BIOLOGY OF REPRODUCTION 69, 718–724 (2003)
Published online before print 16 April 2003.
DOI 10.1095/biolreprod.102.013102
Regulation of FasL/Fas in Human Trophoblasts: Possible Implications
for Chorioamnionitis1
Dhruv R Balkundi,2,3 Judy A Ziegler,3 Jon F Watchko,3 Catherine Craven,4 and Massimo Trucco5
Department of Pediatrics,3 Division of Neonatology and Developmental Biology, University of Pittsburgh,
Magee Womens Research Institute, Pittsburgh, Pennsylvania 15213
Department of Pathology,4 Magee Womens Hospital, Pittsburgh, Pennsylvania 15213
Department of Pediatrics,5 Division of Immunogenetics (MT), University of Pittsburgh, Pittsburgh, Pennsylvania 15213
ABSTRACT
Chorioamnionitis is a common cause of premature birth and
is associated with significant morbidity and mortality in the
mother and infant. Preterm birth shares similarities with rejec-
tion of the fetal allograft, which is characterized by increased
apoptosis of placental trophoblasts. We hypothesized that there
is increased trophoblast apoptosis in chorioamnionitis and that
this increased apoptosis is mediated by the Fas ligand (FasL)/Fas
pathway. To test our hypothesis, we examined placental villous
tissues from patients with chorioamnionitis and used the TUNEL
assay to demonstrate enhanced trophoblast apoptosis in patients
with chorioamnionitis. When the same samples were stained for
Fas, there was increased trophoblast Fas expression in patients
with chorioamnionitis. To define the mechanisms responsible for
this increase in trophoblast apoptosis, we cultured villous ex-
plants from uncomplicated term placentas with proinflamma-
tory cytokines and demonstrated a marked increase in tropho-
blast apoptosis. By blocking FasL, we reduced tumor necrosis
factor a-induced and interferon g-induced apoptosis. These data
suggest that chorioamnionitis is associated with increased tro-
phoblast apoptosis and enhanced trophoblast Fas expression. As
a complement to our in vivo study, we demonstrated that cy-
tokine-induced trophoblast apoptosis is mediated in part by the
FasL/Fas pathway, suggesting that cytokines promote sensitivity
to Fas-mediated apoptosis. These mechanisms may be important
in perpetuating inflammation in the placental microenvironment
and may contribute to the pathogenesis of chorioamnionitis.
apoptosis, cytokines, immunology, placenta, trophoblast
INTRODUCTION
Chorioamnionitis is the most common cause of prema-
ture delivery in gestations of ,30 wk and occurs in ap-
proximately 10% of all births [1, 2]. Histologic signs of
chorioamnionitis [3] and an increase in inflammatory me-
diators [4–6] can be detected in .50% of women who de-
liver prematurely. Intrauterine infection during chorioam-
nionitis activates placental and decidual cells to produce a
number of proinflammatory cytokines at the fetomaternal
interface [6–8], including tumor necrosis factor a (TNFa),
1
Supported by grants from the Magee Womens Health Foundation, the
Mario-Lemieux Centers for Patient Care and Research, and the 25 Club
of Magee Womens Hospital.
2
Correspondence: Dhruv Balkundi, Department of Pediatrics, Magee
Womens Hospital, 300 Halket St., Pittsburgh, PA 15213.
FAX: 412 641 5313; e-mail: dbalkundi@mail.magee.edu
Received: 14 November 2002.
First decision: 12 December 2002.
Accepted: 1 April 2003.
Q 2003 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
718
interleukin (IL) 1b, interferon g (IFNg), IL-6, IL-8, and
granulocyte colony-stimulating factor (G-CSF) [5, 8, 9].
Recent evidence suggests that proinflammatory cytokines
are potentially harmful to pregnancy in mice [10] and hu-
mans [11]. For example, excess production of TNFa and
IFNg is thought to be involved in premature delivery [11,
12]. In addition to influencing the maternal immune re-
sponse, many of these cytokines affect trophoblast physi-
ology, specifically the proliferation and apoptosis of tro-
phoblast cells [13].
Apoptosis is an important process that maintains the ap-
propriate cell numbers by removing excess cells. The Fas
ligand (FasL)/Fas pathway is an important pathway of ap-
optosis that controls cell proliferation and tissue remodeling
[14]. Fas (CD95) is a transmembrane protein of the TNF/
nerve growth factor superfamily that is expressed on both
immune and nonimmune cell types [15]. Fas when bound
by FasL activates a signal transduction pathway that even-
tually results in apoptosis of the cell. FasL/Fas-mediated
apoptosis is essential for immune homeostasis, a process by
which unnecessary peripheral T lymphocytes are removed
from the active repertoire [16]. FasL also plays an impor-
tant role in several immune processes, including immune
privilege [17, 18]. We and others have demonstrated that
FasL present on human trophoblasts may confer immune
privilege to the fetus by destroying maternal T cells. [19–
23].
In addition to FasL, the trophoblasts express its receptor
Fas. Several other tissues, including mammary gland [14],
human ovary [24], and endometrium [25], express both
FasL and Fas. Despite expressing both FasL and Fas, vil-
lous trophoblasts undergo apoptosis in limited numbers dur-
ing normal pregnancy [26]. However, trophoblasts exhibit
increased apoptosis in pregnancies complicated by fetal
growth restriction [26] and preeclampsia [27, 28]. Recent
evidence suggests that the expression of FasL and Fas in
nonimmune cells is correlated with the presence of a tissue-
specific microenvironment [29, 30]. A number of factors in
the placental microenvironment, including hormones [31]
and cytokines [32], modulate the immune response by reg-
ulating the expression of FasL and Fas. Chorioamnionitis
is a commonly occurring inflammatory state associated
with a marked increase in proinflammatory cytokines in the
placental microenvironment. In the present study, we tested
the hypothesis that chorioamnionitis is associated with in-
creased villous trophoblast Fas-mediated apoptosis and that
proinflammatory cytokines regulate the expression and ac-
tivity of FasL and Fas at the fetomaternal interface. We
demonstrated that trophoblast apoptosis is markedly in-
creased in patients with chorioamnionitis in association
with increased Fas expression on trophoblasts. Proinflam-
 FAS-MEDIATED APOPTOSIS IN CHORIOAMNIONITIS
matory cytokines also induce trophoblast apoptosis in part
via the FasL/Fas apoptosis pathway.
MATERIALS AND METHODS
Tissue Collection and Processing
Placentas from patients with chorioamnionitis. This study was ap-
proved by the Institutional Review Board at Magee Womens Hospital.
Paraffin-embedded placental villous tissues from patients (n 5 5) with
grade 3 chorioamnionitis [33] were obtained from an archival collection
in the Department of Pathology at Magee Womens Hospital. Placental
tissues from gestational age-matched patients without chorioamnionitis
were used as controls. The diagnosis of chorioamnionitis was confirmed
by a perinatal pathologist, and the severity categorized as grades 1–3 using
a modification of the Blanc classification [33]. Grade 3, or severe chorio-
amnionitis, is characterized by a dense infiltration of neutrophils (.30
neutrophils/high-power field) that extends from the subchorionic space
throughout the chorion.
Placentas from uncomplicated pregnancies. Human placental tissues
were obtained from normal pregnant women at term (39–41 wk gestation,
n 5 15) after elective cesarean section. Inclusion criteria were 1) a single
fetus, 2) no preexisting clinical conditions such as diabetes, hypertension,
or autoimmune disease, and 3) no clinical evidence of chorioamnionitis
or group B streptococcal infection. Once received, the tissues were pro-
cessed immediately.
In Situ End Labeling of DNA for Cell Death Detection
Trophoblast apoptosis in placental villous tissues from patients with
chorioamnionitis was detected by enzymatic labeling of DNA strand
breaks using the TUNEL assay performed using a cell death detection kit
(Oncogene Research Products, San Diego, CA). Tissue sections were first
treated with 2 mg/ml of proteinase K, followed by quenching with 3%
H2O2 and equilibration with TdT equilibration buffer. The sections were
then blocked with blocking buffer followed by incubation with the sub-
strate diamimobenzidine (DAB) and counterstained with methyl green.
The slides were then examined by one of us (D.R.B.) who was blind to
the diagnosis of chorioamnionitis. Ten high-power fields (2003) were ex-
amined under a light microscope. Only the fields that showed complete
villous structure were included in the study. Villi at the edge of the section
were excluded from the study to avoid false-positive results due to an
edge artifact. Positive staining was defined as brown nuclei. Apoptotic
nuclei were counted in 10 random fields and reported as number of apo-
ptotic nuclei per 10 high-power fields.
Immunohistochemistry for Fas
Trophoblast Fas expression in paraffin-embedded placental villous tis-
sues from patients with chorioamnionitis was determined by immunohis-
tochemistry using the Vectastain ABC kit (Vector Laboratories, Burlin-
game, CA). Tissue sections were deparaffinized and rehydrated through
graded alcohols using standard procedures. Endogenous peroxidase activ-
ity was quenched by a 30-min incubation with 0.3% H2O2 in water. Non-
specific binding sites were blocked by incubation with 5% goat serum.
Samples were incubated with Fas antibody (polyclonal antibody C-20 at
1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at 48C over-
night. Sections were then washed twice and incubated with a biotinylated
secondary antibody diluted at 1:200 in PBS. Two successive washing steps
were followed by incubation with preformed avidin-biotinylated peroxi-
dase complex. Sections were developed with DAB as the chromogen sub-
strate, rinsed, and counterstained with Mayer hematoxylin. Sections in-
cubated with PBS instead of primary antibody served as negative controls.
Culture of Placental Villous Explants with Cytokines
and FasL Blocking Antibody
Placental villous tissues (100 mg) obtained from uncomplicated preg-
nancies were rinsed with warm 0.9% NaCl solution, and explants were
established in six-well culture plates. The explants were cultured with
different concentrations of IL-1b, IFNg, or TNFa (PeproTech, Rocky Hill,
NJ) for 24 h. The culture medium consisted of Dulbecco modified Eagle
medium (DMEM; Mediatech, Herndon, VA) supplemented with 10% fetal
calf serum (FCS; Hyclone, Logan, UT) and antibiotics. The explants were
incubated at 378C in a humidified atmosphere of 5% CO2 and 95% air.
The concentrations of cytokines used were 0.1, 1, 10, and 50 ng/ml. These
719
concentrations are consistent with the amniotic fluid concentrations seen
in patients with chorioamnionitis [4]. After 24 h of culture, the explants
were fixed in formaldehyde for TUNEL assay.
In experiments using FasL-blocking antibody, villous explants from
placentas of uncomplicated pregnancies were processed as above. The ex-
plants were initially treated with a specific FasL-blocking protein Fas-Fc
(Alexis Corporation, San Diego, CA) for 1 h at a concentration of 2 mg/
ml. Pretreatment with Fas-Fc for 1 h completely blocked FasL-induced
apoptosis in an ovarian carcinoma cell line [24]. Proinflammatory cyto-
kines (TNFa, IL-1b, or IFNg) were then added to the six-well plates at a
concentration of 10 ng/ml and cultured under standard cell culture con-
ditions for a further 24 h. Explants pretreated with Fas-Fc were also cul-
tured in 10% serum without cytokines. At the end of 24 h, the explants
were fixed in formaldehyde, and paraffin-embedded sections were pre-
pared. The sections were then stained to reveal apoptotic nuclei using the
TUNEL technique.
Isolation of Cytotrophoblasts
Cytotrophoblasts were isolated and purified using a modified version
of the protocol established by Kliman et al. [34]. Term placental tissues
were obtained after elective cesarean section under sterile conditions and
processed immediately. Several cotyledons from a single placenta were
removed from the underlying fibrous elements and rinsed thoroughly in
0.9% NaCl and penicillin (50 U/ml)/streptomycin (50 mg/ml) (Sigma
Chemical Co., St. Louis, MO). Soft villous material from the fetal surface
was cut away from the connective tissues and blood vessels. Approxi-
mately 30–40 g of tissue was collected from each placenta. The tissue
was coarsely minced and transferred to 150 ml warmed calcium- and mag-
nesium-free Hanks solution (CMF Hanks) containing 25 mM Hepes (Sig-
ma). The tissue was then digested with trypsin (1 mg/ml) and DNase 1 (5
mg/ml; Sigma) at 378C for 20 min each in a shaking water bath. The flask
was then set at an angle, and tissue fragments were allowed to settle for
5 min; the supernatant was then discarded. The tissue fragments were
subjected to the enzymatic digestion three more times as described above,
and the supernatant was collected each time. The supernatant was layered
over 1.5 ml calf serum in a 15-ml polystyrene conical centrifuge tubes
and pelleted. The resultant pellets were resuspended in DMEM containing
25 mM Hepes and 25 mM glucose (DMEM-H-G) at room temperature.
The final pellet was then resuspended in 3 ml DMEM-H-G and layered
on a preformed discontinuous Percoll (Pharmacia, Piscataway, NJ) gradi-
ent made up in CMF Hanks solution. The gradient was centrifuged at
room temperature for 20 min, and a multilayered preparation of eight
bands was obtained. The middle four to six layers from the bottom (25%–
35% density area) containing cytotrophoblasts were then washed with
DMEM-G, and the cells were counted using a hemocytometer. The cell
suspension was then purified by eliminating CD451 cells following cul-
ture with CD45-coated magnetic beads (Polysciences, Warrington, PA).
This step is needed to obtain a pure population of cytotrophoblasts. We
routinely have been able to isolate 1–1.5 3 106 cells/g of placental tissue
with this method. The purity of the cytotrophoblast population (generally
.95%) was ascertained using anti-cytokeratin and anti-CD45 antibodies
(R&D Systems, Minneapolis, MN). The freshly isolated cells were used
for cell culture and Western immunoblotting.
Culture of Cytotrophoblasts with Cytokines
The cytotrophoblasts were cultured for 24 h in DMEM with 10% FCS
in 12-well plates at a cell density of 3 3 106 cells/well. The cells were
then washed with sterile PBS, dead (floating) cells were gently aspirated,
and the adherent cells were incubated with increasing concentrations (0.1,
1, 10, 50, and 100 ng/ml) of individual cytokines (IL-1b, TNFa, or IFNg).
These concentrations were chosen because they represent the range of
cytokines in amniotic fluid in women with proven histologic chorioam-
nionitis [4]. The cell lysates were then subjected to Western blot analysis.
Western Blot Analysis for FasL and Fas Expression
Protein extraction and Western blot analysis of FasL and Fas were
performed as previously described [22]. After 24 h of culture with cyto-
kines, 3 3 106 cytotrophoblasts cultured with cytokines were washed once
in PBS (13) and harvested in PBS containing 1 mM PMSF and 10 mg/
ml leupeptin (Sigma). The resultant cell pellet was resuspended in 15 ml
of Laemmli sample buffer and then sonicated on ice for 15–20 sec. After
centrifugation at 14 000 rpm for 15 min, the supernatants were collected,
and total protein concentrations determined by the Bio-Rad Protein Assay
(Bio-Rad Laboratories, Hercules, CA) with BSA as the protein standard.
720 BALKUNDI ET AL.

FIG. 1. In situ localization of apoptotic

nuclei in placental villi from uncomplicat-
ed (A) and chorioamnionitis (B) pregnan-
cies. Representative sections were stained
using the TUNEL technique. Apoptotic nu-
clei were stained brown (arrow). Original
magnification 3200. The total number of
apoptotic nuclei per 10 high-power fields
was determined in the placental villi in
uncomplicated and chorioamnionitis preg-
nancies (C). The red bar represents the
median number of apoptotic nuclei. Signif-
icant difference (P , 0.001) between un-
complicated and chorioamnionitis preg-
nancies.

Equal amounts of protein extracts were separated by SDS-PAGE using
10% polyacrylamide gels and then transferred to nitrocellulose mem-
branes. After blocking nonspecific binding sites by treatment with 5% milk
powder and 0.05% Tween-20 in PBS, the blots were washed and probed
with anti-FasL (polyclonal antibody C-178 at 1:1000 dilution; Santa Cruz
Biotechnology) or anti-Fas (polyclonal antibody C-20 at 1:200 dilution;
Santa Cruz Biotechnology) for 1 h at room temperature. Following incu-
bation with a donkey anti-rabbit horseradish peroxidase-conjugated sec-
ondary antibody (1:2500 dilution; Amersham), the blots were washed. The
protein bands were visualized using an enhanced chemiluminescence kit
(NEN Life Science Products, Boston, MA) and exposed on BioMax MR
film (Eastman Kodak, Rochester, NY). The films were electronically
scanned, and the band densities were quantified using Molecular Analyst/
MacIntosh software (Bio-Rad). Equal protein loading was confirmed by
Coomassie blue staining of all the gels.
Statistical Analysis
Data were analyzed by chi-square tests and Student t-tests. The differ-
ences were considered significant at P , 0.05. All experiments were per-
formed in triplicate.
RESULTS
fields for patients with chorioamnionitis (Fig. 1B). The me-
dian number of apoptotic nuclei found in chorioamnionitis
was significantly higher than that in the controls (4 vs. 0,
P , 0.001; Fig. 1C).
Chorioamnionitis Is Associated with Increased Villous
Trophoblast Fas Expression
To determine whether the increased trophoblast apopto-
sis in chorioamnionitis is associated with increased Fas ex-
pression, we performed immunohistochemistry on placental
villous tissues from women with chorioamnionitis and com-
pared these tissues with gestational age-matched controls.
There was a marked increase in Fas expression in the tro-
phoblasts (both syncytio- and cytotrophoblasts) of the cho-
rioamnionitis placentas (Fig. 2) compared with low levels
of constitutive Fas expression in placentas from uncompli-
cated pregnancies (Fig. 2B). In contrast, there was a marked
increase in Fas expression in placental villous trophoblasts
in patients with chorioamnionitis (Fig. 2B).

Chorioamnionitis Is Associated with Increased Villous
Trophoblast Apoptosis
To test our hypothesis that chorioamnionitis is associated
with increased villous trophoblast apoptosis, we studied
placental villous tissues from women with chorioamnionitis
(n 5 5). For the negative control, we chose placentas from
otherwise normal pregnancies, and these placentas were
matched for gestational age. The placental tissues from pa-
tients with chorioamnionitis showed increased apoptosis in
the trophoblast layer involving both syncytiotrophoblasts
and cytotrophoblasts, whereas the control tissues were gen-
erally negative for this increase (Fig. 1A). There was a
marked increase in apoptotic nuclei in randomly chosen
Proinflammatory Cytokines Induce Trophoblast Apoptosis
To understand the mechanisms for trophoblast apoptosis
in chorioamnionitis, we performed in vitro studies using
villous tissues from uncomplicated term pregnancies. Vil-
lous explants were established and cultured with proinflam-
matory cytokines TNFa, IL-1b, or IFNg individually. Tro-
phoblast apoptosis was determined by TUNEL assay. There
was marked apoptosis noted in both syncytio- and cytotro-
phoblast layers (Fig. 3, C, E, and G). Figure 3 demonstrates
the trophoblast apoptosis at a cytokine concentration of 10
ng/ml, although apoptosis was noted at lower concentra-
tions of individual cytokines (data not shown). The villous
explants were then cultured with a combination of all three
FAS-MEDIATED APOPTOSIS IN CHORIOAMNIONITIS 721

FIG. 2. Immunolocalization of
Fas-expressing trophoblasts in placental
villi from uncomplicated (A) and chorio-
amnionitis (B) pregnancies. Representative
sections were stained for Fas expression.
Fas expression was increased in cytotro-
phoblasts and syncytiotrophoblasts in pa-
tients with chorioamnionitis. Original mag-
nification 3200.

FIG. 3. FasL-blocking protein Fas-Fc

reduces TNFa- and IFNg-induced apopto-
sis. Placental villi from uncomplicated
term pregnancies were initially treated
with 2 ng/ml of Fas-Fc for 1 h and cul-
tured with TNFa, IFNg, or IL-1b for 24 h.
Fas-Fc does not reduce the spontaneous
apoptosis when villous explants are cul-
tured for 24 h (A and B). Fas-Fc reduces
TNFa-induced (C and D) and IFNg-in-
duced (E and F) but not IL-1b-induced (G
and H) apoptosis. Original magnification
3200.
722 BALKUNDI ET AL.

FIG. 4. Western blot analysis demonstrating the effect of TNFa, IFNg, and IL-1b on cytotrophoblast FasL and Fas expression. Cytotrophoblasts
(3 3 106) were cultured with different concentrations of individual cytokines for 24 h (0, 0.1, 1, 10, and 50 ng/ml). TNFa (A), IFNg (B), and IL-1b (C)
increased Fas expression, but none of the cytokines had an effect on FasL expression. The intensity of the signal was analyzed using a digital imaging
analysis system. Bars represent the mean 6 SEM. The figures are representative of three independent experiments.

cytokines, resulting in marked trophoblast apoptosis (data
not shown). These results suggest that the proinflammatory
cytokines induce trophoblast apoptosis,; however, they do
not elucidate the mechanism of apoptosis.
TNFa and IFNg but Not IL-1b Induce Fas-Mediated
Apoptosis of Trophoblasts
To determine whether the apoptosis induced by the
proinflammatory cytokines was induced by FasL, we cul-
tured the placental villous explants with the specific FasL-
blocking protein Fas-Fc for 1 h prior to culture with TNFa,
IL-1b, or IFN-g. Following 24 h of culture with Fas-Fc and
cytokines, the placental explants were subjected to TUNEL
assay. We and others have noted that placental villous tro-
phoblasts undergo spontaneous apoptosis under culture
conditions [35] (Fig. 3A). However, this apoptosis was not
reduced by blocking FasL (Fig. 3B). In contrast, Fas-Fc
reduced trophoblast apoptosis in explants treated with
TNFa (Fig. 3D) and IFNg (Fig. 3F). Fas-Fc had no effect
on the villous explants treated with IL-1b (Fig. 3H) or a
combination of all three cytokines. These findings suggest
that TNFa and IFNg induce apoptosis by ligation of FasL
with Fas, whereas IL-1b uses a pathway independent of
Fas. Contrary to the studies by others, our results suggests
that trophoblast FasL and Fas are biologically active and
that this activity is dependent on the specific microenviron-
ment in which proinflammatory cytokines are increased.
Effect of Proinflammatory Cytokines on FasL and Fas
Expression in Cytotrophoblasts
To determine the effects of cytokines on trophoblast
FasL and Fas expression, we cultured cytotrophoblasts with
individual cytokines. Because cytotrophoblasts are the stem
cells for the different populations of trophoblasts, they
serve as good models to determine the effects of cytokines
on FasL and Fas expression. Cytotrophoblasts isolated from
placentas obtained from uncomplicated term pregnancies
were cultured with different concentrations of cytokines for
24 h, and then Western blot analysis was performed. FasL
and Fas expression was determined using FasL and Fas
antibodies, respectively. Cytotrophoblasts expressed FasL
and Fas constitutively (Fig. 4). TNFa, IFNg, and IL-1b had
no effect on the expression of FasL. In contrast, all three
cytokines increased Fas expression at concentrations con-
sistent with amniotic fluid concentrations in patients with
chorioamnionitis. However, this increase in Fas expression
was not significant (P 5 0.06).
DISCUSSION
The results of the present study indicate that chorioam-
nionitis is associated with increased trophoblast Fas ex-
pression and apoptosis. When placental villous explants
from uncomplicated pregnancy were cultured with proin-
flammatory cytokines TNFa or IFNg, trophoblast apoptosis
occurred by the FasL/Fas pathway. These findings suggest
that FasL expressed on the trophoblasts and activated ma-
 FAS-MEDIATED APOPTOSIS IN CHORIOAMNIONITIS
ternal lymphocytes can induce trophoblast Fas-mediated
apoptosis by autocrine or paracrine interactions. In addition,
the increased Fas expression on trophoblasts may make
them more sensitive to Fas-mediated apoptosis.
The increase in trophoblast apoptosis associated with
chorioamnionitis provides support for our hypothesis that
immune cells in placenta regulate trophoblast apoptosis via
cytokines at the fetomaternal interface. One possible reason
for the increase in trophoblast apoptosis associated with
chorioamnionitis could be the changes taking place in cy-
tokine composition in the placental microenvironment.
Chorioamnionitis is characterized by the activation of the
placental and decidual immune cells, resulting in the release
of inflammatory mediators, specifically cytokines and che-
mokines, into the placental microenvironment [5]. This re-
lease of inflammatory mediators results in dense neutrophil
and lymphocytic infiltration in the chorion, amnion, and
placental villi, which is the hallmark of chorioamnionitis
[3]. In addition to the immune cells, trophoblasts also pro-
duce cytokines. The proinflammatory cytokines whose in-
creases are associated with chorioamnionitis are cytotoxic
to trophoblasts, resulting in their apoptosis [13, 36].
A possible mechanism for the increased trophoblast apo-
ptosis associated with chorioamnionitis could be the acti-
vation of the FasL/Fas pathway of apoptosis. Cells undergo
apoptosis by either ligation of cell surface death receptors
by their respective ligands or by the mitochondrial pathway
[37]. The Fas L/Fas system has been recognized as an im-
portant pathway of apoptosis [16, 18, 37]. However, the
role of FasL and Fas in trophoblast survival is perhaps more
complex than originally anticipated. We confirmed the pres-
ence of Fas in normal trophoblasts (Figs. 2 and 4), as has
been previously reported [38–40]. FasL and Fas interaction
may occur between neighboring trophoblasts or on the
same cell by paracrine and autocrine mechanisms.
Contrary to studies by other investigators [38], we dem-
onstrated that Fas on trophoblasts is biologically active. By
specifically blocking FasL, we reduced TNFa- and IFNg-
induced apoptosis (Fig. 3). This finding suggests that Fas
is biologically active during inflammation, when proinflam-
matory cytokines are increased in the placental microen-
vironment. A possible explanation for the resistance to Fas-
mediated apoptosis described by other investigators could
be the low levels of trophoblast Fas expression constitu-
tively (Fig. 2A). Recent evidence suggests that cells are
more sensitive to Fas-mediated apoptosis when levels of
Fas expression increase [41]. Our data demonstrating in-
creased Fas expression may therefore explain the increased
sensitivity to Fas-mediated apoptosis in trophoblasts. An-
other explanation for resistance to Fas-mediated apoptosis
is the presence of Th-2 cytokines or inhibitors downstream
of Fas, such as FLICE-inhibitory protein [32] and Bcl-x
[42].
Although our data (Western blots) did not demonstrate
an increase in cytotrophoblast FasL expression following
culture with cytokines, an increase in Fas expression may
predispose cells to apoptosis. The absence of a detectable
increase in FasL expression could be explained by the
cleavage of membranous FasL and its release as the soluble
form (sFasL), which is chemotactic and proinflammatory
[43, 44]. In the presence of increased Fas expression, sFasL
has a synergistic effect, making the cells more susceptible
to Fas-mediated apoptosis [41]. Thus, sFasL secreted into
the placental microenvironment together with the increased
Fas expression may furthur amplify the inflammatory pro-
cess. The increased Fas concentration also may downreg-
723
ulate FasL expression by a negative feedback mechanism.
These questions merit future study.
Generally when cells undergo apoptosis there is no in-
flammatory response; however, when peritoneal macro-
phages [45] and dendritic cells [46] undergo Fas-mediated
apoptosis, cytokines and neutrophil chemotactic factors are
released and inflammation is perpetuated. Because tropho-
blasts can themselves produce cytokines, a similar phenom-
enon may occur in the placental microenvironment in pa-
tients with chorioamnionitis. We propose the following
mechanism for inflammation in chorioamnionitis: bacterial
infection activates placental and decidual immune cells to
produce cytokines, which in turn induce Fas-mediated ap-
optosis of trophoblasts, and more cytokines and chemotac-
tic factors are released, which amplifies the inflammatory
process. The Fas signaling pathway in trophoblasts could
activate cytoplasmic mediators that regulate chemokine
production and release and activate caspases [47, 48].
Therefore, trophoblasts could undergo apoptosis and release
cytokines, functions that are independent of each other [49,
50].
The increased trophoblast apoptosis associated with cho-
rioamnionitis implies that the number of cells necessary to
perform the immune privilege function is diminished, pos-
sibly contributing to fetal allograft rejection, i.e., premature
delivery. Critical events during normal pregnancy, such as
immunoprotection, are regulated by cytokines produced lo-
cally at the mother-fetus interface [51]. The locally pro-
duced cytokines may exert their effect via the FasL/Fas
pathway. Our results shed light on the delicate balance be-
tween immunoprotection and fratricidal trophoblast death.
These data have implications for understanding the patho-
genesis of chorioamnionitis and for the development of fu-
ture therapeutic approaches to this disease.
ACKNOWLEDGMENTS
The authors thank Terry O’Day for help with statistical analysis and
preparation of figures, Dr Jonathan Wispe for critical review of the man-
uscript, and Robert Szmyd and Karen Bellisario for assistance in the pro-
curement of normal term placentas.
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