Chorionic gonadotropin induces dendritic cells to express a
Hui Wan,*,1Marjan A. Versnel,* Lonneke M. E. Leijten,* Cornelia G. van Helden-Meeuwsen,*
Durk Fekkes,†Pieter J. M. Leenen,* Nisar A. Khan,* Robbert Benner,*
and Rebecca C. M. Kiekens*
Departments of *Immunology and†Neuroscience and Psychiatry, Erasmus MC, Rotterdam, The Netherlands
onic gonadotropin (hCG) has been suggested to
play an immunoregulatory role in addition to its
endocrine function, thus contributing to the pre-
vention of fetal rejection. We hypothesized that
hCG is involved in the maternal-fetal immune tol-
erance by the regulation of dendritic cell (DC)
function. Therefore, we studied the effect of hCG
on DC maturation. Upon hCG treatment in combi-
nation with LPS, mouse bone marrow-derived DC
(BMDC) increased the ratio of IL-10:IL-12p70,
down-regulated TNF-?, and decreased antigen-
specific T cell proliferation. Addition of hCG to-
gether with LPS and IFN-? blocked MHC class II
up-regulation, increased IL-10 production, and
decreased the antigen-specific T cell proliferation
by DC. Splenic DC showed similar results. Upon
hCG treatment, IDO mRNA expression and its me-
tabolite kynurenine were increased by LPS- and
IFN-?-stimulated DC, suggesting its involvement in
the decreased T cell proliferation. To study the
effect of hCG on DC differentiation from precur-
sors, BMDC were generated in the continuous
presence of hCG. Under this condition, hCG de-
creased cytokine production and the induction of
T cell proliferation. These data are suggestive
for a contribution of hCG to the maternal-fetal
tolerance during pregnancy by modifying DC to-
ward a tolerogenic phenotype. J. Leukoc. Biol.
83: 000–000; 2008.
The pregnancy hormone human chori-
Key Words: hCG ? DC ? IDO ? tolerance
Immune-stimulating dendritic cells (DC) are potent APC that
process antigens and up-regulate costimulatory molecules to
activate naı ¨ve T cells and induce an adaptive response. In
addition, DC regulate the adaptive immune response by Th1/
Th2 skewing. The production of IL-12 by DC, especially the
increased ratio of IL-12:IL-10, promotes a Th1 response [1–3].
LPS is commonly used to mature DC toward an immune-
stimulating phenotype. These immune-stimulating DC are
characterized by high MHC class II and costimulatory mole-
cule surface expression, increased IL-12 production compared
with tolerogenic DC, and high T cell activation capacity . In
addition to LPS, IFN-? is used to activate myeloid cells such
as DC and macrophages to increase MHC II expression .
Conversely, DC that induce tolerance, the so-called tolerogenic
DC, can have an immature or a mature phenotype. An impor-
tant difference between immunogenic DC and tolerogenic DC
lies in the level of IL-10 production, which is higher in
tolerogenic DC than immunogenic DC [6–9].
Human chorionic gonadotropin (hCG) is a placental glyco-
protein mainly secreted by trophoblasts during pregnancy.
hCG induces the production of progesterone and estrogen
during early pregnancy, but in addition, it is proposed to have
immunosuppressive effects. During pregnancy, the maternal
immune system undergoes alterations that help to tolerate the
fetus during intrauterine life. High hCG levels were found to
coincide with the development of peritrophoblastic immune
tolerance . Meanwhile, pregnancy biases toward a Th2
condition supported by increased systemic levels of Th2-type
cytokines such as IL-4, IL-5, and IL-10. This Th2 condition
shift might be related to the observation that many Th1-type
autoimmune diseases remit during pregnancy and recur after
childbirth. This has led to the proposal that the change of
pregnancy-related factors such as hCG has an immunosuppres-
sive effect and influences the severity of autoimmune diseases
We found that hCG treatment can prevent autoimmune
diabetes . The repeated injection of hCG into 14-week-old
NOD mice, a spontaneous model for type I diabetes, prevented
the occurrence of diabetes in this model and reversed the
inflammatory infiltration of the pancreas by lymphocytes and
macrophages. In addition, the transfer of splenocytes, contain-
ing DC and lymphocytes, from hCG-treated NOD mice into
immunocompromised NOD-scid mice, inhibited the develop-
ment of diabetes . Considering the importance of DC
orchestrating the immune response [14, 15], the effect of hCG
in diabetes development in NOD mice might be a result of a
direct effect of hCG on DC. Interestingly and in line with this
hypothesis, it has been reported that DC bear the receptor for
1Correspondence: Department of Immunology, Erasmus MC, Dr. Molewa-
terplein 50, NL-3015 GE Rotterdam, The Netherlands. E-mail: h.wan@
Received April 27, 2007; revised October 22, 2007; accepted December 1,
0741-5400/08/0083-0001 © Society for Leukocyte Biology
Journal of Leukocyte Biology
Volume 83, April 2008
Uncorrected Version. Published on January 2, 2008 as DOI:10.1189/jlb.0407258
Copyright 2008 by The Society for Leukocyte Biology.
hCG . Earlier, it has been shown that hCG itself has an
activating effect on human peripheral blood-derived DC. A
3-day culture of these cells in the presence of GM-CSF without
other stimulation resulted in increased expression of costimu-
latory molecules, unchanged HLA-DR, increased induction of
allogeneic T cell proliferation, and cytokine secretion (IL-12
and IFN-?) . However, there are no data about the role of
hCG on stimulated DC, and that is important for the under-
standing of the maternal-fetal tolerance during pregnancy.
Furthermore, it is relevant for the putative, therapeutic effect of
hCG in autoimmune diseases.
Ueno et al.  recently reported that hCG injection into
NOD mice inhibited diabetes development, and this protective
effect was associated with an increased expression of IDO by
DC. From this study, it is unclear, however, whether the
up-regulation of IDO is a direct or indirect effect of hCG on DC
. IDO is an immunoregulatory enzyme that degrades tryp-
tophan to the metabolic products known as kynurenines. IDO
has been found to be increased in the placenta in the first
weeks of pregnancy [19, 20]. DC are present at the maternal-
fetal interface, and IDO expression was up-regulated on these
DC during pregnancy . Mature DC (mDC) that express IDO
enzyme activity are potent suppressors of T cell responses and
promote the development of regulatory T cells (Tregs) .
Such Tregs can secrete IL-10, and this cytokine subsequently
helps to sustain expression of functional IDO in mDC .
This loop is supposed to result in the development of immune
tolerance of the maternal immune system against trophoblasts
with paternal antigen expression [21, 24]. Indeed, administra-
tion of an IDO inhibitor caused allogeneic fetal rejection .
Interestingly, recurrent pregnancy loss has been observed in a
patient that developed anti-hCG autoantibodies . The co-
localization of IDO and hCG in trophoblasts and placenta and
their similar role in the immunological tolerance between
mother and fetus suggest that hCG could be a direct inducer of
In this study, we investigated the effect of hCG on DC
function. Upon activation of DC by LPS and IFN-?, additional
hCG treatment resulted in hampered MHC class II expression,
increased IL-10, and increased IDO expression, which all
resulted in a decreased ability to stimulate T cell proliferation.
This is important to understand the role of hCG in successful
pregnancy against rejection and its therapeutic effect on auto-
MATERIALS AND METHODS
Specific pathogen-free C57BL/6 and C3HeB/FeJ female mice were purchased
from Harlan (Horst, The Netherlands) and were housed in microisolator cages
and given mouse chow and water ad libitum in the animal care facility at
Erasmus MC (Rotterdam, The Netherlands). OT-II (OVA-TcR transgenic)
female mice were bred according to standard procedures. Mice were 8 weeks
of age when killed for the isolation of bone marrow (BM) cells and naı ¨ve T cells
from the spleen. All experiments were performed with approval of the Erasmus
MC animal welfare committee.
BM cell isolation
Femora were removed from C57BL/6 mice. BM cells were flushed out of the
bones with culture medium RPMI 1640 (BioWhittaker, Verviers, Belgium),
supplemented with 10% FCS (Perbio, Etten-Leur, Netherlands), 100 U/ml
penicillin, and 100 ?g/ml streptomycin (BioWhittaker). The BM cells were
filtered through a 100-?m sieve (BD Biosciences, Erembodegen, Belgium) and
resuspended in cold culture medium (4°C). Cells were used after storage in
liquid nitrogen with essentially the same results.
BM-derived DC (BMDC) culture and stimulation
BMDC were generated by thawing BM cells and culturing in petri dishes
(Becton Dickinson, Le Pont De Claix, France) at a concentration of 2 ? 106
live cells per petri dish (A 10 cm) in 10 ml culture medium containing 20
ng/ml recombinant mouse (rm)GM-CSF (Biosource International, Camarillo,
TX, USA) in a 37°C, 5% CO2incubator. At Day 3, medium containing
rmGM-CSF was refreshed. At Day 5, another 10 ml medium with rmGM-CSF
was added to the cultures.
At Day 6, nonadherent cells were collected by adding 0.05% EDTA (Fluka,
Buchs, Switzerland) for 20 min at 37°C. The adherent cells were detached and
collected. All cells were incubated with an antibody against CD86 [American
Type Culture Collection (ATCC), Manassas, VA, USA] and anti-rat IgG mi-
crobeads, followed by AutoMACS separation (both from Miltenyi Biotec,
Bergish Gladbach, Germany). The CD86-negative cells, representing immature
DC (iDC), were collected, counted, and used in the following experiments.
For cell activation, iDC were transferred into 96-well flat-bottom plates
(Nunc A/S, Roskilde, Denmark), 0.5 ? 106cells/well, and stimulated with the
following stimuli: 50 ng/ml LPS (Sigma Chemical Co., St. Louis, MO, USA),
100 ng/ml IFN-? (rmIFN-?; Biosource, Nivelles, Belgium), or 50 ng/ml LPS ?
100 ng/ml IFN-?, with or without 150 U/ml hCG (Pregnyl, Organon, Oss, The
Netherlands). After overnight incubation, the culture supernatants were col-
lected, and the cells were harvested for flow cytometry and T cell proliferation
To study the effect of hCG on DC differentiation, C57BL/6 BM cells were
cultured for 6 days in the presence of rmGM-CSF, with or without 150 U/ml
hCG. Cells were stimulated overnight with LPS in the presence or absence of
hCG. At Day 7, the culture supernatants were collected, and the cells were
To exclude the possible contamination of hCG by LPS, polymyxin B (Sigma
Chemical Co.), an antibiotic known to inhibit activities induced by LPS, was
applied. The observed effects of hCG were found not to be caused by LPS
Splenic DC isolation and stimulation
Splenic DC were obtained from C57BL/6 mice. From a spleen cell suspension,
RBCs were lysed by Gey’s medium (Millipore, Billerica, MA, USA), and cells
were incubated with CD11c-labeled microbeads (Miltenyi Biotec), followed by
positive selection using the AutoMACS.
CD11c?DC (0.5?106cells/well) were seeded into 12-well flat-bottom
plates (Nunc A/S) and stimulated with 50 ng/ml LPS ? 100 ng/ml IFN-?, with
or without 150 U/ml hCG. After overnight incubation at 37°C, the culture
supernatants were collected, and the cells were harvested for flow cytometry.
At Day 7, BMDC were collected and labeled with the following antibodies:
ER-TR3-bio (MHC II, BMA, Augst, Switzerland) and streptavidin-allophyco-
cyanin, CD86-FITC, and CD80-PE (all from BD PharMingen, Erembodegen,
Belgium). After labeling, the BMDC were resuspended in 7-aminoactinomycin
D (Molecular Probes, Leiden, Netherlands) to exclude dead cells and analyzed
on a FACSCalibur apparatus. Data were analyzed using CellQuest software
ELISA kits for IL-10, IL-6, and TNF-? (Biosource) and IL-12p40, IL-12p70
(R&D Systems, Oxon, UK) were used according to the protocols supplied by
the manufacturer. Briefly, plates were coated with capture antibody for 18 h
and washed with PBS-Tween (0.05%). Diluted supernatants and standards
were added and incubated for 2 h at room temperature. After that, the
biotin-labeled detection antibody was added, followed by incubation with
streptavidin-HRP for 30 min. Chromogen substrate tetramethylbenzidine was
added for 30 min, followed by the addition of the stop solution. In between
incubations, the plates were washed with PBS-0.05% Tween. The OD of the
2Journal of Leukocyte Biology
Volume 83, April 2008
product solution was measured at 450 or 450 ? 650 nm by an ELISA reader
(Thermo Labsystems, Finland).
Antigen-specific T cell proliferation
For determination of antigen-specific T cell proliferation, naı ¨ve T cells were
obtained from the spleens of OT-II mice, after lysis of the RBCs by Gey’s
medium (Millipore) and incubation with CD11b ? CD45R ? MHC II anti-
bodies (hybridoma supernatant M1/70, B220, and M5/114 for CD11b, CD45R,
and MHC II, respectively, ATCC) and anti-rat IgG microbeads (Miltenyi
Biotec), followed by AutoMACS separation of the negative cells. DC were
collected and pulsed with OVA peptide 323–339 (ISQAVHAAHAEINEAGR,
4 ?M) for 2 h at 37°C, followed by the addition of OT-II T cells that recognize
this peptide. After culturing T cells (1.5?105cells/well) and DC (0.3?105
cells/well) in round-bottom 96-microwell plates (Nunc A/S) for a period of 3
days, proliferation of T cells was measured by uptake of3H-thymidine (1
?Ci/well, DuPont-NEN, Boston, MA, USA) and expressed as cpm. The cocul-
ture supernatants were also collected for IL-10 ELISA.
Naı ¨ve T cells were obtained from spleens of C3HeB/FeJ mice as described
above. BMDC were added to T cells (ratio 1:5). After coculture in RPMI-1640
culture medium containing 10% FCS, 60 mg/ml penicillin, and 100 mg/ml
streptomycin for 3 days, proliferation of T cells was measured by uptake of
3H-thymidine (1 ?Ci/well, DuPont-NEN) over a period of 16 h and expressed
Quantitative real-time PCR (qRT-PCR) of IDO
iDC were stimulated with 50 ng/ml LPS, 100 ng/ml IFN-?, or 50 ng/ml LPS ?
100 ng/ml IFN-?, with or without 150 U/ml hCG. After 0, 2, 4, 6, 8, and 10 h
of incubation at 37°C, cells were collected and lysed. RNA was extracted using
an RNeasy mini kit (Qiagen, Hilden, Germany), according to the protocol
supplied by the manufacturer. cDNA was synthesized using the Superscript
first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA, USA),
according to the manufacturer’s instructions. qRT-PCR was performed and
analyzed using an ABI 7700 sequence detection system (Applied Biosystems,
Foster City, CA, USA) and Taqman probe-based chemistry. Primers for mouse
IDO (Mm00492586_m1) and ABL were obtained from Primer Express™
(Applied Biosystems). Each PCR sample was run in duplicate. The mean value
of the two reactions was defined as representative of the sample. The resulting
IDO cycle threshold (CT) values were corrected relative to ABL CT values. To
facilitate interpretation of results, we have used the following equation for all
figures: 2 (ABL–IDO) ? 100%. Therefore, an increase is proportional to an
increase in expression of the particular target gene.
Concentrations of kynurenine were determined using an isocratic, reversed-
phase HPLC (Agilent, Santa Clara, CA, USA) and fluorometric detection
(Jasco, Tokyo, Japan). The analytical column consisted of a 250 ? 2.1-mm
intradermal (i.d.) column packed with 5 ?m particles of GraceSmart RP-18
(Grace Davison Discovery Sciences, Deerfield, IL, USA), which were protected
by a guard cartridge column (4.0?2.0 mm i.d.) containing Phenomenex C18
material. A Hewlett Packard ChemStation (Hewlett Packard, Agilent) was used
for data collection and handling.
For kynurenine determination, 100 ?l specimen was mixed with 20 ?l 300 ?M
1-methyltryptophan (internal standard) in 14% (w/v) trichloroacetic acid. This
mixture was placed on ice for 10 min and centrifuged at 12,000 g and 4°C for 15
min. Supernatant (80 ?l) was transferred to a HPLC vial and mixed with 20 ?l 0.6
M LiOH. The sample (15 ?l) was injected onto the column, and HPLC was carried
out at a flow rate of 0.4 ml/min and a column temperature of 40°C. Mobile phase
was potassium phosphate buffer (50 mmol/L, pH 3.6) containing 5% (v/v) meth-
9.9 min) were detected via their natural fluorescence at excitation and emission
wavelengths of 363 and 500 nm and 285 nm and 365 nm, respectively. Quanti-
tation was done by measuring peak height relative to a calibration mixture.
Recoveries (mean?SD) of kynurenine and 1-methyltryptophan were 100 ? 13 and
87 ? 4%, respectively. The intra- and interassay coefficients of variation for both
compounds were less than 2% and 4%, respectively.
Data are expressed as mean values ? SEM in all the figures. All statistical
analyses were performed using logarithmic transformation and/or Student’s
paired t-test. P values ?0.05 were considered significant; *, P ? 0.05; **,
P ? 0.01; ***, P ? 0.001.
hCG treatment hampers up-regulation of MHC
class II expression by LPS ? IFN-?-stimulated
The effect of hCG on DC activation was studied by stimulating
iDC with LPS or LPS ? IFN-? in the presence or absence of
hCG. Unstimulated cells exhibited after overnight culture a
small, spontaneously matured population (Fig. 1A). LPS fully
Fig. 1. Hampered up-regulation of MHC
class II expression by BMDC upon hCG
treatment. C57BL/6 BM cells were cultured
for 6 days in the presence of rmGM-CSF.
CD86–iDC were isolated and stimulated
with PBS, LPS, or LPS ? IFN-?, with or
without hCG. After 18 h, the cells were
collected and analyzed by flow cytometry.
(A and B) Dot-plot and histogram of a dou-
ble staining for MHC class II and CD86 by
DC after PBS (control, filled histogram) or
LPS ? IFN-? stimulation in the presence
(dotted line) or absence (dark line) of hCG.
(C) MHC class II expression [mean fluores-
cence intensity (MFI)] by gated CD86high
CD80highmDC after LPS or LPS ? IFN-?
stimulation in the presence or absence of
hCG. The data are the mean of four to six
experiments; *, P ? 0.05.
Wan et al.
hCG induces DC to express a tolerogenic phenotype3
matured DC, resulting in high expression of CD86, CD80, and
MHC class II. The addition of hCG did not change the expres-
sion of these markers; CD40, CD11c, and F4/80 also remained
unchanged upon hCG treatment (data not shown). When LPS
? IFN-? was added, an additional 15% CD86highCD80high
mDC was obtained compared with LPS stimulation alone (Fig.
1A). The expression of CD80, CD86, and CD40 was increased
twofold in a dose-dependent way after stimulation with LPS ?
IFN-? compared with LPS stimulation alone. F4/80 and MHC
class II expression was unchanged (data not shown and Fig.
1C). The addition of hCG hampered the up-regulation of MHC
class II on LPS ? IFN-?-activated DC, seen for percentage of
cells and expression level (measured as MFI; Fig. 1, A–C). No
change in the expression of CD86, CD80, CD40, CD11c,
F4/80, chemokine receptor CCR5, and DC marker DEC205
was observed upon hCG addition (data not shown).
hCG alters cytokine production of BMDC
LPS-activated iDC secreted IL-10, TNF-?, and IL-6. The
addition of hCG resulted in an increase in IL-10 and a de-
crease in TNF-? and IL-6 secretion (Fig. 2A).
Activation of iDC with LPS ? IFN-? resulted in a significant
up-regulation of IL-12p70, IL-10, and TNF-? compared with
LPS stimulation alone (Fig. 2, A and B). hCG treatment of LPS
? IFN-?-stimulated BMDC significantly increased TNF-? and
IL-10 production by 15% and 30%, respectively, and IL-6 for
more than twofold (Fig. 2B). Under both stimulation conditions,
IL-12p70 production did not change upon hCG treatment. It
should be noticed that the absolute production of TNF-? and
IL-6 by LPS- and LPS ? IFN-?-stimulated BMDC differs,
which suggests that the decreases in TNF-? and IL-6 upon
hCG treatment by LPS-stimulated BMDC, although statisti-
cally significant, are biologically irrelevant (Fig. 2, A and B).
To further investigate if the decreased MHC class II by LPS ?
IFN-?-stimulated DC upon hCG treatment is via the induction
of TNF-? or IL-10, BMDC were cultured with neutralizing
anti-TNF-? or neutralizing anti-IL-10. Neutralizing TNF-? or
IL-10 did not result in restoration of MHC II expression (data
not shown). Alternatively, BMDC were cultured with rTNF-? or
rIL-10 to approximate the effects of hCG. No effects on MHC
class II expression were observed upon the addition of TNF-?
or IL-10 (data not shown).
Effect of hCG on splenic DC
To study the effects of hCG on a resident DC population,
splenic CD11c?cells were isolated (purity ?85%) and stim-
ulated by LPS ? IFN-? ex vivo for 24 h followed by gating of
CD86highCD80highmDC. Addition of hCG to these cultures
declined the up-regulation of MHC class II (Fig. 3, A and B).
In line with the previous result obtained from BMDC, a sig-
nificant increase of IL-10 production by LPS- and LPS ?
IFN-?-stimulated, splenic DC was observed in the presence of
hCG (Fig. 3C). Under both conditions studied, IL-12p70 produc-
tion did not alter upon the addition of hCG (data not shown).
hCG treatment of BMDC hampers their ability to
stimulate antigen-specific T cell proliferation
In the next set of experiments, we tested the influence of hCG
treatment during LPS, LPS ? IFN-?, and IFN-? activation of
DC on the induction of OVA-specific CD4?T cell prolifera-
tion. The addition of hCG during DC maturation induced by
any of the tested stimuli resulted in a significant decrease in
their ability to stimulate antigen-specific T cell proliferation.
Treatment of iDC with hCG in the absence of additional
maturation stimuli enabled these cells to stimulate shown
antigen-specific CD4?T cell proliferation to a slightly higher
extent compared with untreated iDC (Fig. 4A).
Th1 and Th2 polarization capacity of DC was determined by
the measurement of the IL-4 and IFN-? production by T cells.
LPS- and LPS ? IFN-?-stimulated DC induced IFN-? but no
IL-4 production. The addition of hCG did not change this
Th1/Th2 balance (data not shown).
To study whether T cells stimulated by hCG-treated DC display
tolerogenic properties, the IL-10 level in the supernatants of
cocultures of T cell and DC was measured. Unstimulated and
PMA-stimulated T cells did not produce IL-10, nor did iDC-
activated T cells (Fig. 4B). LPS stimulated DC-activated T cells to
produce IL-10. hCG treatment to LPS-stimulated DC showed a
similar IL-10 level as LPS-stimulated cocultures without hCG.
The IL-10 production in LPS ? IFN-?-stimulated cocultures with
hCG treatment is significantly higher than LPS ? IFN-?-stimu-
lated cocultures without hCG treatment (Fig. 4B).
IDO involvement in the observed effect of hCG
To investigate whether the hCG-induced inhibition of T cell
proliferation might be mediated directly via the induction of
IDO, the mRNA expression of IDO was studied. IDO mRNA
Fig. 2. hCG altered cytokine production by LPS- or LPS ? IFN-?-stimulated
BMDC. iDC were isolated as CD86–cells from BMDC cultures and stimulated
with LPS or LPS ? IFN-?, with or without hCG. After 18 h, the culture
supernatants were collected and analyzed for the presence of IL-12p70, IL-10,
TNF-?, and IL-6 by ELISA. Cytokine production by (A) LPS (n?3)- or (B) LPS
? IFN-? (n?6)-stimulated DC, with or without hCG treatment, was shown; *,
P ? 0.05; **, P ? 0.01.
4Journal of Leukocyte Biology
Volume 83, April 2008
was found quickly induced from 2 h after LPS or IFN-?
stimulation and peaked at 4 h (data not shown). After 4 h of
stimulation, LPS induced IDO mRNA expression about seven-
fold and IFN-?-stimulated approximately 20-fold. The combi-
nation of LPS and IFN-? resulted in a more than hundred-fold
increased IDO expression compared with unstimulated BMDC
(Fig. 4C). The addition of hCG significantly increased the IDO
mRNA expression by BMDC upon stimulation with LPS or
IFN-? alone but not by LPS ? IFN-?-stimulated and unstimu-
lated BMDC (Fig. 4D). To analyze whether the increased IDO
mRNA expression upon hCG treatment was transcribed into
functional protein, the biological activity of induced IDO was
investigated by quantification of tryptophan and its catabolite
kynurenine in culture medium. hCG treatment significantly
increased the kynurenine level by BMDC upon stimulation
with LPS or IFN-? alone but not by LPS ? IFN-?-stimulated
and unstimulated BMDC (Fig. 4E). The level of tryptophan
remained unchanged upon hCG treatment, probably as a result
of the high concentration of tryptophan in the culture medium
(data not shown).
Effect of hCG on BMDC differentiation from
To investigate the effect of hCG on the differentiation of DC
from precursor cells, hCG was added from the start of the BM
cell cultures and refreshed at the same time when medium was
changed routinely. By applying double staining for Ly6C and
CD31 , the differentiation of BMDC was evaluated. The
continuous presence of hCG did not change the expression of
these differentiation markers as well as MHC class II and
CD11c (data not shown). Without LPS stimulation, DC pro-
duced low levels of cytokines, and the presence of hCG during
their development did not increase cytokine production. LPS
activated DC-secreted IL-12p40, IL-12p70, IL-10, TNF-?, and
IL-6. The continuous presence of hCG in the BMDC cultures
resulted in a decrease in the production of 12p40, IL-12p70,
IL-10, and TNF-? (Fig. 5A), as well as IL-6 (data not shown).
This generalized decrease is not related to increased cell
death, as Rhodamine123 staining revealed that similar, low
frequencies of dead cells were observed upon hCG addition
(data not shown). Different from the hCG effect on the matu-
ration of DC, MHC class II expression by LPS ? IFN-?-
activated BMDC did not change when hCG was added from the
beginning of the BM cell cultures (data not shown).
The allogeneic T cell proliferation after LPS stimulation was
decreased significantly when BMDC were cultured in the con-
tinuous presence of hCG (Fig. 5B).
We studied the effect of hCG on DC maturation and function to
explore its role in the maternal-fetal tolerance and the remis-
sion of several autoimmune diseases during pregnancy .
BMDC were stimulated to mature by LPS, and IFN-? in
combination with LPS was used for further maturation of DC.
hCG addition resulted in hampered MHC class II up-regulation
by DC stimulated with LPS ? IFN-? but not with LPS alone.
Upon stimulation of BMDC with LPS ? IFN-?, hCG signifi-
cantly increased TNF-? production by DC, whereas upon
stimulation with LPS alone, hCG significantly decreased
TNF-? production by DC. It has been shown that TNF-? can
suppress IFN-?-induced MHC class II expression . This
suggests that hCG may influence MHC class II via inhibition of
Fig. 3. Hampered up-regulation of MHC
class II expression and increased IL-10 pro-
splenic CD11c?DC cultured in the pres-
ence of hCG. Splenic CD11c?cells were
isolated from C57BL/6 mice and stimulated
with LPS ? IFN-?, with or without hCG.
Eighteen hours later, the cells were col-
lected and stained for CD86, CD80, and
MHC class II. In dot-plot (A) and histogram
(B), CD86highCD80highcells were gated,
followed by analysis of MHC class II ex-
splenic CD11c?DC with (dotted line) or
without (dark line) hCG treatment. Filled
histogram represents the isotype control. (C)
IL-10 production by splenic CD11c?cells
stimulated with LPS or LPS ? IFN-?, with
or without hCG. The results shown are a
single representative from three indepen-
dent experiments with a similar outcome.
Wan et al.
hCG induces DC to express a tolerogenic phenotype5
IFN-?-induced up-regulation of TNF-?, but our data showed
no effects of TNF-? on hCG-induced suppression of MHC class
II, nor did IL-10. In general, lower levels of MHC class II
expression are correlated with a lower capacity to present
antigen and to stimulate T cell proliferation and/or function.
Indeed, we observed decreased antigen-specific T cell prolif-
eration by LPS ? IFN-?-stimulated DC upon hCG treatment.
However, DC stimulated with LPS also exhibited a decreased,
antigen-specific T cell stimulation, and these particular DC did
not show impaired MHC class II expression. This indicates that
under these conditions, other hCG-induced factors are in-
volved as well.
Fig. 4. DC treated with hCG revealed a decreased ability to induce antigen-specific CD4?T cell proliferation and an increased IDO mRNA expression. (A)
Cultured C57BL/6 BMDC were stimulated with LPS, IFN-?, or LPS ? IFN-?, with or without hCG for 18 h. For OVA antigen-specific CD4?T cell proliferation
induction, DC were collected and pulsed with OVA for 2 h and then added to splenic T cells from OT-II mice. These DC and T cells (ratio 1:5) were cocultured
for 3 days. The proliferation of the T cells was determined by3H-thymidine incorporation. The supernatants from these cocultures were collected and analyzed
for IL-10 production (B). Data were obtained from three individual experiments. (C) iDC were stimulated with LPS, IFN-?, or LPS ? IFN-? for 4 h, and then cells
were collected for IDO qRT-PCR analysis; n ? 3. unst, Unstimulated. In the absence or presence of hCG, iDC were stimulated with LPS, IFN-?, or LPS ? IFN-?
for 4 h and then collected for IDO mRNA measurement by qRT-PCR (D), or supernatants of these cultures were collected at 24 h for measurement of kynurenine
and tryptophan (E). NS, Not significant. The picture was depicted as relative to non-hCG treatment in the same stimulation (n?3); *, P ? 0.05; **, P ? 0.01.
Fig. 5. Continuous presence of hCG during development of DC from BM precursors inhibits their ability to produce cytokines and stimulate T cell proliferation.
BMDC were cultured for 7 days in the presence or absence of hCG and stimulated with LPS, with or without hCG. After 18 h of incubation, the supernatants were
collected and evaluated for IL-12p40, IL-12p70, IL-10, and TNF-? content (A), and the cells were tested in the allogeneic T cell proliferation assay (B). Data
represent the mean of three individual experiments; *, P ? 0.05; **, P ? 0.01.
6 Journal of Leukocyte Biology
Volume 83, April 2008
A candidate hCG-induced factor that may contribute to
decreased APC function is IL-10. This cytokine has important
immunosuppressive functions. High production of IL-10 by DC
is a characteristic feature for tolerogenic DC. IL-10 is also
considered a Th2-type cytokine [3, 8]. In former experiments,
we showed that the splenic CD4?T cells from hCG-treated
NOD mice tend to produce more IL-10 . Here, we observed
an increased production of IL-10 but unchanged IL-12p70,
resulting in an increased IL-10:IL-12p70 ratio, from LPS- and
LPS ? IFN-?-activated BMDC treated with hCG. Increased
IL-10 production was also found in splenic CD11c?DC upon
hCG treatment. The change we observed in the IL-10:IL-12p70
ratio implies that hCG might contribute to a Th2 cytokine
environment and an immunosuppressive state during preg-
nancy. However, results from the in vitro T cell proliferation
assay did not show switching from Th1 to Th2 responses. This
is consistent with data in the literature demonstrating that
pregnancy is a condition characterized by increased Th2-type
cytokine production rather than a change in T cell polarization
[29, 30]. Furthermore, hCG treatment increased the IL-10
production by T cells cocultured with LPS ? IFN-?-stimulated
DC compared with cultures without hCG treatment. These
results suggest that hCG-treated DC can induce T cells with
IL-6 is thought to be a proinflammatory cytokine involved in
chronic inflammatory responses, but IL-6 also plays a crucial
anti-inflammatory role in local and systemic acute inflamma-
tory responses and helps Th0 cells differentiate into Th2 cells
[31, 32]. Depending on the stimulation conditions, hCG mod-
ified IL-6 production by DC differently. Our data indicate that
further studies are needed to elucidate the role of hCG-mod-
ulated cytokine production in immunosuppression during preg-
Stimulation of BMDC and splenic DC with LPS ? IFN-? but
not LPS only revealed a lack of up-regulation of MHC class II
expression upon hCG treatment, suggesting that IFN-? is in-
volved in this effect of hCG. IFN-? was found to trigger IDO
production in DC . IDO is an important down-regulator of
the immune response and is produced in large amounts by
Tregs [34, 35]. Induction of IDO expression in trophoblasts
suppresses T cell activity against fetus . Retarded intra-
uterine development is accompanied with a significantly lower
IDO activity in placenta . Administration of the IDO in-
hibitor 1-methyL-tryptophan caused allogenic fetal rejection
. Furthermore, in pregnant women, kynurenine levels, the
metabolite produced upon IDO activation, increase in blood
and urine in comparison with nonpregnant women of the same
age . The working mechanism of IDO is that it inhibits T
cell proliferation in vitro by rapidly consuming available tryp-
tophan, resulting in anergic T cells and increased levels of
proapoptotic kynurenines [39, 40]. Therefore, we investigated
whether hCG directly influenced the expression of IDO in DC.
Indeed, hCG was able to induce substantially more IDO mRNA
expression in LPS- or IFN-?-stimulated DC compared with
stimulation with LPS or IFN-? alone. This increased IDO
mRNA was accompanied by an increase in kynurenine levels.
These results are consistent with a role of IDO in the observed
hCG-induced inhibition of T cells. By influencing the IDO
expression by trophoblasts and DC, hCG might thus directly
contribute to the maintenance of tolerance at the maternal-fetal
interface, especially under conditions of immunological chal-
lenge [22, 41, 42].
hCG levels are systemically elevated in early pregnancy;
therefore, it is interesting to investigate what the influence of
hCG is on the development of DC from BM precursors. We
cultured BM cells in the continuous presence of hCG, followed
by maturation of BMDC in the presence of hCG. Under these
conditions, BMDC revealed a generally hampered cytokine
production as well as a decreased T cell proliferation, which
was not a result of cell apoptosis, postponed DC differentiation,
or down-regulation of the hCG receptor (unpublished data).
Notably, hCG binds to the G-protein-coupled receptor (GPCR)
that activates the Gs/cAMP pathway, which may be responsible
for the hampered IL-12 production by activated DC. The hCG
receptor is one of the large GPCR superfamilies. Binding of
hCG to the hCG receptor triggers a conformational change of
the transmembrane region of the receptor facilitating binding
and activation of Gs, followed by effector enzyme activation
and a subsequent intracellular adenylyl cyclase/cAMP signal-
ing pathway [43, 44]. The activated cAMP pathway inhibits
IL-12 expression in human DC . All of the above data
indicate that hCG may contribute to the maternal-fetal toler-
ance via modulating DC function.
In conclusion, hCG treatment of activated DC results in
hampered up-regulation of MHC class II expression, as well as
in increased IL-10 and IDO expression, which all lead to the
decreased ability to stimulate T cell proliferation. This modu-
lating influence of hCG on DC differentiation and function may
have an important contribution to maternal-fetal tolerance as
well as the remission of several autoimmune diseases during
This study was financially supported by Biotempt B.V., Koe-
kange, The Netherlands. We highly appreciate Marieke van
der Heide-Mulder for her skillful technical assistance and Tar
van Os for his support in preparation of the figures.
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