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• Original Article •
Expression of BAFF in the
trophoblast and decidua of normal
early pregnant women and
patients with recurrent
spontaneous miscarriage
GUO Wen-jing, QU Xun, YANG Mei-xiang, ZHANG
Wei-dong, LIANG Lu, SHAO Qian-qian, KONG Beihua
GUO Wen-jing Department of Obstetrics and
Gynecology, Qilu Hospital, Shandong University,
Jinan, Shandong 250012, China; QU Xun Basic
Research Institute of Clinic Medicine, Qilu Hospital,
Shandong University, Jinan, Shandong 250012,
China; YANG Mei-xiang Basic Research Institute of
Clinic Medicine, Qilu Hospital, Shandong University,
Jinan, Shandong 250012, China; ZHANG Wei-dong
Department of Pathology, Institute of Basic
Medicine, Shandong Academy of Medical Sciences,
Jinan, Shandong 250012, China; LIANG Lu Basic
Research Institute of Clinic Medicine, Qilu Hospital,
Shandong University, Jinan, Shandong 250012,
China; SHAO Qian-qian Basic Research Institute of
Clinic Medicine, Qilu Hospital, Shandong University,
Jinan, Shandong 250012, China; KONG Bei-hua
Department of Obstetrics and Gynecology, Qilu
Hospital, Shandong University, Jinan, Shandong
250012, China
Correspondence to: KONG Bei-hua Department of
Obstetrics and Gynecology, Qilu Hospital, Shandong
University, Jinan, Shandong 250012, China (Tel:86531-82169008 Email:kongbeihua @yahoo.com.cn )
QU Xun, Basic Research Institute of Clinic Medicine,
Qilu Hospital, Shandong University, Jinan, Shandong
250012, China (Tel: 86-531-82169251. Email:
[email protected]. cn)
udy was supported by : National Natural Science
Foundation of China to Dr. KONG Bei-hua(No.
30571953)
Keywords: B lymphocyte stimulator·recurrent
spontaneous abortion·maternal-fetal immunology
Abstract:
Background BAFF, the B cell activation factor, is a
member of the tumor necrosis factor (TNF) ligand
family that binds to BCMA, TACI, and BAFF-R.
Previous studies have shown that members of the
TNF family are detected in human placental
trophoblast cells, but the expression patterns of
BAFF involved in human decidua and the differential
expression of BAFF between normal pregnancy and
miscarriage are still incompletely documented or
unknown. This study was designed to investigate the
expression of BAFF and BAFF-R in the trophoblast
and decidua of normal early pregnant women and
recurrent spontaneous abortion (RSA) patients.
Methods Forty-five patients with RSA and 45
normal pregnant women were included in this study.
By reverse transcriptase-polymerase chain reaction
(RT-PCR), Western blotting and
immunohistochemical experiments, we explored the
expression of BAFF and BAFF-R in the maternal-fetal
interface of normal early pregnant women and RSA
patients.
Results Analysis by RT-PCR and Western blotting
revealed that BAFF was detected in both trophoblast
and decidua of all the samples, and the expression
level was higher in the tissues of normal early
pregnant women (P<0.05) than that of recurrent
spontaneous abortion patients under the same
gestational weeks. Messages for BAFF-R were
absent. Immunohistochemical experiments showed
that expression of BAFF was cell-specific which was
localized to villous cytotrophoblast and
syncytiotrophoblast cells in trophoblast and to
stromal cells in decidua. Whereas BAFF was
prominent on the trophoblast and decidua of normal
early pregnant women, it was decreased in the
tissues of RSA patients.
Conclusions BAFF might steer maternal leukocytes
away from a harmful immune response and toward
a favorable one and play a potentially vital role for
successful pregnancy.
2008;121(4):309-315
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Maternal immune response during pregnancy is thought to be harmful to the
survival of the fetus, but in fact it may be beneficial. The successful pregnancy
relies on appropriate growth of the placenta and one of the functions of the
placenta is to establish a haven in which the fetus can flourish despite intimate
contact with an immunologically semiforeign environment. In this task, the
trophoblast is the principal player which lines the villous placenta and separates
maternal blood from fetal tissue and has a major role in cellular alterations of
the maternal immune system.1 Evidence presented in several studies indicates
that immune privilege is known to be conferred by members of the tumor
necrosis factor (TNF) superfamily,2-5 which have roles in placental cell growth,
cell death, cell migration and hormone production.6-10 Earlier studies stated that
messages encoding all of the death-inducing TNF superfamily ligands were
detected in placental trophoblast cells.11 Subsequently Phillips et al12
demonstrated that expression of some nonapoptosis- inducing ligands in this
family (APRIL, BAFF and CD30L/CD153) were also examined in the trophoblast
cells. But during the invasive phase of implantation, maternal decidual cells also
secrete products and are proposed to supply nutrition to the growing embryo
until the formation of a functional placenta, and to shield the embryo from the
maternal immune system.13,14 To date, it has not been determined whether any
of these TNF superfamily members and their receptors is expressed in maternal
decidual cells.
BAFF was known as B cell-activating factor belonging to the TNF family, also
called B lymphocyte stimulator (BLyS) TNF- and Apop-releated leukocyte
expressed ligand 1 (TALL-1), TNF homologue that activates apoptosis, nuclear
factor (NF)-κB, and c-Jun NH2-terminal kinase (THANK), and zTNF4.15-19 BAFF
plays an important role in immune responses and overexpression or deficiency of
BAFF is associated with the development of autoimmune diseases.20
Recurrent spontaneous abortion (RSA), defined as two or more consecutive
miscarriages, is a prevalent disorder. The pathogenesis of most cases remains
unknown, but immunological factors are considered to play a vital role in the
pathogenesis of recurrent spontaneous abortion. The above mentioned
experiments revealed that BAFF was readily detected in trophoblasts and
deciduas of normal early pregnant women and recurrent spontaneous abortion
patients. But the expression of BAFF gene and protein was significantly
decreased in trophoblasts and deciduas of recurrent spontaneous abortion
patients than that of normal early pregnant women under the same gestational
weeks. By immunohistochemistry, localization of BAFF protein to specific cell
types was observed. Thus, information on the expression of BAFF in trophoblasts
and deciduas may play an important role in maintenance of successful pregnancy
or pathogenesis of failure pregnancy.
In this study, we analyzed the expression of BAFF and BAFF-R not only in
trophoblasts but also in deciduas of normal early pregnant women and recurrent
spontaneous miscarriage patients by reverse transcriptase-polymerase chain
reaction (RT-PCR) and Western blotting; on the other hand,
immunohistochemical experiments were used to determine cellular locations of
the proteins.
METHODS
Tissues collection
The trial, which enrolled 45 clinically elective women for normal pregnancy
termination and 45 patients with recurrent spontaneous miscarriage (gestational
age, 4−10 weeks), was conducted between January 2005 and October 2006 at
the Department of Obstetrics and Gynecology, Qilu Hospital, Shandong
University. The 45 patients were divided into three groups according to
gestational weeks (group 1: 4−5 (6/7) weeks; group 2: 6−7 (6/7) weeks; group
3: 8−10 weeks; n=15 each group), respectively. The first-trimester human
trophoblasts and deciduas were obtained from the above patients. Each patient
completed a signed, written consent form. The Shandong University Human
Investigation Committee approved the study. The tissues were manually
dissected and immediately frozen, using liquid nitrogen, and stored at −80˚C
until used for isolation of total RNA. For every tissue collected, small pieces were
also placed into Bouin's fixative, processed into blocks, sectioned and stained
with hematoxylin.
Reverse transcription-polymerase chain reaction analysis
Total RNA was isolated using the Rneasy mini kit® (Qiagen Inc., Valencia, CA,
USA) according to the manufacturer's protocol. Sample quality was assessed
with 2100 Bioanalyzer® (Aligent Technologies, Palo Alto, CA, USA) and confirmed
by electrophoresis with an agarose gel. All samples contained 18 S and 28 S
rRNA peaks with no degradation. The yield of total RNA was determined by
absorbance at 260 nm on a DU 640 Spectrophotometer (Beckman Coulter,
Fullerton, CA, USA). The 260/280 nm ratios of the samples were >1.8. To
correct for variations in mRNA recovery and reverse transcription yield, the
amount of BAFF cDNA was normalized with β-actin. One microgram of each RNA
sample was treated with DNase I to remove genomic DNA and then was
denatured, and reverse transcription was performed for 1 hour at 42ºC with 0.5
µg oligo(dT)18, 1.0 mmol/L 4dNTP, 20 U RNasin ribonuclease inhibitor (Promega,
Madison, WI, USA), 200 U Moloney virus-reverse transcriptase (Superscript II;
Life Technologies, Paisley, UK), and 5 × reaction buffer in a total of 20 µl.
Amplification was performed with 5 µl cDNA, 0.2 mmol/L dNTP, 1 mmol/L MgCl2,
2.5 U AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT, USA), 0.8 mmol/L
specific sense and antisense primers, and 10 × reaction buffer in a 50-µl reaction
volume. Amplification primers for the human genes were as follows: BAFF, 134
bp, forward: 5'-TCTTTGAACCACCAGCTCCA-3', reverse: 5'GGCACTTCCCCTTTTAAAGCT-3'; BAFF-R, 144 bp, forward: 5'CAAGGTCATCATTCTGTCTCCG-3', reverse: 5'-GTGGTCACCAGTTCAGTGGA-3'; βactin, 101 bp, forward: 5'- TTGCCGACAGGATGCAGAA-3', reverse: 5'GCCGATCCACACGGAGTACT-3'. After 5-minute precycle at 95ºC, the reaction
was followed by 35 cycles of 45 seconds at 94 ºC, 30 seconds at 55ºC, and 1
minute at 72ºC. When the final cycle was over, samples were kept at 72ºC for
10 minutes to complete the synthesis. Polymerase chain reaction products were
analyzed on 1.5% agarose gels and ethidium bromide-stained bands were
photographed. Authenticity of all of the amplified PCR products was confirmed by
direct sequencing. Three independent experiments were performed, each in
triplicate.
Western blotting
Tissues were lysed in a buffer containing 10 mmol/L Tris (pH 7.4), 10 mmol/L
NaCl, 3 mmol/L MgCl2, 0.5% Nonidet P-40 and protease inhibitors, and then the
supernatants were collected as the total cell lysates. Protein concentrations were
measured using the protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA,
USA). The lysates were electrophoresed on a 12% SDS-PAGE gel before being
transferred to a polyvinylidene fluoride (PVSD) membrane (Amersham Pharmacia
Biotech, Buckinghamshire, UK). The membrane was then blocked in 5% nonfat
milk in TBS-T (Tris buffered saline plus 0.05% Tween-20) and incubated with
goat anti-human BAFF mAb (1:1000, R&D, MAB1241, System, Inc.) or isotype
antibody at 4ºC overnight. Washed the membrane in TBS-T for 4 times, then the
membrane was incubated with horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG (1:2000, Jackson Immuno Research Laboratory, West Grove, PA,
USA). The immunoreactive protein complexes were detected using an enhanced
chemiluminescence detection system (ECL, Amersham) according to the
manufacturer's protocol. Endogenous β-actin expression served as an internal
control.
Immunohistochemistry
Paraffin-embedded sections of deciduas and trophoblasts of normal early
pregnant women and recurrent spontaneous abortion patients were
deparaffinized with xylene and rehydrated to water through graded alcohol
series. Endogenous peroxidase activity was blocked by incubation with 50:50
mixtures of 3% H2O2/methanol for 10 minutes and rinsed well with water. The
sections were pretreated by microwave heating with 10 mmol/L citrate buffer,
pH 6.0, for 30 minutes with a 5-minute cool down before rinsing well in running
tap water. After incubation for 10 minutes at room temperature with bovine
serum albumin in PBS, slides were incubated for 30 minutes in a humid chamber
at 4°C with 10 μg/ml rat anti-human BAFF (Buffy-2; Alexis Biotechnology, San
Diego, CA, USA), followed by a biotinylated secondary antibody and finally
incubated for 30 minutes with the avidin–biotin complex (ABC) reagent (Vector
Laboratories, Burlingame, CA, USA). Isotype-matched Ig control (Dako
Cytomation, Carpinteria, CA, USA) was used at the same concentration as the
matching primary antibody. The sections were counterstained with hematoxylin
and observed with light microscopy (Olympus America).
Statistical analysis
All values are shown as mean ± standard error (SE). Statistical comparison
applied the Mann-Whitney U test and the Wilcoxon matched-pairs test with use
of Analyse-it software (Analyse-it Software Ltd., Leeds, UK) for Microsoft Excel.
Differences were accepted as significant at P<0.05.
RESULTS
Detection of transcripts for BAFF in human trophoblasts and deciduas of
normal early pregnant women and spontaneous abortion patients
Our initial approach to determining whether BAFF is synthesized in human
trophoblasts and deciduas using RT-PCR. Parallel PCR analysis of RT-negative
controls for all samples and primer pairs failed to yield any products, confirming
lack of genomic DNA contamination (data not shown). All amplicons were
sequenced and found to be authentic. Figure 1 shows that transcripts encoding
BAFF were clearly detected in total RNA acquired from first-trimester
trophoblasts of normal early pregnant women and spontaneous abortion
patients. Using RT-PCR and primers for BAFF, a single band of 134 bp was
amplified in cDNAs from different trophoblasts of normal early pregnant women
divided into three groups (Figure 1A, Lanes 1, 3, 5) and other three groups'
different trophoblasts of spontaneous abortion patients (Figure 1A, Lanes 2, 4,
6). We then measured the ratio of BAFF to β-actin after the images on the UV
transilluminator were put into a computer and the band intensity was measured
using densitometrical software. The expression level of BAFF was higher in the
trophoblasts of normal early pregnant women compared with that of
spontaneous abortion patients under the same gestational weeks (P<0.05)
(Figure 1B). There was no significant difference (P>0.05) in the trophoblasts of
pregnant women accompanied by first-trimester gestation progression.
Figure 1. Analysis of mRNAs encoding BAFF by RT-PCR in firsttrimester trophoblasts of normal early pregnant women and
spontaneous abortion patients. (A) RT-PCR products are shown on
a gel when amplified with specific primer sets for BAFF. Samples
of RNA from different trophoblasts were amplified with primers
designed for BAFF (Lanes 1, 3, 5: normal early pregnant women;
Lanes 2, 4, 6: spontaneous abortion patients; 1, 2: 4−5 (6/7)week pregnancy; 3, 4: 6−7 (6/7)-week pregnancy; 5, 6: 8−10view in a week pregnancy), a single band of 134 bp was detected in both
new window normal pregnancy and abortion tissues. (B) RT-PCR data of BAFF
cDNA was normalized with β-actin in each group and is shown as
relative gene expression ± SE. *Significantly (P<0.05) lower BAFF
expressed in the trophoblasts of spontaneous abortion patients
compared to that of normal early pregnant women under the
same gestational weeks. 1: 4−5 (6/7)-week pregnancy; 2: 6−7
(6/7)-week pregnancy; 3: 8−10-week pregnancy; N: normal early
pregnant women; A: spontaneous abortion patients.
The expression of BAFF gene in human deciduas of normal early pregnant
women and spontaneous abortion patients are shown in Figure 2. Messages
encoding BAFF were also detected in first trimester deciduas of normal early
pregnant women (Figure 2A, Lanes 1, 3, 6) and spontaneous abortion patients
(Figure 2A, Lanes 2, 4, 6). Figure 2B shows that the level of expression of the
BAFF gene in the decidual tissues from normal early pregnant women was
significantly more than that of spontaneous abortion patients under the same
gestational weeks (P<0.05). There was no obvious difference (P>0.05) in the
trophoblasts as gestation progressed to 10 weeks.
Figure 2. RT-PCR detection of transcripts for BAFF in first
trimester deciduas of normal early pregnant women and
spontaneous abortion patients. (A) The size of BAFF product,
estimated by its migration relative to size markers, is indicated on
a gel. Transcripts for BAFF were readily detected in the deciduas
of normal early pregnant women (Lanes 1, 3, 5) and spontaneous
abortion patients (Lanes 2, 4, 6) (1, 2: 4−5 (6/7) weeks; 3, 4:
6−7 (6/7) weeks; 5, 6: 8−10 weeks). (B) Ratios of BAFF mRNA to
view in a
β-actin mRNA are expressed as means ± SE. *Significantly
new window
(P<0.05) lower BAFF expressed in the deciduas of normal early
pregnant women than that of spontaneous abortion patients
under the same gestational weeks. 1: 4−5 (6/7)-week pregnancy;
2: 6−7 (6/7)-week pregnancy; 3: 8−10-week pregnancy; N:
normal early pregnant women; A: spontaneous abortion patients.
And then we detected the concentration of BAFF protein in human trophoblasts
and decidual tissue samples by Western blot analysis with a monoclonal antibody
specific for BAFF. There was no detectable expression of BAFF protein in the
human decidual tissues lysis buffer. The concentration of BAFF protein was
significantly reduced in the spontaneous abortion groups than in the normal early
pregnancy group (Figure 3).
Figure 3. Expression of BAFF protein in human trophoblasts and
decidual tissues. Total proteins prepared form pregnancy
view in a
termination patients and the spontaneous abortion patients were
new window
separated on a SDS polyacrylamide gel and immunoblotted with
anti-BAFF mAb. Lane 1: the trophoblasts of the representative
normal early pregnant group; Lane 2: the trophoblasts of the
spontaneous abortion group; Lane 3: the decidua of the
representative normal early pregnant group; Lane 4: the decidua
of the spontaneous abortion group.
Expression of BAFF-R in human trophoblasts and deciduas
To establish the potential for BAFF ligand to influence cells development and
function at the maternal-fetal interface, we examined the same RNA samples
from early gestation trophoblasts and deciduas for BAFF-R. But we failed to
detect specific messages encoding BAFF-R in first-trimester trophoblasts and
deciduas of all the normal early pregnant women and spontaneous abortion
patients.
Localization of BAFF protein to specific cells in human trophoblasts and
deciduas by immunohistochemistry
To determine which cells were the source of the BAFF proteins, we performed
immunohistochemistry on paraffin-fixed tissue sections taken from first-trimester
trophoblasts and deciduas of all the samples (n=at least 3 each). And negative
controls did not demonstrate positive staining. Figure 4 illustrates the
immunostaining patterns. In first-trimester trophoblasts of normal early
pregnant women (Figure 4A), BAFF protein was localized to the cytotrophoblast
and syncytiotrophoblast cells. In the same cells of spontaneous abortion patients
(Figure 4B), immunostaining was weak. These results show a decrease in BAFF
at the trophoblasts of spontaneous abortion patients. As illustrated in Figure 4D,
BAFF in early deciduas of normal early pregnant women was localized to stromal
cells and blood vessel endothelium cells. As in first-trimester trophoblasts, BAFF
declined as immunohistochemistry was performed in decidual cells of
spontaneous abortion patients (Figure 4E). However, we failed to find the
localization of BAFF protein in decidual blood vessel endothelium cells of
spontaneous abortion patients (Figure 4E). The protein was identified in thirty
first-trimester trophoblasts and deciduas, the positive cells were of the same
subpopulation (cytotrophoblast and syncytiotrophoblast cells) as that in
trophoblasts, and BAFF protein was in the same location (predominantly stromal
cells) in deciduas, either of normal early pregnant women or of spontaneous
abortion patients. These observations of BAFF are consistent with the results of
gene expression reported in Figures 1, 2 and 3.
Figure 4. Immunohistochemical detection of BAFF in firsttrimester trophoblasts (A and B) and deciduas (D and E) of a
representative normal early pregnant woman(A and D; n=at least
view in a 3 each) and a spontaneous abortion patient (B and E; n=at least
new window 3 each), respectively. The cytotrophoblast and syncytiotrophoblast
cells were stained well in two samples, with more prominently in
trophoblasts of normal early pregnant woman compared to in that
of spontaneous abortion patient (A and B). D shows BAFF protein
was localized to the stromal cells and blood vessel endothelium
cells in deciduas of normal early pregnant woman. E shows lighter
staining in decidual stromal cells of spontaneous abortion patient,
while the staining in the blood vessel endothelium cells can not be
seen. The BAFF antibodies were tested against trophoblast and
decidua sections taken from the same patients; first trimester was
4−10 weeks. Negative staining by control IgG is shown in C (in
trophoblast) and F (in decidua). This was noticeable in other
tissues, as in the one shown here (Original magnification ×200).
DISCUSSION
The results of this study demonstrate that BAFF, both gene and protein, is well
expressed in trophoblasts and deciduas of normal early pregnant women and
recurrent spontaneous abortion patients. Additionally, it is expressed at relatively
low levels in the tissues of recurrent spontaneous abortion patients. In view of
these observations, expression of BAFF in trophoblasts and deciduas confirms
that BAFF is an important factor in the implantation of blastocyst and
maintenance of pregnancy, the lower expression of BAFF may be involved in the
pathogenesis of unexplained recurrent spontaneous abortion.
During the process of successful pregnancy, the coordinated development of the
implanting trophoblast and the maternal decidua is believed to be a critical
event.21,22 Trophoblast cells, constituting the interface between the fetus and
maternal blood and tissues, have an important role in successful pregnancy.
Additionally, the importance of decidual cells in ensuring appropriate endometrial
functions to support pregnancy is beyond doubt. So the study of human
trophoblasts and deciduas is thought to be important. Previous studies obtained
using commercially available organ blots have shown relatively high levels of
BAFF gene expression in term placentas, peripheral blood leukocytes, lymphoid
tissues, bone marrow, and several other organs and tissues.15,16,18 Other two
recent reports indicate that human placentas are identified as rich sources of TNF
family ligand and receptor transcripts.11,12 Here we found transcript encoding
BAFF was detected in trophoblasts of gestated women, and BAFF protein was
localized to specific types of cells, cytotrophoblast and syncytiotrophoblast cells,
agreeing well with the previous studies above. In addition, our studies expanded
other reports and added detections in deciduas, and demonstrated the lower
expression of BAFF in trophoblasts and deciduas of recurrent spontaneous
abortion patients.
Recurrent spontaneous abortion is defined as two or more consecutive
pregnancy losses. Although the pathogenesis of most cases remains unknown,
several studies have indicated that the majority of these cases are caused by
immunological disturbances.23,24 Locksley et al25 have reported that some of the
TNF superfamily members that do not mediate apoptosis are key regulators of
immune cell development. BAFF, B lymphocyte stimulator, is a member of the
TNF superfamily members that do not mediate apoptosis and plays a vital role in
immune responses. The study investigated the expression of BAFF in recurrent
spontaneous abortion patients and found that BAFF was decreased more in those
than in normal early pregnant women. This observation confirms the idea that
placental cytokines synthesized at the maternal-fetal interface preferentially
divert immunity away from the cell-mediated arm of the immune response,
which is associated with cytotoxic T lymphocyte activity, toward the humoral or
antibody-mediated arm, which is associated with B lymphocyte activity, as
originally postulated by Wegmann et al.26
In the previous study, investigators identified multiple downstream genes
transcriptionally induced by BAFF, including the cytokine IL-10 and other
factors.27 It has been shown that IL-10 can suppress cytokine production and
several accessory cell functions by Th1 cells and is regarded as a potent
suppressor of the effector functions of Th1 cells.28 As is known that Th2
immunity is beneficial to pregnancy while Th1 immunity is harmful, fetus
acceptance has been associated with a bias toward Th2 cytokine production
(e.g., IL-4, IL-10, transforming growth factor-β (TGF-β)) and away from Th1type proinflammatory cytokines (e.g., interferon-γ (IFN-γ), tumor necrosis
factor-α (TNF-α)) at the maternal-fetal interface.26,29,30 Th1/Th2 cytokine ratios
are significantly elevated in women with RSA, hence Th1 cytokine production
may up regulated in these women.31,32 In this study, we found expression of
BAFF gene and protein was significantly diminished in trophoblasts and deciduas
of recurrent spontaneous abortion patients than that of normal early pregnant
women under the same gestational weeks. The observation is consistent with the
above ideas that BAFF possibly induces the signature Th2 cytokine IL-10, so that
the lower expression of BAFF during pregnancy might shift the balance from
protective Th2 to a harmful Th1 cytokine bias associated with recurrent
pregnancy loss. But the intracellular signaling mechanisms of BAFF lower
expression in spontaneous abortion patients are not clear. For the phenomenon
there is another possible explanation that BAFF signaling initiates the antiapoptotic activity that is associated with the activation of NF-κB at the maternalfetal interface. Earlier study indicates that BAFF is a cytokine that belongs to the
TNF family and activates NF-κB through a distinct receptor.18 Thus, the lower
expression of BAFF induces the excessive apoptosis of the cells at the maternalfetal interface associated with recurrent fetal loss.
By immunohistochemistry, we found expression of BAFF protein was cell-specific.
BAFF was mainly localized to stromal cells of deciduas and cytotrophoblast and
syncytiotrophoblast cells of villi in both normal pregnant women and abortion
patients. Generally speaking, BAFF ligand is believed to be produced mainly in
leukocytes.15,16,18 Our detection of BAFF protein present in decidual stromal cells
and cytotrophoblast and syncytiotrophoblast cells was somewhat surprising, but
this detection agreed with the previous experiment reported by Phillips et al12
that BAFF was detected primarily in villous term cytotrophoblasts cells. The
immunostaining pattern of protein light expression in the tissues of abortion
patients was very similar to the pattern of gene expression shown by RT-PCR. It
is noticeable that the staining of BAFF in the blood vessel endothelium cells of
abortion patients can not be seen, while the protein was localized to that of
normal pregnant women. It is possible that BAFF play an important role in the
procedure of angiogenesis. Angiogenesis, the formation of new blood vessels
from pre-existing capillaries, is of importance in pregnancy.33,34 Typically, it
involves activation of quiescent endothelial cells, proliferation and differentiation
of endothelial cells, and formation of a new lumen and maturation of the
endothelium.35-39 In this study, we can not make sure the specific functions of
BAFF found in the blood vessel endothelium cells of normal pregnant women, it
may be a critical player in the formation and maintenance of endothelial integrity
and reorganization of the vascular bed in pregnancy.
It should be noted that messages encoding BAFF-R were undetectable in all the
samples, which supports the idea that BAFF-R has been detected only in
secondary lymphoid organs and not in any other tissues, including placenta.40 It
is already known that BAFF also has other two receptors, BCMA and TACI, but
we did not investigate expression of the two receptors in our study. Probably
BAFF may bind to BCMA and TACI or target other maternal tissues and/or
embryonic tissues.
In summary, our findings on BAFF detection in trophoblasts and deciduas and
lower expression of BAFF in recurrent spontaneous abortion women support the
postulation that BAFF is likely to be of profound importance to reproductive
success in women and contribute to cells growth, angiogenesis and immune
privilege at the maternal-fetal interface.
Acknowledgement: We are grateful to Dr. GAO Wen-juan and Dr. LIU Jia for
the technical support.
REFERENCES
1. Hunt JS, Petroff MG, McIntire RH, Ober C. HLA-G and immune tolerance in
pregnancy. FASEB J 2005; 19: 681-693. [PubMed]
2. Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligandinduced apoptosis as a mechanism of immune privilege. Science 1995; 270:
1189-1192. [PubMed]
3. Guller S, LaChapelle L. The role of placental Fas ligand in maintaining immune
privilege at maternal-fetal interfaces. Semin Reprod Endocrinol 1999; 17: 39-44.
[PubMed]
4. Choi C, Benveniste EN. Fas ligand/Fas system in the brain: regulator of
immune and apoptotic responses. Brain Res Brain Res Rev 2004; 44: 65-81.
[PubMed]
5. Niederkorn JY. See no evil, hear no evil, do no evil: the lessons of immune
privilege. Nat Immunol 2006; 7: 354-359.[PubMed]
6. Chen HL, Yang YP, Hu XL, Yelavarthi KK, Fishback JL, Hunt JS. Tumor necrosis
factor alpha mRNA and protein are present in human placental and uterine cells
at early and late stages of gestation. Am J Pathol 1991; 139: 327-335. [PubMed]
7. Runic R, Lockwood CJ, Ma Y, Dipasquale B, Guller S. Expression of Fas ligand
by human cytotrophoblasts: implications in placentation and fetal survival. J Clin
Endocrinol Metab 1996; 81: 3119-3122. [PubMed]
8. Hunt JS, Chen HL, Miller L. Tumor necrosis factors: pivotal components of
pregnancy? Biol Reprod 1996; 54: 554-562. [PubMed]
9. Chung IB, Yelian FD, Zaher FM, Gonik B, Evans MI, Diamond MP, et al.
Expression and regulation of vascular endothelial growth factor in a first
trimester trophoblast cell line. Placenta 2000; 21: 320-324. [PubMed]
10. Leisser C, Saleh L, Haider S, Husslein H, Sonderegger S, Knofler M. Tumour
necrosis factor-alpha impairs chorionic gonadotrophin beta-subunit expression
and cell fusion of human villous cytotrophoblast. Mol Hum Reprod 2006; 12:
601-609. [PubMed]
11. Phillips TA, Ni J, Hunt JS. Death-inducing tumor necrosis factor (TNF)
superfamily ligands and receptors are transcribed in human placentas,
cytotrophoblasts, placental macrophages, and placental cell lines. Placenta 2001;
22: 663-672. [PubMed]
12. Phillips TA, Ni J, Hunt JS. Cell-specific expression of B lymphocyte (APRIL,
BLyS)- and Th2 (CD30L/CD153)- promoting tumor necrosis factor superfamily
ligands in human placentas. J Leukoc Biol 2003; 74: 81-87. [PubMed]
13. Salamonsen LA, Dimitriadis E, Jones RL, Nie G. Complex regulation of
decidualization: a role for cytokines and proteases-a review. Placenta 2003; 17:
S76-S85. [PubMed]
14. Kammerer U. Antigen-presenting cells in the decidua. Chem Immunol Allergy
2005; 89: 96-104. [PubMed]
15. Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, et al. BLyS:
member of the tumor necrosis factor family and B lymphocyte stimulator.
Science 1999; 285: 260-263. [PubMed]
16. Schneider P, MacKay F, Steiner V, Hofmann K, Bodmer JL, Holler N, et al.
BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell
growth. J Exp Med 1999; 189: 1747-1756. [PubMed]
17. Shu HB, Hu WH, Johnson H. TALL-1 is a novel member of the TNF family that
is down-regulated by mitogens. J Leukoc Biol 1999; 65: 680-683. [PubMed]
18. Mukhopadhyay A, Ni J, Zhai Y, Yu GL, Aggarwal BB. Identification and
characterization of a novel cytokine, THANK, a TNF Homologue that activates
apoptosis, nuclear factor-κB, and c-Jun NH2-terminal kinase. J Biol Chem 1999;
274: 15978-15981. [PubMed]
19. Gross JA, Johnston J, Mudri S, Enselman R, Dillon SR, Madden K, et al. TACI
and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune
disease. Nature 2000; 404: 995-999.[PubMed]
20. Mackay F, Schneider P, Rennert P, Browning J. BAFF and APRIL: a tutorial on
B cell survival. Annu Rev Immunol 2003; 21: 231-264. [PubMed]
21. Paria BC, Reese J, Das SK, Dey SK. Deciphering the cross-talk of
implantation: advances and challenges. Science 2002; 296: 2185-2188.
[PubMed]
22. Lyall F. Mechanisms regulating cytotrophoblast invasion in normal pregnancy
and pre-eclampsia. Aust N Z J Obstet Gynaecol 2006; 46: 266-273.[PubMed]
23. Coulam CB. Understanding the immunobiology of pregnancy and applying it
to treatment of recurrent pregnancy loss. Early Pregnancy 2000; 4: 19-29.
[PubMed]
24. Christiansen OB, Nielsen HS, Kolte AM. Future directions of failed
implantation and recurrent miscarriage research. Reprod Biomed Online 2006;
13: 71-83.[PubMed]
25. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies:
integrating mammalian biology. Cell 2001; 104: 487-501. [PubMed]
26. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine
interactions in the maternal-fetal relationship: is successful pregnancy a TH2
phenomenon? Immunol Today 1993; 14: 353-356. [PubMed]
27. Xu LG, Wu M, Hu J, Zhai Z, Shu HB. Identification of downstream genes upregulated by the tumor necrosis factor family member TALL-1. J Leukoc Biol
2002; 72: 410-416. [PubMed]
28. Moore KW, O'Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin10. Annu Rev Immunol 1993; 11: 165-190.[PubMed]
29. Shurin MR, Lu L, Kalinski P, Stewart-Akers AM, Lotze MT. Th1/Th2 balance in
cancer, transplantation and pregnancy. Springer Semin Immunopathol 1999; 21:
339-359. [PubMed]
30. Ostensen M, Forger F, Villiger PM. Cytokines and pregnancy in rheumatic
disease. Ann N Y Acad Sci 2006; 1069: 353-363.[PubMed]
31. Emmer PM, Nelen WL, Steegers EA, Hendriks JC, Veerhoek M, Joosten I.
Peripheral Natural Killer cytotoxicity and CD56 (pos) CD16 (pos) cells increase
during early pregnancy in women with a history of recurrent spontaneous
abortion. Hum Reprod 2000; 15: 1163-1169. [PubMed]
32. Kwak-Kim JY, Gilman-Sachs A, Kim CE. T helper 1 and 2 immune responses
in relationship to pregnancy, nonpregnancy, recurrent spontaneous abortions
and infertility of repeated implantation failures. Chem Immunol Allergy 2005;
88: 64-79. [PubMed]
33. Ahmed A, Perkins J. Angiogenesis and intrauterine growth restriction.
Baillière's Best Pract Res Clin Obstet Gynaecol 2000; 14: 981-998. [PubMed]
34. Nardo LG. Vascular endothelial growth factor expression in the endometrium
during the menstrual cycle, implantation window and early pregnancy. Curr Opin
Obstet Gynecol 2005; 17: 419-423.[PubMed]
35. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000;
6: 389-395. [PubMed]
36. Folkman J, D'Amore PA. Bloods vessel formation: what is its molecular basis?
Cell 1996; 87: 1153-1155. [PubMed]
37. Risau W. Mechanisms of angiogenesis. Nature 1997; 386: 671-674.[PubMed]
38. Carmeliet P. Angiogenesis in health and disease. Nat Med 2003; 9: 653-660.
[PubMed]
39. Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med
2003; 3: 643-651. [PubMed]
40. Thompson JS, Bixler SA, Qian F, Vora K, Scott ML, Cachero TG, et al. BAFFR, a newly identified TNF receptor that specifically interacts with BAFF. Science
2001; 293: 2108-2111. [PubMed]