This information is current as

of December 9, 2014.

Exosomes Secreted by Human Placenta

Carry Functional Fas Ligand and TRAIL
Molecules and Convey Apoptosis in Activated
Immune Cells, Suggesting Exosome-Mediated
Immune Privilege of the Fetus
Ann-Christin Stenqvist, Olga Nagaeva, Vladimir Baranov
and Lucia Mincheva-Nilsson

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J Immunol 2013; 191:5515-5523; Prepublished online 1

November 2013;
doi: 10.4049/jimmunol.1301885
http://www.jimmunol.org/content/191/11/5515

The Journal of Immunology

Exosomes Secreted by Human Placenta Carry Functional Fas

Ligand and TRAIL Molecules and Convey Apoptosis in
Activated Immune Cells, Suggesting Exosome-Mediated
Immune Privilege of the Fetus
Ann-Christin Stenqvist, Olga Nagaeva, Vladimir Baranov, and Lucia Mincheva-Nilsson

ammalian reproduction has to face and solve the immunological challenge of accepting a semiallogeneic
fetus and supporting its development and growth. The
human hemochorial placenta, known to preferentially express
paternal Ags, is in direct contact with the maternal blood, and thus,
the syncytiotrophoblast (STB) covering the chorionic villi is
physically exposed to a risk of a potential attack from the maternal
immune system. Despite this, the human placenta and the fetal
allograft evade a harmful maternal immune attack and enjoy an
immunologic privilege in the uterine cavity.
Multiple maternal and fetal mechanisms of tolerance work in
concert to prevent fetal rejection (reviewed in Ref. 1). Several of
these mechanisms are mediated by the placenta that not only
Division of Clinical Immunology, Department of Clinical Microbiology, Umea University, S-90185 Umea, Sweden
Received for publication July 16, 2013. Accepted for publication September 25,
2013.
This work was supported by the Swedish National Research Foundation (Vetenskapsradet,
Grant K2013-54X-22341-01-05); the Swedish National Cancer Research Foundation
(Cancerfonden Grant CAN2010/495); Central ALF funding and Spjutspetsanslag,
County of Vasterbotten; and the Insamlingsstiftelsen at the Medical Faculty, Umea
University.
Address correspondence and reprint requests to Dr. Lucia Mincheva-Nilsson, Division of Clinical Immunology, Department of Clinical Microbiology, Umea University, S-90185 Umea, Sweden. E-mail address: lucia.mincheva-nilsson@climi.umu.se
The online version of this article contains supplemental material.
Abbreviations used in this article: APO, apoptosis Ag; CTB, cytotrophoblast; DAB,
3,39-diaminobenzidine tetrahydrochloride; DISC, death-inducing signaling complex;
EM, electron microscopy; FasL, Fas ligand; IEM, immunoelectron microscopy;
mFasL, membrane-bound Fas ligand; MMP, matrix metalloproteinase; MVB, multivesicular body; sFasL, soluble FasL; STB, syncytiotrophoblast.
Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301885

supplies hormonal, nutritional, and oxygen support but also stands

out as an important immunomodulatory organ. Apoptosis, or programmed cell death, is one of the proposed maternofetal mechanisms of tolerance that plays an important role in implantation and
pregnancy (2). Apoptosis is detected in the placenta throughout
pregnancy and has been shown to participate in trophoblast invasion,
differentiation, and turnover during placental formation (3, 4) and in
the creation of maternal immune tolerance toward the fetus (5, 6).
Apoptotic cell death can be initiated through an intrinsic pathway by
intracellular signals sensed by mitochondria or an extrinsic pathway
via death receptors, members of the TNF superfamily (7, 8). Of all
members, the death messengers Fas ligand (FasL) and the TRAIL
are physiologically the most relevant.
FasL (apoptosis Ag [APO]-1L and CD95L), a homotrimeric
37-kDa type II membrane protein, signals cell death by engagement of its cognate receptor Fas (APO-1 and CD95). Initially,
FasL was described as an apoptosis-inducing protein expressed on
activated immune cells. Later studies revealed that it is involved in
organ development and homeostasis and is expressed on various
other cells and tissues including cells associated with immune
privileged sites such as the eye, brain, testis, and placenta (9, 10).
The intracellular and extracellular domains of FasL are located in
the N- and C-terminal regions, respectively. The proapoptotic
activity is associated with membranal expression of FasL where
oligomerization of the molecule is needed in the formation of the
death-inducing signaling complex (DISC) (11). A cleavage of the
extracellular domain by the metalloprotease metralysin produces
a 26-kDa soluble form of FasL (12) that loses its apoptotic activity
and can even inhibit the action of membrane-bound (m)FasL.
TRAIL (APO-2L and CD253) was first identified as a TNF
superfamily member capable of inducing apoptotic cell death

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Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We investigated the expression and
in vitro apoptotic activity of two physiologically relevant death messengers, the TNF family members Fas ligand (FasL) and TRAIL
in human early and term placentas. Both molecules were intracellularly expressed, confined to the late endosomal compartment of
the syncytiotrophoblast, and tightly associated to the generation and secretion of placental exosomes. Using immunoelectron microscopy, we show that FasL and TRAIL are expressed on the limiting membrane of multivesicular bodies where, by membrane
invagination, intraluminal microvesicles carrying membranal bioactive FasL and TRAIL are formed and released in the extracellular space as exosomes. Analyzing exosomes secreted from placental explant cultures, to our knowledge, we demonstrate
for the first time that FasL and TRAIL are clustered on the exosomal membrane as oligomerized aggregates ready to form
death-inducing signaling complex. Consistently, placental FasL- and TRAIL-carrying exosomes triggered apoptosis in Jurkat
T cells and activated PBMC in a dose-dependent manner. Limiting the expression of functional FasL and TRAIL to exosomes
comprise a dual benefit: 1) storage of exosomal FasL and TRAIL in multivesicular bodies is protected from proteolytic cleavage
and 2) upon secretion, delivery of preformed membranal death molecules by exosomes rapidly triggers apoptosis. Our results suggest that bioactive FasL- and TRAIL-carrying exosomes, able to convey apoptosis, are secreted by the placenta and tie up the
immunomodulatory and protective role of human placenta to its exosome-secreting ability. The Journal of Immunology, 2013,
191: 55155523.

5516

PLACENTAL EXOSOMES EXPRESS APOPTOSIS-INDUCING FasL AND TRAIL

Materials and Methods

Samples and Abs
Early and term placental specimens were donated by healthy women undergoing elective termination of pregnancy (814 wk of gestation) or
normal delivery after ethical committee approval and informed consent.
Clones and specificities of Abs were as follows: mouse anti-human FasL
(G247-4; BD Pharmingen), goat anti-human TRAIL (K-18), mouse antihuman CD63 (MX-49.129.5; Santa Cruz Biotechnology), mouse antihuman pan MIC (clone 6D4; BD Biosciences), FITC-conjugated mouse
anti-human CD63 (Immunotech), mouse anti-human CD95 (EOS9.1;
eBioscience), mAbs against TRAIL-R1 (clone number 69036) and
TRAIL-R2 (clone number 71908) (R&D Systems), CD56 (My31; BD
Biosciences), mAbs against CD4, CD8, CD19, and isotype-matched control mAbs IgG1, IgG2a, and IgG2b (DakoCytomation), normal goat serum
(Vector Laboratories), and HRP-conjugated secondary Abs (Jackson
ImmunoResearch Laboratories).

RNA extraction, RT-PCR, and quantitative PCR from isolated

and laser-microdissected STB
Trophoblast from placental villi (three early and three term placental samples)
was isolated using a mild optimized protocol as described in Ref. 26. STB was
separated and enriched from the isolated trophoblast by positive selection
with anti-human pan MICcoated Dynabeads as previously described (26),
put immediately in dissolving solution and frozen for total RNA isolation. In
addition, STB was obtained by laser microdissection of chorion villi (two
early and two late placental samples) and dissolved for RNA extraction (27).
Total RNA extraction and real-time quantitative RT-PCR were performed as
described previously (5, 26, 27). In brief, RNA was extracted according to
the acid guanidinum thiocyanate-phenol-chloroform method, and reverse
transcription was performed at 40C for 15 min using random hexamer
primers (Life Technologies/Applied Biosystems) and murine leukemia
virus reverse transcriptase (Promega). Assays-on-Demand Gene Expression probes (Applied Biosystems) (FASLG, assay ID Hs00181226_g1; and
TNFSF10, assay ID Hs00921974_m1) were used. Multiplexed real-time
PCR assay with 18S rRNA as an endogenous control (Life Technologies/
Applied Biosystems) was performed on a 7900HT instrument with the
factory default thermocycle conditions for 40 cycles, and raw data were
analyzed using a relative quantitation method. Gene expression levels are
presented as a relative fold change between the studied samples. Each
gene test included PBMC stimulated by PMA/ionomycin as a positive
control and a negative control omitting addition of template.

Immunohistochemistry
Chorionic villi were fixed in 4% paraformaldehyde for 2 h, washed in PBS
with 3.5% sucrose and 1% fish gelatin, and snap-frozen in liquid nitrogen.
Eight-micrometer cryosections were stained as described previously (22, 26).
FcgRs were blocked by a mixture of horse serum (ImmPRESS reagent kit)
and 10% normal human serum (AB+), aldehyde groups were blocked by PBS
with 0.1 M glycine, and 0.03% H2O2 in PBS was used for extinguishing the
endogenous peroxidase activity. ImmPRESS anti-mouse or anti-goat Reagent
kit (Vector Laboratories) were used for enhanced detection with 3,39-diaminobenzidine tetrahydrochloride (DAB) as substrate. Serial sections were
included as negative controls stained by exchanging the first Ab with isotypematched control mAbs or normal goat serum for the polyclonal primary Abs.

IEM of chorionic villi

Freshly isolated placental samples were immediately fixed in 4% paraformaldehyde for 4 h, washed in 0.1 M phosphate buffer containing 7%
sucrose and 0.05% saponin at 4C overnight, and snap-frozen. Cryosections
were processed by indirect immunoperoxidase method, as previously described (22, 27) using mAb clone G247-4 against FasL and goat antihuman Ab (K18) against TRAIL. ImmPRESS anti-mouse or anti-goat
Reagent kit (Vector Laboratories) was used for enhanced detection with
DAB as substrate. One-percent normal human serum (AB+) and 0.5%
BSA in PBS was used to block FcRs on STB in all blocking steps. Antigoat and anti-rabbit ImmPRESS reagents and DAB were used to reveal
specific staining. Ultrathin sections were examined in a Zeiss EM 900
electron microscope (Carl Zeiss) without additional staining. Sections incubated with isotype-matched control mAbs or affinity purified normal
goat serum were used as negative control.

Placental explant cultures

Explants of freshly obtained early placentas were immediately processed
and put in culture within 1 h after extraction as described in Ref. 28. In brief,

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mainly in tumor cells. TRAIL, a type II membrane protein, spontaneously trimerizes around a centrally positioned Zn atom important for its stability, solubility, and biological activity as a death
ligand (13) and binds to four membrane-bound receptors
TRAIL-R1/death receptor 4, TRAIL-R2/death receptor 5, TRAILR3/decoy receptor 1, and TRAILR4/decoy receptor 2 (3); and the
soluble receptor osteoprotegerin (14). The ability of TRAIL to kill
transformed cells by apoptosis is well established, but the overall physiological role of TRAIL is not fully defined; however,
TRAIL-induced apoptosis has also been implied in other processes such as activation-induced cell death, homeostasis, intrathymic negative selection, suppression of autoimmunity, and immune
surveillance (14). Studies of the cytotoxic immune response showed
that both FasL and TRAIL could be recovered from the supernatant
of cultured activated T cell blasts and leukemia cells in a bioactive
microvesicular form (15). These findings were supported by our
morphological studies of the cytotoxic machinery of activated
decidual gdT and NK cells, which revealed that FasL and perforin
were preformed and stored in microvesicles in multivesicular body
(MVB)like membrane-bound cytotoxic granules (5).
Both FasL and TRAIL were found to be constitutively expressed
by the placenta and proposed as cooperating players for inducing
apoptosis in activated lymphocytes (1618) that might pose a potential threat to the fetus. As stated above, membrane-bound Fas
ligand (mFasL) but not soluble FasL (sFasL) promotes apoptosis
of Fas-bearing cells (19, 20). Convincing reports by us and others
have shown that the human placenta lacks cell surface membranal
FasL expression (21, 22). Instead, the FasL expression is located
inside the cytoplasm in the endosomal compartment of the STB of
the chorionic villi and could be secreted in active microvesicular
form (22).
Exosomes are 30- to 100-nmsized membrane-bound microvesicles formed and released through the late endosomal compartment by a variety of cells. They contain both cytosolic and
transmembrane proteins, and mRNA and microRNA and can be
viewed as a way of intercellular communication without the
need for cellcell contact, thus conveying a potential biologic
activity to more distant targets. Depending on their origin and
composition, the exosomes can have either immunostimulatory
or immunosuppressive properties (2325). Lately, the role of
microvesicles/exosomes in the multifactorial ways of promoting immunotolerance has gained elevated focus in many different areas, including tumor escape and fetal survival during
pregnancy (2325).
In previous immunoelectron microscopy (IEM) studies of the
human placenta, we showed that FasL expression takes place in the
STB and is solely confined to the endosomal compartment where
FasL-expressing microvesicles were revealed in the MVBs as well
as signs of MVB fusion with the plasma membrane, suggesting
a release of FasL-carrying exosomes (22). In the current study, we
have extended and deepened our investigation to include the
biogenesis, expression, and precise intracellular location of the
other major proapoptotic TNF superfamily member TRAIL. In
this study, we provide evidence that similarly to FasL, TRAIL
expression in the human placenta is restricted to STB and is
present in MVBs on microvesicles released in the extracellular
space as exosomes. Furthermore, to our knowledge, we show for
the first time that FasL and TRAIL were expressed on the exosomal membrane in an oligomerized complex of two homotrimeric FasL and/or TRAIL molecules required for DISC formation
and triggering apoptosis, and show evidence for their biologic
activity in exosome-induced apoptosis in Jurkat T cells and activated PBMC from healthy donors.

The Journal of Immunology

10 mg wet weight pieces of chorion villi were cultured in ultracentrifuged
medium of RPMI 1640 supplemented with 0.5% BSA (Sigma Aldrich) and
antibiotics at 37C in humid atmosphere with 5% CO2. Supernatant was
harvested after 24 h in culture and used for exosome isolation.

Isolation of exosomes
Supernatants from short-term placental explant cultures from individual
donors were cleared from cell debris and larger particles by sequential
centrifugations at 4,000 3 g for 30 min and 17,000 3 g for 25 min. After
filtration through a 0.2-mm filter, the supernatant was ultracentrifuged at
110,000 3 g for 2 h. The pellet was resuspended in sterile, particle-free
PBS and ultracentrifuged through a 20 and 40% discontinuous sucrose
gradient; the exosomes were collected and washed a couple of times with
sterile PBS. The yield of exosomes was estimated with bicinchoninic acid
protein assay kit (Pierce). Isolated exosomes were stored at 280C in PBS
or radioimmunoprecipitation assay buffer (Pierce) supplemented with
protease inhibitor mixture (Roche Diagnostics) until use.

Western blot analysis

Immunoflow cytometry of protein expression on the surface of

isolated placental exosomes
For analyzing the expression of proapoptotic molecules on the surface of
isolated placental exosomes, surfactant-free ultraclean 4-mm sulfate latex
microbeads (Interfacial Dynamics) were used as described previously (27,
29). The beads were coated with antiFasL-, antiTRAIL-, or isotypematched control Abs. After blocking of uncoupled sites, isolated exosomes were added and incubated 4C overnight with end-to-end rotation.
After washing, FITC-conjugated CD63 mAb was incubated for 30 min
before final washing. The beads were analyzed by flow cytometry on
FACScan (BD Biosciences) using CellQuest software.

IEM of protein expression on the surface of isolated placental

exosomes
Isolated exosomes were absorbed on formvar/carbon-coated nickel grids,
washed with PBS, and fixed with 2% paraformaldehyde in PBS for 10 min.
Negative staining was performed on the grids using 1.9% methylcellulose
containing 0.3% uranyl acetate. Excess fluid was removed and allowed to
dry before examination with electron microscope. For IEM, after blocking
in 0.1 M glycine and 0.3% BSA, grids with exosomes were incubated with
appropriate monoclonal or polyclonal Abs, isotype-matched control Abs, or
ultracentrifuged goat serum for 1 h in wet chamber. After washing, the grids
were incubated with secondary Abs conjugated with 5- or 10-nm gold particles for 1 h. After washing and additional fixation with 2.5% glutaraldehyde,
the grids were negatively stained as described previously (27). In double IEM
staining after completed first staining with anti-Fas and after a blocking step
with goat serum, the second IEM staining for TRAIL was performed.
Conjugation of the secondary Abs with 5- or 10-nm gold particles was used
to distinguish between exosomal FasL and TRAIL staining.

Isolation and activation of PBMC and culture of Jurkat cells

PBMC from healthy donors were isolated by Lymphoprep (Nycomed)
gradient centrifugation and used for detection of exosome-induced apoptosis. One million isolated PBMC were activated for 24 h with PMA/
ionomycin as previously described (27, 28), and the PBMC activation
was measured by flow cytometry of Fas and TRAIL-R1 and TRAIL-R2
expression. Jurkat cells, purchased from the American Type Culture Collection, were cultured in RPMI 1640 medium, supplemented with 0.5%
BSA (Sigma Aldrich) and antibiotics at 37C in atmosphere of 5% CO2
and humidity and used in apoptosis detection tests.

Assessment of the apoptotic effect of placental exosomes

Annexin V:PE Apoptosis Detection Kit I (BD Biosciences) and immunoflow cytometry were used for detection of exosome-induced apoptosis on
Jurkat cells, according to the manufacturers instructions. In brief, Jurkat

cells (1 3 106/ml) were cultured in a 96-well plate, treated with different

concentrations of isolated placental exosomes for 24 h, harvested, washed
with PBS, stained with Annexin V/7-aminoactinomycin D according to the
manufacturers description, and analyzed by flow cytometry on FACScan
(BD Biosciences). Activated Fas-expressing PBMC were incubated with
exosomes for 24 h in RPMI 1640 medium, supplemented with 0.5% BSA
(Sigma Aldrich) and antibiotics and thereafter analyzed by electron microscopy (EM) to assess apoptotic signs in their morphology.

Statistical analysis
The results, appropriate for statistical analysis, were subjected to two
statistical testsStudent t test and two-way ANOVA performed with GraphPad Prism 5 program.

Results
FasL and TRAIL are transcribed and expressed in early and
term human normal placentas
Fig. 1A shows the relative mRNA expression of FasL and TRAIL
in STB of early and term pregnancy placentas assessed by realtime quantitative RT-PCR. STB from five individual samples of
early and term normal pregnancy placentas were analyzed. As can
be seen, mRNA for both FasL and TRAIL was found in all
samples confirming these molecules expression in early and term
placentas. FasL showed higher mRNA expression level in early
pregnancy placentas, whereas TRAIL mRNA dominated in expression in term placentas; however, these differences were not
statistically significant (Fig. 1A). Fig. 1B illustrates FasL and
TRAIL expression at the protein level, revealed by immunohistochemical staining and light microscopy of serial sections of
chorionic villi. Positive staining for both molecules was seen in
the villous trophoblast, preferentially in the STB of the chorionic
villi and on some Hofbauer cells (22) in the villous stroma. The
staining was strong and seemed to be intracellularly localized. In
light microscopy, the DAB-reaction product gave the impression
that both STB and cytotrophoblast (CTB) were stained; however,
the following ultrastructural analysis by IEM excluded staining of
CTB. No staining was observed in negative controls performed
with isotype-matched control Abs. These results were consistent
in all stained samples (n = 4). Similar results were obtained in
term placentas (n = 3; data not shown). From these experiments,
we conclude that FasL and TRAIL are constitutively transcribed
and intracellularly expressed as proteins in the STB of human
early and term placentas.
IEM revealed FasL and TRAIL expression on intraluminal
microvesicles in the MVB of STB, consistent with the
endosomal pathway of exosome biogenesis
We assessed the precise localization of FasL and TRAIL in placentas on the ultrastructural level by indirect immunoperoxidase
staining and EM. Four individual early placental samples were
examined, and no significant differences in the staining patterns
were observed. Representative photomicrographs, illustrating IEM
staining for FasL and TRAIL, are shown in Fig. 2. Both molecules
exhibited a similar intracytoplasmic staining pattern. The staining
of FasL (Fig. 2A, 2B) and TRAIL (Fig. 2C, 2D) in the placenta
was mainly restricted to the STB of the chorionic villi and in
scarce individual Hofbauer cells (22) in the villous stroma (data
not shown). No staining was observed in the CTB cells. Furthermore, the apical microvillous (Fig. 2A, arrowheads) and the basal
membranal surfaces of STB were generally devoid of staining
indicating that there was no expression of these molecules on the
apical and basal syncytiotrophoblastic membrane. Instead, the
limiting membrane of cytoplasmic vacuoles and numerous microvesicles of 406080 nm in diameter tightly packed inside the
vacuoles were positively stained for FasL (Fig. 2A, 2B) and

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Isolated placental exosomes, snap-frozen pieces of chorion villi, and cultured

Jurkat cells were solubilized with radioimmunoprecipitation assay buffer
(Pierce). After separation on 12% SDS-PAGE, proteins were transferred to
polyvinylidene difluoride membranes (GE Healthcare). Membranes were
blocked for 1 h with 5% fat-free powdered milk in PBS with 0.05% Tween
20 (PBST) before incubation overnight with the appropriate Abs. HRPconjugated secondary Abs were applied for 1 h. PBST was used in all
washing steps. Signals were detected with Amersham ECL plus Western blot
detection system with Amersham ECL developing films (GE Healthcare).

5517

5518

PLACENTAL EXOSOMES EXPRESS APOPTOSIS-INDUCING FasL AND TRAIL

TRAIL2 (Fig. 2C, 2D). The stained cytoplasmic vacuoles displayed the classical morphology of MVBs (Fig. 2A, 2B, 2C, 2D).
In all identified MVBs, the number of intraluminal FasL- or
TRAIL-stained microvesicles was by far higher than 9, a number
defined as the lower limit of vesicle content distinguishing between early and late endosomes (30). The MVBs were located at
different levels in the syncytioplasm and were between 600800
nm and up to 1.52 mm in size. Several of them were fused with
the apical microvillous membrane of the STB, as illustrated in Fig.
2D, where a MVB, positively stained for TRAIL on its limiting
membrane, is opening to the apical intervillous space and releasing its microvesicle content. The stained limiting membrane of
the MVBs together with the stained intraluminal vesicles suggest
vesicle generation by inward budding of the MVBs limiting
membrane consistent with biogenesis of exosomes. In summary,
our IEM analyses demonstrated that both FasL and TRAIL are
typically localized on intraluminal vesicles with the size and
morphology of exosomes enclosed in the MVBs of the late

endosomal compartment. Moreover, there were frequent morphological signs of microvesicle secretion/release in the intervillous apical space of the STB.
Secreted placental exosomes express exclusively bioactive
aggregated form of FasL and TRAIL
To further confirm the IEM results of generation and secretion of
FasL- and TRAIL-bearing exosomes, 24-h cultures of placental
explants were established in the presence of metalloprotease inhibitors. The short time of culture was chosen to ensure the explants
good condition throughout the culture avoiding microvesicle release
because of necrosis. Thus, the vast majority of nanovesicles in
the culture supernatant emanate from exosome secretion. The exosomes, isolated and enriched by sucrose gradient ultracentrifugation
from the culture supernatant, were subjected to analysis for FasL
and TRAIL with three different methodsWestern blot analysis,
immunoflow cytometry, and IEM. The results from representative
experiments are summarized in Fig. 3.

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FIGURE 1. FasL and TRAIL are constitutively

expressed by human normal placenta throughout
pregnancy. (A) Quantitative real-time RT-PCR
mRNA expression of FasL and TRAIL in STB,
isolated or laser microdissected from early (n = 5)
and term (n = 5) placenta. The relative mRNA
expression was determined by comparison of
mRNA expression in term pregnancy placenta to
mRNA expression in early pregnancy placenta
(relative expression = 1). PMA/ionomycin-stimulated PBMC served as positive control. All
samples were normalized to 18S rRNA as described in Materials and Methods. (B) Immunohistochemical staining of early normal placenta
showing the presence of FasL and TRAIL in the
villous trophoblast and in separate Hofbauer cells
in the stroma of the chorionic villi. Serial sections
stained with isotype-matched irrelevant mouse
anti-human mAb and normal goat serum served as
negative controls. Note the dotted staining of the
trophoblast that seems to be intracellularly localized. Arrows point to the chorionic villi covered
with stained villous trophoblast; arrowheads
point to Hofbauer cells and stars to the stromal
tissue composed of loosely connected stromal
cells. Original magnification 3400. V, Placental
vessel located in the stroma of a chorionic villous.

The Journal of Immunology

5519

Isolated placental exosomes, placental tissue, and a positive

control of lysed Jurkat T cells were used in the Western blot
analyses of FasL and TRAIL molecules (Fig. 3A). The tetraspanin
CD63 was analyzed as an exosomal control. As can be seen, FasL
was expressed in all exosomal fractions exclusively as a single
protein band of 75 kDa corresponding to a membranal hexameric form of FasL, composed of two trimeric molecules (11),
whereas in placental tissue, both a 37-kDa and a hexameric 75kDa FasL were revealed (Fig. 3A). In contrast, lysed Jurkat cells
showed a dominating band of 37 kDa. Similarly, TRAIL was
enriched in the exosomal fractions and placentas as a 70-kDa
band, whereas the major amount of TRAIL in Jurkat was expressed as a 34-kDa band and only a smaller amount as a 70-kDa
protein band. To our knowledge, this is the first demonstration of
FasL and TRAIL in human placental exosomes, showing that both
FasL and TRAIL, the major apoptosis-inducing molecules of the
TNF superfamily, are not only secreted by exosomes but exclusively enriched as an oligomerized membranal bioactive form.
To further prove that the localization of FasL and TRAIL is
indeed on the exosomal membrane, experiments were performed
by immunofluorescence staining and immunoflow cytometry with
exosomes loaded on latex beads (Fig. 3B) and by immunogold
staining of exosomes and IEM (Fig. 3C). Representative histograms of the flow cytometric analysis, shown in Fig. 3B, revealed
membranal FasL and TRAIL expression. In Fig. 3C, using negative contrast staining, we illustrate the typical exosomal size and
cup-shaped morphology of the isolated microvesicles, thus proving that they are exosomes. Besides a morphological characteristic, the positive immunogold staining of CD63 and placental
alkaline phosphates further depict them as exosomes of human
placental origin. IEM staining of FasL and TRAIL was revealed
on the surface of the exosomes, confirming that these molecules

FIGURE 3. FasL and TRAIL are enriched on the membrane of placental

exosomes. (A) Western blot analyses of FasL and TRAIL protein expression by exosomes from two donors, isolated from supernatants of early
placenta explant cultures, compared with expression in early placenta
tissue and Jurkat cells. CD63 was used to confirm the exosomal origin of
the isolated microvesicles. Protein load, 40 mg/well. (B) Immunoflow
cytometry of purified placental exosomes captured on latex microbeads
coated with mAbs against FasL and TRAIL and revealed by fluorescence
staining with mAbs against the exosomal marker CD63. Superimposed
shaded histogram represents negative controls with isotype-matched
mAbs. (C) IEM of whole-mount placental exosomes. The typical cupshaped morphology and size of 30100 nm is shown by negative contrast
staining seen to the left. Specific mAbs and immunogold labeling with
5- or 10-nm gold particles were used to reveal surface protein expression.
Anti-CD63 staining confirms the exosomal nature of the isolated microvesicles, and their placental origin is revealed by staining for placental
alkaline phosphatase (PLAP). FasL staining was performed with 5-nm gold
particles and TRAIL with 10-nm particles. Note that in the double staining,
FasL and TRAIL are present on separate exosomes. Scale bars, 100 nm.

were expressed as membranal proteins. We made a double IEM

staining with FasL and TRAIL using 5- and 10-nm gold particles,
conjugated to secondary Abs to distinguish between FasL and
TRAIL localization. Interestingly, we found FasL (stained by
5-nm gold particles) and TRAIL (stained by 10-nm gold particles)
on different exosomes, indicating that at least to a certain extend
they might be generated in separate MVBs.
In summary, with three independent methodsWestern blot,
immunoflow cytometry with latex beads, and IEMwe demonstrate that the placenta secretes exosomes that carry FasL and
TRAIL, enriched on the exosomal membrane as single protein
bands with sizes consistent with their bioactive membranal form.

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FIGURE 2. Immunoelectron microscopic localization of FasL and

TRAIL in STB of human early placentas. (A) Low-power micrograph of
STB stained for FasL. The electron-dense reaction product labels MVB in
the syncytioplasm (arrow). Note that the apical surface membrane is not
stained (arrowheads). (B) High-power micrograph of FasL-positive MVB
containing tightly packed and intensively stained intraluminal microvesicles (arrowheads). (C) Micrograph of TRAIL-positive MVB (arrow) in
perinuclear location showing stained limiting membrane and internal
microvesicles. (D) High-power micrograph of an apically located MVB,
which exhibits staining of the limiting membrane and internal microvesicles (arrowheads) and forms an opening to the intervillous space (star).
Note that apical plasma membrane is TRAIL negative (arrow). Scale bars,
1 mm (A); 400 nm (B); and 200 nm (C, D). N, Nucleus.

5520

PLACENTAL EXOSOMES EXPRESS APOPTOSIS-INDUCING FasL AND TRAIL

FasL- and TRAIL-expressing placental exosomes induce

apoptosis in vitro in a dose-dependent manner

Discussion
Three main points summarize the results presented in this paper: 1)
In the STB of human placenta, the apoptosis-inducing molecules
FasL and TRAIL are constitutively transcribed and expressed as
proteins on the MVBs membrane and on the membrane of
intraluminal vesicles that are released in the extracellular space as
exosomes. The protein expression of FasL and TRAIL is, thus,

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We studied the proapoptotic ability of exosomes from separate

placental samples in vitro in two model systemsJurkat leukemia
T cells and activated PBMC from healthy donors. The apoptosis
was revealed by Annexin V:PE Apoptosis Detection Kit I (BD
Bioscience) and by EM looking for early signs of apoptosis in
activated PBMCs incubated with placental exosomes. Fig. 4A and
4B summarizes the results of Jurkat cell experiments with exosomes isolated from culture supernatants of individual placental
explants and Fig. 4CG the results from experiments with activated PBMC. As can be seen, the placental exosomes induced
statistically significant apoptosis in Jurkat cells in a dose-dependent
manner (n = 7; Fig. 4A). In contrast, the explant culture supernatants depleted of microvesicle after gradient ultracentrifugation
were unable to trigger apoptosis of the target cells indicating that
the apoptotic effect was exosome specific (data not shown). The
in vitro proapoptotic effect on the Jurkat cells could differ between
individual exosome samples (Fig. 4B). Although most exosomal
samples had a good proapoptotic capacity, some had lower intensity, and two samples had very low effect. The reason for the
variation in the apoptotic capacity is at present not known but may
be explained by several factorsfor example, less concentration
of apoptotic molecules on the surface of some isolated exosomes
because of insufficient protective effect of the protease inhibitor
mixture during isolation and storage; variations because of differences in the STB or in the gestational stage of the placental
samples; variations because of the condition of the samples and
of the explant cultures performance; and/or biological differences
between the donors. Exosomal samples, unprotected by matrix
metalloproteinase (MMP) inhibitors lost their FasL and TRAIL
expression and their apoptotic ability as shown in Supplemental
Fig. 1. The presence of metalloprotease inhibitors in the culture
medium during storage and experimental procedures with exosomes is essential for the preservation of their proapoptotic effect.
Despite a biological variation in the apoptotic potency in individual
samples, the mean value 6 SD of induced apoptosis was statistically significant and dose dependent (Fig. 4A).
In addition, we studied the in vitro apoptotic effect of exosomes
on PBMC from healthy donors (n = 5) activated by PMA/
ionomycin. The activation was assessed by measurement of Fas
and TRAIL receptors by immunoflow cytometry as illustrated in
a typical experiment in Fig. 4CE where Fas and TRAIL receptor expression is shown on different subpopulations of activated
PBMC from one donor. Apoptosis was assessed by Annexin V:PE
Apoptosis Detection Kit (Fig. 4F) and EM (Fig. 4G). As can be
seen in a representative PBMC experiment (Fig. 4F), the number
of Annexin Vexpressing apoptotic cells is doubled in the presence of 40 mg exosomes. In the EM analyses, we saw cells with
intact cellular membrane but with clear early apoptotic signs like
fragmentation of the nucleus, vacuolization of the cytoplasm, and
membrane blebbing as illustrated in Fig. 4G. Taken together, our
results indicate that human placenta secretes exosomes with
membranal expression of preformed functional FasL and TRAIL
molecules that are able to rapidly trigger apoptosis in vitro in
Jurkat T cells and in activated PBMC from healthy donors.

strictly confined to the endosomal compartment of STB, whereas

the apical and basal syncytiotrophoblastic membranes are completely devoid of FasL and TRAIL expression. 2) The STB-derived
exosomes enrich FasL and TRAIL on their membrane as bioactive
oligomerized molecules able to form DISCs. 3) FasL- and TRAILcarrying placental exosomes trigger apoptosis in Jurkat T cells and
activated PBMC from healthy donors in a dose-dependent manner.
Taken together, these results indicate that human early and term
placenta constitutively secrete functional FasL and TRAIL on exosomes that are able to convey apoptosis and thus might contribute
to protection of the fetoplacental unit from activated maternal immune cells.
The intracellular localization of FasL and TRAIL in human
placenta is intimately bound to the biogenesis of exosomes. To our
knowledge, this is the first demonstration of TRAIL protein expression on the ultrastructural level and the first report that TRAIL
is solely exosomally expressed in STB of human placenta. Furthermore, a constitutive release of FasL- and TRAIL-expressing
exosomes from the apical microvillous surface of STB was demonstrated. Our results are in tune with previous investigations by
others and us (22, 31, 32) showing that FasL is targeted to the MVB
of secretory lysosomes and is expressed on exosome-like microvesicles (5, 15, 33, 34). Two independent pathways that control
the internalization of FasL on microvesicles in MVBs have been
identifiedphosphorylation of FasL by binding of its prolinerich domain to Fgr, Fyn, and Lyn tyrosine kinases and a direct
ubiquitinylation at the lysine residues flanking the proline-rich
domain (3537). The extracellular domain of FasL, TRAIL, and
other members of the TNF family contain cleavage sites for
MMPs that generate soluble proteins by proteolytic shedding and
substantially reduce the proapoptotic activity of the shed proteins.
The mFasL is the proapoptotic molecule, whereas sFasL is 1000
times less active in inducing apoptosis (38, 39). Translocation of
FasL to the plasma membrane as a way to provide mFasL will
expose the molecule to proteolytic cleavage reducing apoptotic
activity. Moreover, FasL expressed on the cellular membrane,
instead of dampening the immune responses, induces inflammation and promotes allograft rejection (19, 3942) as compared
with sFasL that was proposed to be anti-inflammatory (40, 41, 43).
Thus, different molecular forms of FasL do mediate different
functions. The exact mechanisms are not completely understood,
but compartmental expression, levels of membranal and/or shed
form, and the surrounding microenvironment where it is expressed
seem to act together to influence its biological functions (10).
Similarly to FasL there are differences in TRAIL activity depending on whether it is in a membrane-bound form or cleaved to
generate a soluble form. Initially, it was found that both forms
were equally effective (44), whereas later reports singled out
membrane-bound TRAIL as the molecular form preferably inducing cell death (4547). Recently, it was reported that TRAIL
protein in STB cultures could be at least partly localized to the
cytoplasm (18). Previously proposed function as a protein repairing syncytial damage and maintaining the placental barrier
integrity could not be confirmed. Instead, a similar function for
TRAIL as for FasL as a contributor to immune privilege was suggested, and an intracellular compartmental restriction of TRAIL
expression in the placenta similar to that of FasL was assumed but
not documented (18). In line with this assumption, we show that
TRAIL is restricted to STBs endosomal compartment and expressed and released on exosome-like microvesicles. The previously described cell membrane expression on CTB and STB (18)
could not be confirmed despite detailed IEM analyses. Similar
results of exosomal TRAIL and FasL expression have been shown
in other cells and sites such as dendritic and T cells (5, 48, 49),

The Journal of Immunology

5521

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FIGURE 4. Human placenta-derived exosomes carry functional FasL and TRAIL on their membrane. (A and B) FasL- and TRAIL-bearing placental
exosomes trigger apoptosis of Jurkat T cells in a dose-dependent manner. (A) Staple diagram showing average percentage 6 1 SD of dose-dependent apoptosis
in Jurkat cells induced by different concentrations of exosomes isolated from supernatant of placental explant cultures (n = 7). (B) Induction of apoptosis by
individual exosomal preparations derived from explant cultures from seven consecutive samples of human early placenta from normal pregnancies showing
biological variation in apoptotic activity. (CG) FasL- and TRAIL-expressing exosomes trigger apoptosis in activated PBMC from healthy donors. (CE)
Immunoflow cytometry showing Fas- and TRAIL-R1 and -R2expressing cells in subpopulations of PBMC from healthy donors, activated by PMA/ionomycin. (F) Immunoflow cytometry analysis of apoptotic cells in activated PBMC before and after addition of 20 mg placental exosomes. Note that the
percentage of the apoptotic PBMC is doubled in the presence of FasL- and TRAIL-bearing placental exosomes. (G) Electron micrograph of a representative
experiment out of three, showing classical apoptotic signs of nuclear fragmentation, vacuolization of the cytoplasm and plasma membrane blebbing of activated
PBMC incubated with FasL- and TRAIL-bearing placental exosomes. Original magnification 35000. ***p = 0.0001; **p = 0.0016; *p = 0.0498.

melanoma, and ovarian cancer cells (50, 51) and in joint fluid of
rheumatoid arthritis patients (52). Interestingly, our double immunogold staining of FasL and TRAIL revealed these molecules
on separate exosomes, suggesting that in human placenta these

molecules might be enriched and processed through separate

MVBs. This is consistent with a previous finding of only partial
colocalization of endosomal FasL and TRAIL expression in human melanoma (46).

5522

PLACENTAL EXOSOMES EXPRESS APOPTOSIS-INDUCING FasL AND TRAIL

explanation could be that we have studied different microvesicles
because they were isolated from different sources and by different
isolation procedures.
The physiological importance of apoptosis in normal pregnancy
is associated with the fetal immune privilege in the pregnant uterus
a phenomenon similarly regulated as other immune privileged sites,
the eye being the one more thoroughly studied (9, 58, 59). We can
speculate that the release of proapoptotic placental exosomes is in
highest concentration near the placenta and builds an exosomal
gradient in the maternal blood nearest the placenta, which functions as a protective shield of the fetoplacental unit. The results
presented in this study suggest that the placenta secretes exosomes
expressing preformed bioactive FasL and TRAIL molecules able
to convey apoptosis and might partly contribute to the immune
privilege of the fetus. Thus, the immunomodulatory and protective
role of the placenta seems at least partly associated to its exosomesecreting ability.
Several published reports (reviewed in Ref. 25), including this one,
concern normal placenta and depict exosomal secretion as beneficial
to pregnancy. A logical question is how the exosomal secretion is
affected in pathological pregnancies. So far, there are few conclusive
exosome studies regarding such conditions. In preterm pregnancy,
exosomal concentration in the maternal peripheral blood was shown
to be lower compared with that in women with normal term delivery
and nonpregnant women (60). Previous studies (61) of preeclampsia
highlighted another type of extracellular microvesicles, the much
larger shedding microvesicles generated from the apical microvillous
syncytial membrane, and showed that they are highly elevated in the
circulation of preeclamptic women and seemed to be involved in the
enhanced inflammation, cytokine production, and endothelial destruction and dysfunction that are cardinal characteristic for this
enigmatic disease. One can speculate that there may be a balance
in the STB between generating shedding vesicles and generating
exosomes, and a disturbance of this balance by overproducing or
underproducing either type of extracellular STB vesicles might
affect the fine-tuning of the placental function. Many more welldesigned future studies of syncytiotrophoblast microparticles and
exosomes in pathological pregnancy conditions are needed to
elucidate this question.

Acknowledgments
We thank the donors and the colleagues at the Department of Obstetrics
and Gynecology and the staff of the operation theater Centralop II at
Umea University Hospital, Umea, Vasterbotten, Sweden, for providing
placental samples.

Disclosures
The authors have no financial conflicts of interest.

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Soluble and membranal FasL and TRAIL molecules have

separate biologic activities; thus, exosomally expressed FasL and
TRAIL may be viewed as a soluble form that preserves membranal expression and a proapoptotic biologic activity. Using the
highly specific anti-FasL mAb G247-4 (53), we revealed a single
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would have a m.w. between 75 and 80 kDa depending on glycosylation level. We suggest that the single 75 kDa FasL and the
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the exosomal membrane, the proapoptotic ability was lost as well
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superfamily ligands, able to form DISC (11). Limiting the expression of oligomerized FasL and TRAIL to exosomes in MVB
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emanates from cross-linking or trimerization of the Triton treatmentproduced sFasL. It has been reported that, if the cleavage of
mFasL occurs by MMP7 at a particular protease cleavage site, the
generated sFasL may form functional trimers (11, 56, 57). Another

The Journal of Immunology

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