Relationship of Abortion and the Expression of Indoleamine 2,3- dioxygenase (IDO) in Villus and Syncytiotrophoblasts

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Journal of Reproduction & Contraception (2005) 16 (4):235-242

Relationship of Abortion and the Expression of Indoleamine
2,3- dioxygenase (IDO) in Villus and Syncytiotrophoblasts

Xue-lian LI, Sui-qi GUI, Hai-yan WANG
Department of Gynecology of Integrated Traditional Chinese and Western Medicine, The Hospital of Obstetric &
  Gynecology, Fudan University, Shanghai 200011, China

Objective To study the relationship of abortion and the expression of indoleamine 2,
3- dioxygenase (IDO) in villus and syncytiotrophoblast in vitro.
Methods RT-PCR was applied to analyze the mRNA transcription of IDO in villus of
normal pregnancy and inevitable abortion and JAR cells as well. Immunohistochemistry
was applied to analyze the expression of IDO protein in villus. Western blot was applied
to determinate the expression of IDO protein on cultured syncytiotrophoblast. High-
performance liquid chromatography was applied to determinate whether there was
kynurenine in cell culture medium of syncytiotrophoblast.
Results The expression of IDO mRNA and protein in villus of inevitable abortion was
lower than that of normal pregnancy; IDO mRNA did not express in JAR cells. IDO
protein expressed on cultured syncytiotrophoblast, and there was kynurenine in cell
culture medium of syncytiotrophoblast.
Conclusion Appropriate expression of IDO in villus is necessary for maintenance of
normal pregnancy and an active IDO protein expresses in syncytiotrophoblast.

Key words: indoleamine 2,3-dioxygenase; syncytiotrophoblast; villus; abortion

      There is complicated pathogeny involved in spontaneous abortion, and the modulatory
abnormity of incretion and immunity accounts for 80%-94%. Why is not the fetus, a homo-
geneous implant, excluded by the mother? At present, the study has been to the trophoblasts,
the interface of mother and fetus. How is the immunological relationship of trophoblasts and
lymphocytes came from the mother? Indoleamine 2,3-dioxygenase (IDO), which mostly
consists in the antigen-presenting cells of lymphoid organs and the syncytiotrophoblasts and
macrophages of gestation, is the first and regulatory enzyme of the kynurenine pathway

Corresponding author: Sui-qi GUI; Tel: +86-21-63455050-420; E-mail: sqgui@hotmail.com

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(KP), the major route of L-tryptophan catabolism. The tryptophan catabolites produced through
the KP induce immunosuppression of T lymphocytes[1], and may play an important role in
the balance of Th cytokine at materno-fetal interface and the materno-fetal tolerance. IDO
inhibitor induces the loss of fetus[2,3]. Here we mainly study the relationship of abortion and
the expression of indoleamine 2,3- dioxygenase (IDO) in villus and syncytiotrophoblasts.

Materials & Methods

Materials
Tissue collection
      Villus specimens were donated by healthy women undergoing elective termination of
pregnancy at 6-12 weeks of gestation in Hospital of Obstetric & Gynecology, Fudan University.
JAR cells were purchased from Institute of Biochemistry and Cell Biology (Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences).
Chemicals
      Trypsin (Amresco), deoxyribonuclease I (Sigma), DMEM and bovine calf serum(Gibco),
recombinant Human epidermal growth factors (rhEGF, Peprotech), Percoll(Gibco),
histochemical ABC kit (Huamei), RNAex Reagent, revertAidTM first strand cDNA synthe-
sis kit, Taq DNA polymerase (Fermentas), BCA-100 protein quantitative analysis kit
(Shenergy), visual AP Western blotting reagent set (Ab Minus) SNBC, mouse anti- IDO
monoclonal antibody (Chemoicon), mouse anti-vimentin, cytokeratin and hCG-β monoclonal
antibody (Zhongshan), L-tryptophan, kynurenine (Sigma), primer (designed personally with
the help of primer premier 5.00 according to gene sequence and synthesized by Bioasia).
IDO forward: 5'-TCCGTGAGTTTGTCCTT-3'
      reverse: 5'-GCATAGTATTAGTTTGTGGC-3', product 361 bp
β-actin forward: 5'-GAGCGGGAAATCGTGCGTGACATT-3'
         reverse: 5'-GATGGAGTTGAAGGTAGTTTCGTG-3', product 240 bp
Instruments
      UNICOTM UV-2102C ultraviolet spectrophotometer, Perkin elmer DNA Thermal
cycler, SCR-4 high-pressure electrophoretic instrument, H6-1 electrophoretic slot, Tanon
GIS2010, GIS gel image disposal system, MIAS-2000 immunohistochemical measure sys-
tem for colorized pathological images, Waters high-performance liquid chromatography, C-
18 column (Zorbax C18, 250 × 4.6 mm, 5 µm), Waters 2996 PDA detector.
Methods
RNA isolation and RT-PCR of IDO mRNA on human syncytiotrophoblasts of normal
pregnancy and inevitable abortion and JAR cells
      Total RNA was prepared by RNAex reagent, treated with RNase-free DNase and
quantified spectrophotometrically. RT products were prepared in accordance with the manu-

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facturers’ instructions of the RevertAidTM first strand cDNA synthesis kit. The PCR reac-
tion system involved 1-5 µl template DNA, 2.5 µl PCR buffer (1.5 mol/L MgCl2), 0.5 µl
dNTP mix, 1 µl forward primer, 1 µl reverse primer, 0.5 µl Taq DNA polymerase and DEPC
water filled in a final volume of 25 µl was incubated at 95℃ for 3 min, followed by incubation
at 94℃ for 60 s, 47℃ for 60 s, and 72℃ for 60 s for a total of 30 cycles and 72℃ for 15 min.
Samples were subjected to 1% agarose gel electrophoresis, stained with ethidium bromide,
and photographed under UV light. As a positive control, human β-actin RNA was
retrotranscribed and amplified in parallel for each sample, and genomic DNA was used as a
positive control for the PCR. As a negative control, an identical amount of RNA for each
sample was amplified without being retrotranscribed.
Immunohistochemical measure (ABC)
       The tissue specimen was embedded by paraffin, sliced up for 4 µm, and immunostained
for IDO according to the guide of the histochemical ABC kit. Negative comparison was
done by PBS replacing the primary antibody. The positive absolute proportion, positive rela-
tive proportion and average absorbency of ten areas were measured stochastically in each
slice.
Isolation, culture and identify of syncytiotrophoblast
       The villus tissue was collected sterilely, rinsed in D-Hanks, cut into smaller pieces, and
digested with 0.125% trypsin and 0.02% deoxyribonuclease I 5 min × 3 at 37℃. The
supernatants were filtered over a succession of 200 µm and 60 µm pore size sieves to
remove aggregates. The cells were then centrifuged at 350 × g for 10 min, resuspended in
DMEM, layered over a continuous Percoll gradient (30%-70% in 5% steps of 2 ml
each), then centrifuged at 37℃ and 1 200 × g for 20 min to separate different cell
types. Cytotrophoblast cells between the density markers of 1.049 g/ml and 1.062 g/ml
were collected, suspended in DMEM containing FCS (10%), penicillin (100 U/ml), streptomycin
(100 µg/ml), plated in 24-well plates at a density of 106 cells/ml/well or in 5 ml vase at
(1-5)× 10 6 cells/ml, cultured in a humidified atmosphere (95% air-5% CO 2 at 37℃)
with 10 ng/ml rhEGF to promote the congregation and amalgamation, fixed and immunostained
for cytokeratin, vimentin and hCG-β according to the guide of the histochemical ABC kit.
Western blot analysis for expression of IDO protein in human syncytiotrophoblast
cultured in vitro
       The protein was distilled and quantified according to the BCA-100 kit. Polyacrylamide
slab gel of 10% for separation and 5% gel for concentration were used, 20 µl of sample was
loaded into each well with equal SDS and protein, and subjected to electrophoresis for 1.5-2.0 h.
Electrophoretic transfer of the protein onto nitrocellulose paper was done at 250 mA
constant current for 1.5-2.0 h in ice. The membrane was then washed with TBS buffer and
placed in 4% skim milk powder-TBS for 1.5-2.0 h to block all nonspecific binding sites on the
membrane. The membrane was removed, washed, placed in a buffered solution of primary
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monoclonal IDO antibody (1:1 000 dilution), and then left overnight at 4℃. The membrane
was removed, washed, and placed in secondary antibody (alkaline phosphatased goat anti-
mouse IgG, 1:2 000) for 1 h at room temperature. The membrane was washed and incu-
bated for 10-30 min at room temperature with AP substrate solution(3 ml AP substrate
solution+20 µl NBT solution+20 µl BCIP solution).
IDO activity of syncytiotrophoblasts detected by high-performance liquid
chromatography (HPLC)
      Samples of culture media were deproteinized with 4% trichloroacetic acid and total
free tryptophan and kynurenine were assayed by HPLC on a C-18 column eluted isocratically
using a solvent of 0.1 mol/L phosphate buffer (pH 4.0 with sodium hydroxide). The ratio of
kynurenine/tryptophan was used to assess the IDO activity.
Statistical analysis
      Results were analyzed statistically by t test using SPSS11.0 software package. P
1.0
                                                                         *: P
Figure 3 IDO protein expression in normal villus (A) and villus of inevitable abortion (B)

      A: vimentin-negative; B: cytokeratin-positive;     C: hCG-β-positive; D: IDO-positive
             Figure 4 Syncytiotrophoblasts judged by immunocytochemistry (200 ×)

       Figure 5 Western blot reveals IDO protein (46 kD) expressed in syncytiotrophoblasts

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medium of syncytiotrophoblasts cultured in vitro. This fact reveals that there are IDO activ-
ity in syncytiotrophoblasts cultured in vitro. We suppose that normal expression and activity
of IDO in villus is an important qualification of maintaining pregnancy, and singularly lower
expression or activity of IDO may be one of the reasons of abortion. Our findings could be
explained by the reports that IDO expressed in trophoblasts and defends the conceptus
against rejection by reducing the tryptophan level and suppressing the T cell activity[1], tryp-
tophan is a necessary amino acid of cell proliferation, IDO-expressing cells could creat a
local microenvironment in which levels of tryptophan are low, block the cell cycle at mid-G1,
make T cells be prone to apoptosis especially apoptosis via Fas, regulate T cell proliferation
and activation[4,5] IDO is inducible by cytokines such as interferon-gamma and plays a role in
the balance of Th cytokine at materno-fetal interface and the inflammation and maternal
tolerance of fetal allografts[6]. Blocking tryptophan catabolism during murine pregnancy
allows maternal T cells to provoke fetal allograft rejection. Cells expressing IDO, which
catabolizes tryptophan, prevent T cell cycle progression, enhance activation, induce T cell
death, regulate maternal T cell immunity during pregnancy and might contribute to immuno-
logical discrimination by promoting T cell tolerance in other circumstances[7,8]. The mecha-
nisms of IDO mediating tolerance are not well understood, but recent findings have impli-
cated tryptophan catabolism through the kynurenine metabolic pathway as one of many
mechanisms involved. It has recently been found that inhibition of IDO can result in the
rejection of allogenic fetuses, suggesting that tryptophan breakdown is necessary for
maintaining aspects of immune tolerance. Two theories have been proposed to explain how
tryptophan catabolism facilitates tolerance. One theory posits that tryptophan breakdown
suppresses T cell proliferation by dramatically reducing the supply of this critical amino acid.
The other theory postulates that the downstream metabolites of tryptophan catabolism act to
suppress certain immune cells, probably by pro-apoptotic mechanisms[3].
      It was reported that IDO reactivity could be presented by the ratio of kynurenine/
tryptophan[9]. Our research reveals that there are both kynurenine and tryptophan in the
culture medium of syncytiotrophoblasts cultured in vitro, that is, there is IDO reactivity in
syncytiotrophoblasts cultured in vitro. So, we speculate that the expression and reactivity in
villus of early pregnancy is due to syncytiotrophoblasts. And improving the expression and
activity of IDO on the materno-fetal interface may further modulate the materno-fetal im-
munity to the normal level and maintain normal pregnancy. These findings provid some
academic foundations for the study of pathogenesis and therapy of abortion. But further
studies are still needed to make clear through which route that IDO affect materno-fetal
immunity.

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                                                                                (Received on June 21, 2005)

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