Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan
* Author for correspondence (e-mail: fuji{at}kuhp.kyoto-u.ac.jp.)
Accepted 18 July 2003
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SUMMARY |
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Immunohistochemistry demonstrated that C-C chemokine receptor 1 (CCR1) was
hardly detected on cytotrophoblasts and syncytiotrophoblast but was expressed
on EVTs in the cell column. In vitro, CCR1 protein was also present on the
surface of EVTs that grew out from chorionic villous explants cultured under
20% O2. Chemokines that can bind to CCR1 (CCR1 ligands), such as
regulated on activation, normal T cell expressed and secreted (RANTES) and
macrophage inflammatory protein-1 (MIP-1
), were confirmed in the
decidual tissues by RT-PCR and immunohistochemistry. These CCR1 ligands
promoted the migration of the EVTs that were isolated from the explant
cultures in vitro. These results indicate that CCR1 is expressed on
trophoblasts as they differentiate to EVTs and that CCR1 ligands produced from
the decidual tissue induce EVT migration.
By contrast, CCR1 was scarcely expressed on EVTs that grew out from villous explants cultured in 1% O2, indicating that a relatively high oxygenic environment is needed to induce CCR1 expression. Moreover, CCR1 expression on the isolated EVTs was significantly reduced in the presence of decidua-conditioned medium. Such regulation of CCR1 by surrounding oxygenic and decidual environments supports a close correlation between EVT invasion and their expression of CCR1.
This study demonstrates that trophoblasts acquire CCR1 as they differentiate to an invasive phenotype at the villus-anchoring sites and indicates a novel role for the chemokine-CCR1 system in the initial step of trophoblastic invasion towards the maternal tissue.
Key words: CCR1, Chemokine, Extravillous trophoblast, Migration, Cell column, Endovascular trophoblast
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Introduction |
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We previously reported a characteristic expression profile of DPPIV
(EC.3.4.14.5) on EVTs (Sato et al.,
2002). DPPIV, a membrane-bound peptidase, can metabolize its
substrates and regulate their bioactivity on the cell surface. DPPIV was
expressed intensely on EVTs in the proximal part of the cell column, but it
was downregulated in the distal part where EVTs acquired invasive activity.
This suggests that DPPIV modulated the bioactivity of some molecule(s) that
affect trophoblastic migration in situ. Supporting this idea, in JEG-3 cells
(a DPPIV-positive choriocarcinoma cell line), inhibition of cell-surface DPPIV
activity enhanced invasive activity without affecting proliferation. Recently,
DPPIV was demonstrated to metabolize several chemokines in vitro
(Van et al., 1999
). RANTES, a
member of the chemokine family, is a representative substrate of DPPIV and the
chemotactic activity of RANTES is abrogated by digestion with DPPIV
(Oravecz et al., 1997
).
Because RANTES is produced in human placental tissues
(Denison et al., 1998
) and can
induce the migration of leukocytes and other cell types
(Youngs et al., 1997
), we
speculated that this chemokine is a candidate for a DPPIV substrate that can
affect trophoblastic migration. To substantiate this possibility, we initially
examined the expression of RANTES receptors on trophoblasts. C-C chemokine
receptor 1 (CCR1), CCR3 and CCR5 are reported to be receptors for RANTES
(Murphy et al., 2000
), and
immunohistochemical experiments revealed that EVTs expressed CCR1.
Several chemokines, including CCR1 ligands, have been demonstrated in human
placental tissue (Denison et al.,
1998; Drake et al.,
2001
; Red-Horse et al.,
2001
). They are considered to be involved in either recruiting
specific leukocyte populations to the feto-maternal interface
(Drake et al., 2001
) or
modulating trophoblastic function (Ishii
et al., 2000
). The latter function is supported by the fact that
human choriocarcinoma cell lines as well as human placental
syncytiotrophoblast express functional chemokine receptors
(Ishii et al., 2000
;
Douglas et al., 2001
). To
date, however, no chemokine receptor has been demonstrated on migrating
trophoblasts (i.e. EVTs). Accordingly, few reports have referred to the effect
of chemokines on trophoblastic migration.
Therefore, in this study, we investigated the possible involvement of a
chemokine receptor, CCR1, and its ligands in trophoblastic migration. First,
we confirmed the CCR1 expression on EVTs in early human placental tissues.
Second, using EVTs isolated from chorionic villous explant cultures
(Yagel et al., 1989), we
examined the expression of several chemokine receptors, including CCR1. Third,
the expression of chemokines that bind to CCR1 (CCR1 ligands) was confirmed in
human placental tissues. Fourth, the effects of recombinant CCR1 ligands on
proliferation and migration of isolated EVTs were examined to understand the
function of trophoblastic CCR1. Last, to clarify the possible mechanism(s) of
the regulation of trophoblastic CCR1, we examined the effect of hypoxia and
decidua-conditioned medium on CCR1 expression of EVTs in vitro.
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Materials and methods |
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Placental tissues for villous and decidual explant cultures were aseptically obtained from legal abortions of normal pregnancies (6-9 weeks of gestation, n=15). Fresh tissues were placed in ice-cold RPMI (Gibco, Grand Island, NY) and used for cultures within 2 hours. In some cases, parts of the chorionic and the decidual tissues were taken separately, washed extensively with PBS and snap-frozen in liquid nitrogen for subsequent RNA extraction.
The gestational age was calculated from the date of the last menstrual period and, if necessary, was adjusted according to ultrasonic measurements of the gestational sac and the fetal crown-rump length. Informed consent for use of these tissues was obtained from all donors. The use of the materials was also approved by the Ethics Committee of Kyoto University Hospital.
Reagents and antibodies
Recombinant human RANTES, MIP-1, monocyte chemoattractant protein 2
(MCP-2) and hemofiltrate C-C chemokine 1 (HCC-1) were purchased from PeproTech
(London, UK). Goat anti-human RANTES, MIP-1
, MCP-2 and HCC-1 polyclonal
antibodies (pAbs, IgG fraction) were obtained from Santa Cruz (Santa Cruz,
CA). Two mouse anti-human CCR1 monoclonal antibodies (mAbs), clone 53504.111
(IgG1) and clone 141-2 (IgG1), were purchased from Genzyme (Cambridge, MA) and
MBL (Nagoya, Japan), respectively. Mouse anti-human cytokeratin 7 mAb (clone
OV-TL12/30, IgG1) and FITC-conjugated mouse anti-human cytokeratin 7 mAb
(clone LP5K, IgG2b) were obtained from Dako (Glostrup, Denmark) and Cymbus
Biotechnology (Hants, UK), respectively. Mouse anti-human von Willebrand
factor mAb (clone F8/86, IgG1) and FITC-conjugated sheep anti-human von
Willebrand factor pAb were obtained from Dako and Binding Site (Birmingham,
UK), respectively. Mouse anti-human vimentin mAb (clone V9, IgG1) and
FITC-conjugated mouse anti-human vimentin mAb (clone 3B4, IgG2a) were obtained
from Dako and Progen Biotechnik (Heidelberg, Germany), respectively. Mouse
anti-human CD45 mAb (clone T29/33, IgG1) was obtained from Dako. Mouse
anti-human melanoma cell adhesion molecule (MCAM) mAb (clone F4-35H7, IgG1)
was purchased from Alexis (San Diego, CA). Mouse anti-integrin
1 mAb
(clone MAB1973, IgG1) and FITC-conjugated integrin
5 mAb (clone SAM-1,
IgG2b) were obtained from Chemicon (Temecula, CA) and Southern Biotechnology
(Birmingham, AL), respectively.
FITC-conjugated and nonconjugated mouse IgG1 (clone DAK-GO1), mouse IgG2a
(clone DAK-GO5) and mouse IgG2b (clone DAK-GO9) for negative controls were
obtained from Dako. Normal goat IgG (Santa Cruz) and FITC-conjugated sheep
antibody raised against rat IgG (Binding Site) were also used for negative
control staining. For the blocking antibody in double immunochemistry,
anti-trinitrophenyl (TNP) mAb (unrelated mouse mAb,
Tsujimura et al., 1990) was
employed. For the secondary antibody, FITC-conjugated rabbit anti-goat
immunoglobulin (Ig) pAb (Dako), FITC-conjugated rabbit anti-mouse Ig pAb
(Dako) and rhodamine-conjugated goat anti-mouse Ig pAb (Santa Cruz) were
used.
Single immunohistochemistry
The paraformaldehyde-treated placental tissues were cut into 7-µm-thick
sections, air-dried on Neoprene (Nisshin, Tokyo, Japan)-coated slide glasses
and fixed with acetone (Nakalai, Kyoto, Japan) at 20°C for 5
minutes. The serial sections were incubated with goat anti-human RANTES,
MIP-1, MCP-2 and HCC-1 pAb (IgG, 10 µg ml1) or
normal goat IgG (10 µg ml1) as a negative control for 1
hour. After washing in PBS, the sections were incubated with FITC-conjugated
rabbit anti-goat Ig pAb (diluted 1:100) for 30 minutes. After the sections
were thoroughly washed in PBS, they were mounted with Immunon (PermaFluor,
Pittsburgh, PA) and examined under a confocal laser scanning microscope (Carl
Zeiss, Jena, Germany). Some serial sections were stained with hematoxylin and
eosin (HE) and examined under a light microscope.
Double immunohistochemistry
The 7-µm-thick sections of frozen placental tissues were fixed with
acetone and incubated with mouse anti-human CCR1 mAb (clone 53504.111, 10
µg ml1 and clone 141-2, 10 µg ml1)
or isotype-matched control mAb (10 µg ml1) for 1 hour.
After washing in PBS, the sections were incubated with rhodamine-conjugated
goat anti-mouse Ig pAb (diluted 1:100) for 30 minutes, then washed in PBS and
blocked with anti-TNP mAb (20 µg ml1) for 30 minutes.
After washing in PBS, they were incubated with FITC-conjugated mouse
anti-human cytokeratin 7 mAb (diluted 1:10), FITC-conjugated sheep anti-human
von Willebrand factor pAb (diluted 1:100) or FITC-conjugated negative control
antibodies for 1 hour. After the sections were thoroughly washed in PBS, they
were mounted with Immunon. To observe the coexpression of integrins that are
differentiation markers for EVTs (Damsky et
al., 1992), serial sections were double-stained with the anti-CCR1
mAb (clone 53504.111, 10 µg ml1 and clone 141-2, 10 µg
ml1) followed by rhodamine-conjugated goat anti-mouse Ig pAb
and FITC-conjugated anti-intgerin
5 mAb (diluted 1:10). Alternatively,
sections were single-stained with anti-intgerin
1 mAb (5 µg
ml1) followed by FITC-conjugated rabbit anti-mouse Ig pAb.
These sections were examined under a confocal laser scanning microscope. Some
serial sections were stained with HE and examined under a light
microscope.
Human chorionic villous explant culture and isolation of EVTs
EVTs were isolated from human villous explant cultures as described
(Yagel et al., 1989).
Placental tissues (6-9 weeks of gestation, n=15) were washed with
sterile RPMI and dissected aseptically to remove decidual tissues and fetal
membrane. Small fragments of chorionic villi (
2 mm in diameter) were
teased apart and soaked in culture medium [RPMI containing 10% FCS (Gibco),
100 U ml1 penicillin and 100 µgml1
streptomycin (Gibco)]. About 20 pieces of the villous fragments were put on
10-cm dishes coated with collagen type I (Iwaki, Chiba, Japan). After
incubation for 4 hours under standard conditions (37°C, 20% O2,
5% CO2 and 75% N2) to allow the explants to adhere, 10
ml of culture medium was gently added and the explants incubated under
standard conditions for an additional 48 hours. The formation of cell sheets
and migration of spindle-shaped cells were observed from the adherent villous
tips (a representative example is shown in
Fig. 9A). After they were
washed gently with PBS, the outgrown cells were dispersed with 0.05% trypsin
(Difco, Detroit, MI) and 0.05% EDTA (Nakalai) solution, passed through a 40
µm pore Nylon Cell Strainer (Becton Dickinson, Bedford, MA) to remove the
chorionic villous parts, and replated in collagen type I-coated 6-well plates
(Iwaki). After 4-hour incubation, nonadherent cells and debris were removed by
washing with RPMI. The cells that remained attached were defined as `isolated
EVTs' and used for further experiments as described below.
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Preparation of decidua-conditioned medium
To obtain a mixture of soluble factors derived from the decidua, 10 pieces
of 2 mm diameter decidual fragments (6-9 weeks of gestation,
n=10) were soaked in culture medium and put on a 3.5-cm dish coated
with collagen type I. After incubation for 4 hours to allow adherence of the
explants, they were washed gently with PBS and then cultured in 1 ml of RPMI
for an additional 24 hours under standard conditions. After this incubation,
the supernatant was collected as decidua-conditioned medium, passed through a
0.45 µm-pore filter (Millipore, Bedford, MA), and stored at 80°C
until use.
RT-PCR analysis
Total RNA was extracted from the isolated EVTs (6-9 weeks of gestation,
n=5) and human peripheral blood mononuclear cells (PBMC; used as a
positive control for chemokine receptors) using Trizol (Gibco) as recommended
by the manufacturer. Total RNA was also extracted from chorionic and decidual
tissues (both 6-9 weeks of gestation, n=5). Two micrograms of total
RNA were reverse-transcribed with random primers using a First Strand cDNA
Synthesis kit (Amersham Biosciences, Buckinghamshire, UK) and diluted with
distilled water to a final volume of 200 µl. In some experiments, reverse
transcriptase was omitted as a control for the amplification of contaminating
genomic DNA. PCR was performed in 20 µl of Taq buffer (+Mg2+),
containing 1 µl of the indicated cDNA solution, 2 mM dNTPs and 0.5 U of
rTaq DNA polymerase (Toyobo, Osaka, Japan) with 0.5 mM each of sense and
antisense primers for chemokines, chemokine receptors and S26 as a
housekeeping probe (Table 1)
(Kojima et al., 1994;
Jordan et al., 1999
;
Ishii et al., 2000
;
Middel et al., 2001
;
Huber et al., 2002
;
Kawaguchi et al., 2002
). The
conditions for amplification were: 5 minutes at 94°C, 30 cycles of 1
minute at 94°C, annealing for 1 minute at 56-64°C and incubation for 1
minute at 75°C, followed by a final extension for 5 minutes at 75°C.
PCR products were resolved by electrophoresis on 1-1.5% agarose gels and
viewed by ethidium bromide staining. Because the genes for chemokine receptors
do not contain intron sequences that can be used to control for genomic
contamination, an identical, a parallel PCR was performed containing starting
material that had not been reverse-transcribed.
|
To evaluate the purity of the isolated EVTs, they were double-immunostained using mouse anti-human vimentin mAb (diluted 1:40), anti-human CD45 mAb (diluted 1:50), anti-human von Willebrand factor mAb (diluted 1:40) and anti-human MCAM mAb (diluted 1:20), followed by rhodamine-conjugated goat anti-mouse Ig pAb (diluted 1:100). After blocking with anti-TNP mAb (20 µg ml1), the EVTs were additionally stained with FITC-conjugated mouse anti-human cytokeratin 7 mAb (diluted 1:10).
To examine the expression of CCR1 on the isolated EVTs, they were double-immunostained with anti-human CCR1 mAb (clone 53504.111, 10 µg ml1) or isotype-matched control antibody (10 µg ml1) followed by rhodamine-conjugated goat anti-mouse Ig pAb (diluted 1:40). After blocking with anti-TNP mAb (20 µg ml1), the cells were additionally stained with FITC-conjugated mouse anti-human cytokeratin 7 mAb (diluted 1:10), FITC-conjugated mouse anti-human vimentin mAb (diluted 1:10) or FITC-conjugated negative control antibodies.
Samples were examined under a confocal laser scanning microscope.
Flow cytometry
The isolated EVTs (6-9 weeks of gestation, n=5) were trypsinized
and washed in Hanks' Balanced Salt Solution (HBSS, Gibco) containing 0.1%
bovine serum albumin (Nakalai) and 0.1% NaN3 (Nakalai). The
precipitated cells (2 x 104 cells tube1)
were incubated with anti-human CCR1 mAb (clone 141-2, 100 µg
ml1, 10 µl) or isotype-matched control mAb (100 µg
ml1, 10 µl) for 30 minutes at 4°C. After the cells
were washed twice with HBSS, they were incubated with FITC-conjugated rabbit
anti-mouse Ig pAb (diluted 1:40, 20 µl) at 4°C for 30 minutes in the
dark. The cells were then washed twice and resuspended in 300 µl of HBSS.
Cell surface labeling was analyzed by FITC fluorescence detection using a
FACScalibur (Becton Dickinson). Flow cytometric data were obtained from the
analysis of 5 x 103 cells per sample.
To examine the effect of decidua-derived soluble factors on trophoblastic CCR1 expression, the isolated EVTs (6-9 weeks of gestation, n=5) were trypsinized and 1 x 105 cells ml1 of RPMI with 1% FCS were plated in each well of a collagen type I-coated 24-well plate (Iwaki). Decidua-conditioned medium (100 µl), prepared as above, was added to the wells. Heat-inactivated decidua-conditioned medium was used as control. In each experiment, we used the decidua-conditioned medium derived from the patient from whom the EVTs were isolated. After incubation for 24 hours under standard conditions, the cells were trypsinized and treated for flow cytometric analysis as described above. Differences between percent positivity for CCR1 were analyzed by the two-tailed paired t test.
Laser-capture microdissection
The frozen placental tissues were cut into 8-µm-thick sections, mounted
on foil-covered glass slides (Digital Micro Systems, Kyoto, Japan) and fixed
immediately in 100% methanol (Nakalai) for 3 minutes at room temperature. The
sections were counterstained with 1% toluidine blue (Nakalai) in
diethylpyrocarbonate-treated (DEPC, Nakalai) water for 10 seconds and then
washed in DEPC-treated water at 4°C. Using a Leica AS LMD (Leica
Microsystems, Tokyo, Japan), 30 cell columns were cut out from the
sections, taking care not to contaminate the sample with immune cells,
decidual cells and syncytiotrophoblast. These cell columns were collected and
total RNA was extracted as described above. Half of the sample was
reverse-transcribed to synthesize cDNA and the remainder incubated similarly
but without reverse transcriptase to control for the amplification of
contaminating genomic DNA. The resulting mixtures were subjected to 35 cycles
of PCR amplification with either human CCR1 primers or S26 primers
(Table 1) as described
above.
Invasion assay
Invasion assays were carried out as previously described
(Sato et al., 2002) with
slight modifications. A cell-culture insert (6.4 mm diameter) that contained a
polyethylene terephthalate membrane filter with 8 µm-diameter pores (Becton
Dickinson) was placed in each well of a 24-well companion plate (Becton
Dickinson). The lower surface of the filter was precoated with Matrigel (200
µg ml1, Becton Dickinson) according to the manufacturer's
instructions. The lower well was filled with 800 µl of serum-reduced medium
(RPMI with 1% FCS) containing either intact or heat-inactivated chemokine (50
ng ml1 of either RANTES or MIP-1
, 100 ng
ml1 of MCP-2 and 500 ng ml1 of HCC-1). The
well containing serum-reduced medium without additive was used for control.
The isolated EVTs were trypsinized and 2 x 104 cells 200
µl1 of serum-reduced medium were plated in the upper
well. The cells were allowed to migrate through the pores for 4-6 hours and
those that remained on the upper surface of the filter were thoroughly removed
with a cotton swab. The cells that reached the lower surface were fixed with
100% methanol at 20°C for 5 minutes and immunostained with mouse
anti-human cytokeratin 7 mAb (diluted 1:40) followed by FITC-conjugated rabbit
anti-mouse Ig pAb (diluted 1:40) to visualize trophoblasts. The filters were
mounted with Immunon and examined under a confocal laser scanning microscope.
The numbers of cytokeratin 7-positive cells were counted using NIH Image
1.61.
This experiment was performed in duplicate to determine the average number of cells that migrated under each experimental condition. The result was expressed as a percentage of number of cells that migrated in the control (without additive). Five independent experiments were performed using the EVTs isolated from different chorionic samples (6-9 weeks of gestation, n=5). The differences were analyzed by one-way analysis of variance followed by Scheffe's F-test for multiple comparisons.
Cell-proliferation assay
The isolated EVTs were trypsinized and 1 x 104 cells 100
µl1 of RPMI plus 1% FCS were plated in each well of a
collagen type I-coated 96-well plate (Iwaki). Intact or heat-inactivated
chemokine (50 ng ml1 of either RANTES or MIP-1, 100
ng ml1 of MCP-2 and 500 ng ml1 of HCC-1)
was added to the well. The well without additive was used for control. After
24 hours incubation under standard conditions, the number of viable cells in
each well was assessed using Premix WST-1 Cell Proliferation Assay System
(Takara, Kusatsu, Japan) according to the manufacturer's instructions.
Briefly, 10 µl of Premix WST-1 were added to each well and the cells were
incubated under standard conditions for 1 hour. WST reduction was determined
with automated enzyme-linked immunosorbent assay plate reader (Molecular
Device, Menlo Park, CA) at an optical density of 450-650 nm.
This experiment was performed in duplicate and the average was defined as the WST reduction value under each experimental condition. The result was expressed as the percentage of the value in the control (without additive). Five independent experiments were performed using the EVTs isolated from different chorionic samples (6-9 weeks of gestation, n=5). The differences were analyzed by one-way analysis of variance followed by Scheffe's F-test for multiple comparisons.
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Results |
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Next, we examined the effect of decidua-derived soluble factors on trophoblastic CCR1 expression. To exclude the influence of chorionic villus-derived factors, EVTs were isolated from the villous explant cultures (6-9 weeks of gestation, n=5) and further incubated in the presence of either intact or heat-inactivated decidua-conditioned medium. As shown in Fig. 9E, flow cytometry revealed that the percentage of CCR1-positive cells in EVTs treated with intact decidua-conditioned medium (26.0±10.9%) was significantly lower than in EVTs treated with heat-inactivated medium (43.1±3.6%).
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Discussion |
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In primary explant cultures of human chorionic villi obtained during the
first trimester of pregnancy, cell sheets and migrating spindle-shaped cells
grew out from the explanted villous tips. Because these structures resemble
the cell column and the interstitial trophoblasts in vivo, this villous
explant culture has been proposed to represent trophoblastic differentiation
towards EVTs that occurs at the villus-anchoring sites
(Genbacev et al., 1992;
Vicovac et al., 1995
;
Irving et al., 1995
). We
confirmed that the outgrown cells were positive for cytokeratin 7, an
epithelial cell marker that distinguishes trophoblasts and decidual glandular
cells from other cell types present at the feto-maternal interface
(Haigh et al., 1999
). We also
observed that MCAM, a specific marker for EVTs and endothelial cells
(Shih and Kurman, 1996
), was
expressed on these cells. These results indicate that the cells that grew out
from the explanted villous tips are composed mainly of EVTs, and support the
proposal that the cell sheets and migrating spindle-shaped cells correspond to
the cell column and interstitial trophoblasts in vivo. In this system, CCR1
protein was expressed clearly on the outgrown EVTs. For further analyses in
vitro, we isolated the outgrown EVTs by a previously reported method
(Yagel et al., 1989
). RT-PCR
showed expression of mRNA encoding CCR1 in the isolated EVTs, and
immunocytochemistry and flow cytometry confirmed that CCR1 protein is
expressed on the surface of these cells. Based on these findings and the above
immunohistochemical results, we conclude that trophoblasts acquire a chemokine
receptor, CCR1, as they differentiate towards EVTs at the villus-anchoring
sites.
We then examined whether chemokines that bind to CCR1 are produced at the
human feto-maternal interface. Among reported ligands for CCR1, RT-PCR
analysis of early placental samples confirmed the expression of mRNAs encoding
RANTES, MIP-1, MCP-2 and HCC-1, all of which can stimulate CCR1 to
induce an increase in cytosolic Ca2+ concentration and/or cell
migration (Neote et al., 1993
;
Gao et al., 1993
;
Gong et al., 1997
;
Tsou et al., 1998
).
Interestingly, mRNA for these chemokines was expressed predominantly in the
decidual samples and almost undetectable in their chorionic counterparts.
Using RT-PCR, Ishii et al. demonstrated mRNAs for RANTES, MIP-1
and
MCP-3 in chorionic samples from the first trimester
(Ishii et al., 2000
). This
apparent discrepancy might be due to differences in PCR conditions. In this
study, we used a lower amount of cDNA (cDNA reverse-transcribed from 10 ng of
total RNA in 20 µl PCR solution) for a fewer amplification cycles (30
cycles) compared to cDNA reverse-transcribed from 1 µg of total RNA in 20
µl PCR solution for 35 PCR amplification cycles
(Ishii et al., 2000
). In this
respect, our PCR data do not indicate the absolute absence of other CCR1
ligands, such as MCP-3, MPIF-1 and Lkn-1, in these chorionic and decidual
samples nor do they mean that MIP-1
, MCP-2 and HCC-1 are absolutely
absent from chorionic samples. Rather, they indicate the relative abundance of
mRNA encoding CCR1 ligands in decidual samples as compared with their
chorionic counterparts. Immunohistochemistry of the feto-maternal interface
showed the expression of RANTES, MCP-2 and MIP-1
proteins in the
decidual tissue. By contrast, the expression of these chemokines was weak or
almost absent in the chorionic villi, which supports the above PCR data. In
this immunohistochemistry, we failed to determine the localization of HCC-1
protein. However, it was demonstrated by in situ hybridization that mRNA for
HCC-1 is expressed diffusely in the decidual stromal cells and invading EVTs
(Red-Horse et al., 2001
).
These findings indicate that CCR1 ligands are produced at the feto-maternal
interface. Moreover, it is also implied that these CCR1 ligands are more
abundantly produced in the decidual (maternal) compartment than in the
chorionic (fetal) compartment.
A study of CCR1-knockout mice showed that CCR1 is not required for normal
mouse development (Gao et al.,
1997). However, it was recently reported that mouse ectoplacental
cone-derived trophoblasts express CCR3, CXCR4 and CCR5
(Athanassakis et al., 2001
).
This does not agree with our RT-PCR data showing that human EVTs expressed
CCR1, CCR10 and XCR1 because it suggests that mouse trophoblasts do not
express CCR1. In this respect, the data from mouse knockouts do not negate the
importance of CCR1 in human placentogenesis and it might be difficult to
perform in vivo experiments using mice to extrapolate the functional relevance
of chemokine-CCR1 system in human EVTs. Therefore, in this study, we used the
isolated human EVTs to examine the effects of CCR1 ligands in vitro. In the
invasion assay, RANTES, MIP-1
, MCP-2 and HCC-1, whose expression was
detected in the decidual tissue, promoted the migration of the isolated EVTs.
By contrast, the proliferation of isolated EVTs was not affected by these
chemokines. Because other chemokine receptors that are known to interact with
these chemokines (i.e. CCR3 and CCR5 for RANTES; CCR5 for MIP-1
; CCR2,
CCR3 and CCR5 for MCP-2) (Murphy et al.,
2000
) were hardly detectable in the isolated EVTs by our RT-PCR
analysis, these migration-promoting effects are considered to be mainly
mediated through CCR1. Taking into consideration that CCR1 ligands were
detected predominantly in the maternal side of the feto-maternal interface,
the chemokine-CCR1 system might contribute to the initial step of
trophoblastic invasion towards maternal tissue.
Trophoblastic invasion and/or differentiation is thought to be influenced
by the surrounding oxygen tension
(Genbacev et al., 1997). These
authors demonstrated that trophoblasts do not become invasive in hypoxic
conditions in vitro and proposed that a relatively O2-rich
environment, such as in the maternal arteries, stimulates trophoblastic
invasion and encourages their arterial infiltration. The factors derived from
the decidual tissue are also thought to regulate trophoblastic invasion and
differentiation (Bischof et al.,
2000
). In either ectopic pregnancy or placenta accrete where
development of decidual tissue is insufficient, excess trophoblastic invasion
has been observed. From these observations, it has been suggested that
decidual tissue has a protective effect on trophoblastic invasion. In fact, it
was reported that supernatant derived from decidual cells in culture inhibits
trophoblastic gelatinolytic activity in vitro
(Bischof et al., 1998
). We also
observed that relatively high O2 reduced the expression of DPPIV on
the outgrown EVTs in the villous explant cultures
(Sato et al., 2002
), and that
decidua-conditioned medium increased the MCAM expression on the isolated EVTs
(Higuchi et al., 2003
). In
this study, therefore, we examined the effects of these two factors, the
surrounding O2 tension and decidua-derived factors, on the
expression of CCR1 on EVTs in vitro. Unlike the findings in villous explant
cultures under normoxic conditions, CCR1 expression was hardly induced on the
outgrown EVTs under hypoxic conditions. This indicates that a relatively high
O2 level in the environment is needed to induce CCR1 expression. By
contrast, the induction of CCR1 expression on outgrown EVTs decreased
significantly after they were isolated from chorionic villous parts and
treated with decidua-conditioned medium. This indicates that undefined soluble
factor(s) that are derived from decidua reduce the expression of CCR1 on EVTs.
Based on the concept that trophoblastic invasion is encouraged by high
O2 concentrations and inhibited in the presence of decidual tissue,
the findings on CCR1 regulation support a close correlation between
trophoblastic invasion and expression of CCR1.
After trophoblasts acquired CCR1 in the cell column, CCR1 expression
rapidly diminished on the EVTs that were migrating into the decidual tissue.
Such downregulation of CCR1 was not observed in villous explant cultures,
where the outgrown cell sheets as well as the migrating spindle-shaped cells
expressed CCR1 constantly. This might be caused, at least in part, by the
absence of decidua-derived factor(s) in explant cultures. By contrast, CCR1
expression was maintained on the EVTs that were migrating from the
trophoblasic shell into the maternal arteries, where O2 tension is
expected to be relatively high. This suggests that high O2 tension,
which is necessary for the induction of CCR1 expression on trophoblasts in
vitro, is also important for maintaining CCR1 expression. Because the
expression of chemokine receptors is likely to reflect the responsiveness of
the cell to chemokine stimulation, it is fascinating to speculate that the
chemokine-CCR1 system is involved in the mechanism that leads trophoblasts
towards the maternal arteries. To substantiate this speculation, there must be
a source of CCR1 ligands in the maternal arteries that provides directional
cues that guide EVTs. Unfortunately, our immunohistochemistry failed to show
the expression of CCR1 ligands was highest in maternal endothelial cells.
However, there is another important cell component that could be the source of
CCR1 ligands in the maternal vessels; circulating blood cells. Before 10-weeks
gestation, uterine arteries are occluded with aggregates of endovascular
trophoblasts, which dramatically slows the arterial blood flow
(Drake et al., 2002). This
might promote the attachment of blood cells to the vessel wall where they are
known to transiently enhance the production of various cytokines and
chemokines (Kasahara et al.,
1991
). RANTES is one of the chemokines produced by cultured human
PBMC (Denison et al., 1997
).
It has also been reported that platelets release RANTES and MIP-1
following activation (Boehlen and
Clemetson, 2001
). Moreover, we recently demonstrated that
PBMC-conditioned medium can promote the invasion of BeWo cells, a
trophoblastic cell line (Egawa et al., 2000). In this context, it is possible
that some circulating blood cells produce chemokines as they attach to the
vessel wall and attract EVTs towards the maternal arteries.
In summary, we have demonstrated that trohoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype at the villus-anchoring sites. In vivo, CCR1 was not expressed on cytotrophoblasts or syncytiotrophoblast but was expressed on EVTs in the cell column. In vitro, CCR1 was induced on EVTs that grew out from the villous explants cultured under normoxic conditions. Such CCR1 induction was not observed under hypoxic conditions and the induced CCR1 expression was reduced by treatment with decidua-derived factor(s), which supports a close correlation between trophoblastic invasion and CCR1 expression. CCR1 ligands were confirmed in the maternal tissue and could induce the migration of the isolated EVTs in vitro. From these findings, we propose a novel role for chemokine-CCR1 interactions (a well-known regulatory system for leukocyte trafficking) in the initiation of trophoblastic invasion of maternal tissue. In this study, we could not determine the significance of CCR1 expressed on endovascular trophoblasts. The relevance of other chemokine receptors in trophoblastic invasion also remains to be explored. Future work on understanding the possible participation of chemokines and chemokine receptors in trophoblastic arterial infiltration might contribute to clarifying the pathophysiology of preeclampsia and intrauterine fetal growth retardation, condition in which there is insufficient maternal arterial remodeling.
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ACKNOWLEDGMENTS |
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