-2-induced cell damage, because decidual stromal cells exposed to IL-2 do not release lactate dehyrogenase and alkaline phosphatase, both of which are sensitive indicators of damage to decidual cells in vitro.
The investigations with [35S]methionine incorporation into PRL- and TCA-precipitable proteins are consistent with earlier studies from our laboratory indicating that decidual PRL, unlike pituitary PRL, is not stored in a large intracellular pool and is rapidly released from decidual cells (17, 23, 24). During the fourth day of culture, the control cells released 82.1% of the newly synthesized PRL and 22.9% of the newly synthesized TCA-precipitable proteins. IL-2 resulted in a 65.7% decrease in PRL synthesis and a 78.3% decrease in the amount of newly synthesized PRL released into the medium. IL-2, however, had no effect on the amount of newly synthesized PRL stored in the cells, indicating that the decrease in PRL release in response to IL-2 is secondary to a decrease in PRL synthesis.
The effect of IL-2 on the synthesis and release of decidual PRL is opposite that on the synthesis and release of pituitary PRL (7, 8). However, IL-2 is not the only factor that has different effects on the expression of decidual and pituitary PRLs. For example, arachidonic acid stimulates the synthesis and release of pituitary PRL, but inhibits the synthesis and release of decidual PRL (25). In addition, TRH, which stimulates pituitary PRL expression, and dopamine, which inhibits pituitary PRL expression, have no effects on the expression of decidual PRL, even at concentrations 1000-fold greater than the half-maximal concentrations that affect pituitary PRL (26).
The reason why IL-2 has opposite effects on the expression of decidual and pituitary PRL is unknown. However, the difference is probably due at least in part to the fact that the expression of the decidual PRL gene is regulated by a different promoter than that which regulates expression of the decidual PRL gene (19, 27). Although both PRLs are products of the same gene, decidual PRL is regulated by a distinct promoter (exon 1a) that is located 5.3 kb upstream of the transcription initiation site. The exon 1a promoter also regulates PRL gene expression in other nonpituitary cells that express PRL, such as T lymphocytes, brain, myometrium, dermal fibroblasts, and lacrimal gland.
The effect of IL-2 on the synthesis of decidual PRL appears to be primarily at the level of transcription. IL-2 inhibited trans-activation of the PRL exon 1a promoter, and the magnitude of the inhibition of promoter activity was identical or nearly identical to the magnitude of the inhibition of PRL synthesis and release. The mechanism by which IL-2 inhibits trans-activation of the exon 1a promoter is unknown.
Several recent studies suggest a possible role for IL-2 in the pathogenesis of preeclampsia, one of the most common pathological conditions of pregnancy. Serum concentrations of IL-2 in women with preeclampsia are significantly higher during the first trimester than in women with normal pregnancies (28). In addition, IL-2 is detected in decidualized endometrial stromal cells and lymphocytes in decidua of women with preeclampsia, but not in decidua of normal pregnant women (29). A possible role for IL-2 in the pathophysiology of preeclampsia is suggested by the recent finding that IL-2 may reduce angiogenesis in the placenta of patients with preeclampsia by inducing lymphokine-activated killer cells (30). Studies by Golander et al. have also demonstrated that decidual tissue of women with preeclampsia releases less PRL than decidual tissue from women with normal pregnancies (31). As IL-2 inhibits decidual PRL release, the decrease in decidual PRL release in women with preeclampsia may be due at least in part to the increased serum concentrations of IL-2 in these patients and to an autocrine/paracrine effect of IL-2.
In summary, IL-2 inhibits PRL gene expression and the synthesis and
release of decidual PRL. The effect of IL-2 in decidua is opposite that
observed in the pituitary. The findings that IL-2, IL-1, and TNF
inhibit the synthesis and release of decidual PRL suggest a major role
for cytokines in the regulation of decidual PRL expression.
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Acknowledgments |
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Footnotes |
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2 Present address, Department of Obstetrics and Gynecology, Osaka
University Medical School, Osaka, Japan.
Received July 8, 1998.
Revised October 27, 1998.
Accepted October 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulates prolactin release from anterior
pituitary cells: a possible involvement of intracellular calcium
mobilization. Endocrinology. 128:2785–2790.[Abstract]
inhibits the synthesis and release of human decidual
prolactin. Endocrinology. 134:353–357.[Abstract]
levels before the clinical manifestations of preeclampsia. Am J
Reprod Immunol. 38:89–93.
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