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Original Studies |
B Activity in Cytotrophoblasts by Dexamethasone: A Potential Mechanism for Antiinflammatory Action of Glucocorticoids in Human Placenta1
Departments of Obstetrics and Gynecology (T.R., G.K., Y.M., E.-Y.W., C.J.L., S.G.) and Biochemistry (S.G.), New York University Medical Center, New York, New York 10016
Address all correspondence and requests for reprints to: Dr. Seth Guller, Department of Obstetrics and Gynecology, New York University Medical Center, Tisch Hospital, Room 531, 550 First Avenue, New York, New York 10016. E-mail: seth.guller{at}mcobg.med.nyu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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B (NF-B)
family on cytokine gene expression. In the present study we tested the
hypothesis that during pregnancy, glucocorticoids chronically suppress
inflammation in the human placenta. Cytotrophoblasts obtained from
human term placentas were maintained for 48 h in culture medium
supplemented with 10% charcoal-stripped calf serum with and without
100 nmol/L dexamethasone (DEX). Enzyme-linked immunosorbent assay
studies revealed that cytotrophoblasts constitutively express
interleukin-8 (IL-8), a known mediator of placental inflammation,
between 24–96 h of culture. A 48-h treatment of cytotrophoblasts with
100 nmol/L DEX significantly reduced the production of IL-8 to 24
± 1% of control levels (P < 0.01). DEX and
cortisol mediated a dose-dependent inhibition of IL-8 expression, with
ED50 values of 5 and 50 nmol/L, respectively. DEX treatment
also significantly reduced levels of IL-6 and tumor necrosis factor-
in culture medium, suggesting that glucocorticoids coordinately reduce
cytokine levels in cytotrophoblasts. As cytokine expression is
regulated by NF-B and activator protein-1 (AP-1) transcription
factors, electrophoretic mobility shift assays (n = 4) were used
to determine whether DEX treatment altered the binding of nuclear
proteins from cytotrophoblasts to labeled oligonucleotides
corresponding to the B and AP-1 response elements. We observed that
a 48-h treatment of cytotrophoblasts with 100 nmol/L DEX markedly
reduced binding of nuclear extracts from cytotrophoblasts to the B
response element. DEX treatment promoted a relatively smaller reduction
of binding to the AP-1 response element. Northern blotting experiments
revealed that DEX treatment did not alter the level of IB, p50, or
p65 messenger ribonucleic acid, suggesting that the antiinflammatory
action of glucocorticoid in cytotrophoblasts did not directly involve
alterations in the level of NF-B proteins. Our results demonstrate a
novel chronic suppressive action of glucocorticoid on cytokine
production and nuclear binding of NF-B and AP-1 proteins in
cytotrophoblasts, providing a potential mechanism through which
glucocorticoids may suppress inflammation at maternal-fetal interfaces
across gestation. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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B (NF-B) and activator protein-1 (AP-1) (7), a clear
antiinflammatory role of glucocorticoid in placenta has not been
established. Our working hypothesis is that glucocorticoids maintain
human pregnancy by chronically suppressing the proinflammatory activity
of the transcription factors NF-B and AP-1 in placenta. Histological examination of human placenta as well as the detection of inflammatory cytokines at this site (8, 9, 10) suggest that inflammatory processes may be activated at this site even in the absence of intrauterine infection. We suggest that elevated levels of glucocorticoids present during pregnancy may play a role in suppressing inflammation in the uterus, placenta, and fetal membranes and thereby contribute to the homeostasis necessary for the maintenance of gestation.
Antiinflammatory actions of glucocorticoid appear to result from
inhibition of transcription of genes that code for proteins involved in
inflammatory processes (7, 11). Many of these genes are controlled by
the transcription factors AP-1 and NF-B (7). Interleukin-8 (IL-8) is
a member of the chemokine family, identified initially as a neutrophil
chemotactic factor (12, 13), whose genomic DNA contains binding sites
for AP-1 and NF-B in its 5'-flanking region (14). High levels
of IL-8 expression by the placenta have been documented in both
in vivo (15) and in vitro (16) studies.
The purpose of the present study was to examine the role of
glucocorticoids in placental inflammation by assessing their chronic
effects on mediators of inflammation in cytotrophoblasts isolated from
human term placentas. Cytotrophoblasts can be obtained in high yield
with excellent purity (17) and have been used by our group (4, 18, 19)
and others (20, 21) as a model of human placental protein and gene
expression. Our results indicate that glucocorticoids chronically
suppress both the nuclear binding of NF-B and AP-1 as well as the
production of IL-8 in cytotrophoblasts. In this cytotrophoblast model,
we have demonstrated that glucocorticoids may modulate inflammation in
the placenta across gestation via their chronic negative action on
these proinflammatory nuclear transcription factors.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Culture media and calf serum were obtained from Flow
Laboratories (McLean, VA). FCS was obtained from HyClone
Laboratories, Inc. (Logan, UT). Laboratory plasticware was
purchased from Falcon, Becton Dickinson and Co. Labware
(Lincoln Park, NJ). The medium supplement ITS+ was obtained
from Collaborative Research (Bedford, MA). All other
materials used in the isolation of cytotrophoblasts were obtained from
previously described sources (4). GeneClean II kits, used to purify
plasmids and inserts, were purchased from BIO 101, Inc. (La Jolla, CA).
The Ultraspec ribonucleic acid (RNA) isolation system was purchased
from Cinna/Biotecx Laboratories, Inc. (Houston, TX).
[-32P]Deoxy-CTP was obtained from New England Nuclear
(Boston, MA). Deoxyribonuclease and the random primed DNA labeling kit
were purchased from Boehringer Mannheim (Indianapolis, IN).
Complementary DNAs (cDNAs) to the p105 and p65 subunits of NF-B were
gifts from Drs. Craig Rosen and Charles Kunsch of Human Genome
Sciences, Inc. (Rockville, MD) (22). The cDNA clone to mouse IB
was a gift from Dr. Shigeki Miyamoto of the University of
Wisconsin-Madison and was originally cloned by Dr. Inder M. Verma of
the Salk Institute (San Diego, CA) (23). The enzyme-linked
immmunosorbent assay (ELISA) kits used for quantifying levels of IL-8,
IL-6, and tumor necrosis factor- (TNF) were purchased from
R&D Systems (Minneapolis, MN). The gel shift assay systems
including oligonucleotides corresponding to NF-B and AP-1 response
elements were purchased from Promega Corp. (Madison, WI).
Antibodies against p65 (SC-109X), Fos (SC-52X), and Jun (SC-45X) were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA).
Cell culture
We have previously (4, 18) isolated cytotrophoblasts from human term placentas with purities of 95% or more by the method of Kliman et al. (17), as modified by Douglas and King (24). For experiments, placentas were obtained from women undergoing elective, uncomplicated cesarean section between 37–42 weeks gestation. Briefly, approximately 90 g villous tissue were collected and digested with deoxyribonuclease and trypsin and separated on a continuous Percoll gradient as previously described (4). Cytotrophoblasts sedimented as a ring of cells at a density of approximately 1.05 g/mL. The yield of Percoll-purified cytotrophoblasts was 4–6 x 108 cells/150 g villous tissue. Cells were washed and resuspended in a 1:1 mixture containing phenol red-free Ham’s F-12-DMEM supplemented with 10% charcoal-stripped calf serum (4) and ITS+ (a supplement used to obtain a final concentration of 6.25 µg/mL insulin, 6.25 µg/mL transferrin, 6.25 ng/mL selenous acid, 1.25 mg/mL BSA, and 5.35 µg/mL linoleic acid), i.e. SCS medium. Cells were inoculated in SCS medium with or without 100 nmol/L dexamethasone (DEX) at a density of 0.5 x 106 cells/well of a 24-well dish for ELISA studies and 15 x 106 cells/10-cm dish for electrophoretic mobility shift assays (EMSAs) and Northern blotting procedures. Cells were maintained at 37 C in a humidified atmosphere of 5% CO2-95% air.
EMSA
Nuclear protein extracts from cytotrophoblasts were prepared by
the method of Hoppe-Seyler et al. (25). The concentration of
nuclear protein was quantitated using the DC Protein Assay from
Bio-Rad Laboratories, Inc. (Hercules, CA). Between 2–8
µg total nuclear protein were preincubated in binding buffer
consisting of 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L
ethylenediamine tetraacetate, 0.5 mmol/L dithiothreitol, 50 mmol/L
NaCl, 50 µg/mL poly(dI-dC), and 10 mmol/L Tris-HCl (pH 7.5) in a
reaction volume of 10 µL. The samples were then incubated for 20 min
at room temperature with approximately 5000 cpm 32P-labeled
oligonucleotides corresponding to NF-B and AP-1 response elements
(Promega Corp.). End-labeling reactions were performed
using T4 polynucleotide kinase. The reaction was halted by adding 1
µL 10 x gel loading buffer consisting of 0.2% bromophenol
blue, 0.2% xylene cyanol, 40% glycerol, and 250 mmol/L Tris-HCl (pH
7.5). The samples were then loaded onto a 5% nondenaturing
polyacrylamide gel. To assess the specificity of nuclear binding,
reactions were carried out with a 10-fold excess of unlabeled
competitor oligonucleotide or in the presence of 3 µL antibody to
p65, c-Fos, or c-Jun (Santa Cruz Biotechnology).
Northern blotting
RNA was extracted from cytotrophoblasts using the Ultraspec RNA
isolation system according to instructions provided by the
manufacturer. Approximately 15–20 µg RNA/sample were separated on a
1% (wt/vol) agarose gel containing 2.2 mol/L formaldehyde (26). After
transfer of RNA to nylon membranes (Zeta-Probe, Bio-Rad
Laboratories, Hercules, CA), levels of p65, p105 (the p50
precursor), and IB messenger RNA (mRNAs) were detected using
32P-labeled cDNA probes generated by random primer
synthesis (26). Plasmid DNA was isolated using the Wizard Maxiprep kit
(Promega, Madison, WI).
Enzyme-linked immunoassay
Levels of IL-8, IL-6, and TNF in culture media were
determined by immunoassay (Quantikine, R&D Systems). For
experiments, media were added to 96-well dishes coated with cytokine
antibody, and washing and treatment with secondary antibody were
performed as described by the manufacturer. Levels of cytokines were
derived from optical densities using a microtiter plate reader and the
Soft Max software program (Molecular Devices, Menlo Park,
CA) and were normalized to total cell protein. Statistical analysis
using Student’s t test was carried out with the SigmaStat
software package (Jandel, San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To investigate the chronic effect of glucocorticoid treatment on
cytokine expression in human placenta, cytotrophoblasts isolated from
human term placentas were maintained for 1–3 days in SCS medium with
or without 100 nmol/L DEX, and levels of IL-8 in culture media were
determined by ELISA (Fig. 1
). DEX
treatment reduced levels of IL-8 to 18%, 25%, and 26% of control
values on days 1, 2, and 3, respectively. We found that DEX and
cortisol mediated a dose-dependent inhibition of medium levels of IL-8,
with ED50 values of approximately 5 and 50 nmol/L,
respectively (Fig. 2). The observation
that DEX was approximately 10-fold more potent than cortisol could
reflect oxidation and inactivation of cortisol by placental
11ß-hydoxysteroid dehydrogenase (27).
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were then measured in the culture medium. As
shown in Table 1, DEX treatment
significantly reduced the expression of all three cytokines compared to
the control values, suggesting that glucocorticoids coordinately
suppresses cytokine expression in cytotrophoblasts.
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B element in
cytotrophoblasts
As IL-8 gene expression is known to be regulated by the nuclear
binding proteins NF-B and AP-1 (11, 12), EMSAs were carried out to
examine the effect of glucocorticoid treatment on nuclear binding
activity in cytotrophoblasts. Cells were maintained for 48 h in
SCS medium with and without 100 nmol/L DEX, nuclear binding proteins
were extracted, and EMSA was performed in four independent experiments.
In two representative experiments, shown in Fig. 3, we observed that DEX treatment
markedly reduced binding to the 32P-labeled oligonucleotide
corresponding to the B response element. Binding to the response
element was inhibited by unlabeled specific oligonucleotide as well as
by the presence of anti-p65 antibody, indicating that specific binding
of NF-B protein(s) to the B response element was measured (Fig. 3, lower panel). Conversely, the presence of unlabeled
nonspecific oligonucleotide did not affect binding to the B response
element (not shown). Our finding that the presence of antibodies to
NF-B proteins disrupted and did not hypershift DNA-protein complexes
has been observed by others (28).
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), these effects were not as pronounced
as those on B binding. Competition by antibodies to c-Fos and c-Jun
or by an unlabeled specific oligonucleotide reduced binding to the AP-1
response element in both DEX-treated and control samples, demonstrating
the specificity of this effect (Fig. 4).
In the present study, treatment of cytotrophoblasts for 2 days with 100
nmol/L DEX did not affect levels of total nuclear protein (Table 2), suggesting that the
DEX-mediated suppression of nuclear binding to NF-B and AP-1
response elements was not due to a nonspecific reduction in the level
of nuclear protein. The presence of 125 ng/mL cycloheximide did not
affect glucocorticoid-mediated suppression of NF-B or AP-1 nuclear
binding (data not shown).
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B mRNA are unaffected by DEX
treatment
Northern blot analysis was performed to test the effect of DEX
treatment on levels of NF-B family mRNA in cytotrophoblasts
maintained for 1–4 days in SCS medium with and without 100 nmol/L DEX.
We observed that levels of IB and p65 mRNA were unaffected by DEX
treatment on all days studied when normalized to levels of GAPDH mRNA
(Fig. 5). The level of p105 (p50
precursor) mRNA was also not affected by glucocorticoid treatment (not
shown). This indicated that DEX suppression of B binding activity in
cytotrophoblasts was not mediated at the level of NF-B family mRNAs,
suggesting that direct interaction between the glucocorticoid receptor
and NF-B proteins may be responsible for the chronic
antiinflammatory action of glucocorticoid in the human placenta.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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NF-B, a transcription factor involved in the regulation of many
genes involved in inflammation, is predominantly formed as a
heterodimer from subunits p50 and p65 (11, 29). In quiescent cells,
NF-B is complexed in the cytoplasm to an IB protein that prevents
nuclear translocation and thus inhibits the transcriptional effects of
NF-B (11, 30). In activated cells, IB is degraded, and NF-B is
translocated to the nucleus to activate target genes (11, 30).
Glucocorticoids have been shown to inhibit NF-B activity in a
variety of cells and cell lines, including peripheral monocytes (31)
and endothelial cells (32). In separate reports, Scheinman et
al. working with HeLa cells (33) and Auphan et al.
working with Jurkat cells (34) found that induction of IB
synthesis was associated with glucocorticoid-mediated reduction in
NF-B activity. However, Brostjan (32) found that IB synthesis
was not required for this glucocorticoid effect in endothelial
cells.
In this study, we have shown by EMSA that NF-B and, to a lesser
extent, AP-1 nuclear binding were chronically (2–4 days) suppressed by
DEX treatment in cytotrophoblasts isolated from human term placenta.
The production of IL-8, which appears to be regulated primarily by
NF-B (35, 36), was also found to be chronically suppressed by
glucocorticoids in cytotrophoblasts. It is not surprising that DEX also
inhibited the expression of IL-6 and TNF in cytotrophoblasts,
because these cytokines contain B-binding sites in their respective
genes (37, 38). We also observed that glucocorticoid treatment did not
alter levels of p65, p105 (i.e. the p50 precursor), and
IB mRNAs, as assessed by Northern blotting.
Glucocorticoid-mediated inhibition of NF-B nuclear binding was not
inhibited by cycloheximide treatment. This indicates that chronic
suppression of IL-8 expression and NF-B nuclear binding in
cytotrophoblasts by DEX was not mediated by changes in the production
of NF-B, IB, or perhaps any protein. Direct protein-protein
interaction among the glucocorticoid receptor, NF-B, and AP-1 has
been implicated in glucocorticoid-mediated down-regulation of cytokine
gene expression in other cell types (37, 39, 40, 41). In light of our
finding that de novo synthesis of NF-B proteins was not
required for the chronic suppressive effects of glucocorticoid, we
suggest that protein-protein interactions between glucocorticoid
receptor and NF-B may be a mechanism for the chronic action of
glucocorticoid in placenta.
The release of NF-B from IB and its subsequent translocation to
the nucleus do not require protein synthesis, which allows for rapid
activation of its target genes (42). Rapid activation may be critical
to survival of the organism, because this family of genes is intimately
involved in host defense processes. For this reason, many studies on
the control of the Rel/NF-B/IB family have examined short time
courses, i.e. minutes to hours (31, 32, 33). More recently,
attention has been focused on the role of NF-B in chronic
inflammatory processes (43, 44). These results may be more relevant
toward elucidating the mechanism of the chronic antiinflammatory action
of glucocorticoid in placenta during human gestation, a process that
lasts 9 months.
It is interesting in our studies that IL-8 was constitutively
expressed, and NF-B and AP-1 nuclear binding occurred in cultured
cytotrophoblasts, in the absence of classical acute inflammatory
stimuli (e.g. lipopolysaccharide and IL-1). In most cell
types, IL-8 production is not constitutive; an inflammatory stimulus is
required for production (45). However, Shimoya and colleagues (16) also
showed IL-8 production in unstimulated placental explants from first,
second, and third trimester pregnancies, and more recently, IL-8 mRNA
was detected by PCR in human gestational tissues from both laboring and
nonlaboring patients (9).
In most cells, NF-B is sequestered in the cytoplasm and remains
inactive unless the cell is appropriately stimulated. However,
constitutive activation of NF-B is known to occur in mature B cells
(46), breast cancer cells, and Hodgkin’s disease tumor cells (47, 48).
Our study has demonstrated that in the absence of glucocorticoid,
cytotophoblasts produce IL-8 constitutively, and the most likely
mechanism for this production is chronic activation by NF-B. The
elevated glucocorticoid levels present during pregnancy may be
essential in suppressing NF-B and possibly AP-1 activities and
ensuing inflammation in the placenta.
Histological studies revealed that the incidence of placental inflammation in uncomplicated deliveries at term was extremely low (8). Therefore, it is likely that parturition is not associated with alterations in the antiinflammatory action of glucocorticoid in human placenta, but, rather, is promoted by glucocorticoid stimulation of placental CRH synthesis (49) and/or antagonism of progesterone action (5).
In summary, our results document a chronic antiinflammatory action of glucocorticoid in human placental cytotrophoblasts. Glucocorticoid-mediated suppression of inflammation at uterine-placental interfaces may play an important role in the maintenance of human pregnancy.
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Acknowledgments |
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Footnotes |
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Received March 23, 1998.
Revised June 24, 1998.
Accepted July 1, 1998.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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mediation of immunosuppression by glucocorticoids. Science. 270:283–286.B activity through induction of IB. Science. 270:286–289.B and the glucocorticoid receptor. Proc Natl Acad Sci USA. 91:752–756. gene promoter. J Exp Med. 178:1365–1379.B by phosphorylation of its inhibitor IB. Nature. 344:678–682.[CrossRef][Medline]
B: a
pivotal transcription factor in chronic inflammatory diseases. N
Engl J Med. 336:1066–1071.B/Rel family proteins during B-cell
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B/Rel expression and the pathogenesis of breast
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). Medium levels of IL-8 were determined by ELISA
and are expressed as the mean ± SE for triplicate
culture wells. SEs that fell within the symbols are not
presented.
