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Departments of
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Anatomy and Cell Biology,
Pathology and Laboratory Medicine, and
Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160;
§
Human Genome Sciences, Rockville, MD 20850; and
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Genentech, South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. By contrast, cell lineage-specific
differences were observed in expression of the TRAIL-R genes. Although
all four lines contained mRNA encoding the apoptosis-inducing DR5
receptor, only trophoblast cells contained mRNA encoding the DcR1 decoy
receptor and only macrophages contained DcR2 decoy receptor
transcripts. DR4 mRNA was present only in THP-1 cells and was the only
TRAIL-R transcript increased by IFN-. Cytotoxicity assays revealed
that the two trophoblast cell lines were resistant, whereas the two
macrophage lines were partially susceptible to killing by rTRAIL.
Collectively, the results are consistent with a role for the
TRAIL/TRAIL-R system in the establishment of placental immune
privilege. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo-2L) is a newly identified TNF superfamily member with high homology to FasL (9, 10, 11, 12). TRAIL and FasL cooperate in limiting lymphocyte proliferation following activation and use some common pathways leading to apoptotic cell death (11, 12, 13, 14, 15, 16, 17, 18). Unlike FasL and other apoptosis-inducing TNF family members, TRAIL transcripts are detectable in many normal organs and tissues, including human placentas (9, 10). In the TRAIL/TRAIL-R system, control over apoptosis relies on differential display of receptors (TRAIL-R). These include DR4 (TRAIL-R1) and DR5 (TRICK2/TRAIL-R2), which transduce apoptotic signals, and DcR1 (TRID/LIT/TRAIL-R3) and DcR2 (TRUNDD/TRAIL-R4), which lack functional death domains and act as decoys (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). TRAIL-R are widely expressed, and Northern blots have detected messages encoding all four receptors in human placentas (19, 20, 21, 22, 23, 24, 30, 32).
Because TRAIL expressed in trophoblast cells might contribute to immune privilege by killing activated lymphocytes and TRAIL-R expression would determine the vulnerability of trophoblast to killing by TRAIL, we undertook evaluation of this system in human placentas and cell lines. Specific transcripts were identified in human placentas, and TRAIL protein was immunolocalized to a restricted number of cell types, which included trophoblast and macrophages. We then documented TRAIL and TRAIL-R mRNAs in trophoblast and macrophage cell lines. Having learned that receptor expression differed in the two lineages, we compared their respective abilities to resist killing by TRAIL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sections of human first trimester (n = 3) and term placentas (n = 2) as well as extraplacental membranes (n = 2) were obtained from elective pregnancy terminations and normal cesarean section deliveries, respectively, in accordance with a protocol approved by the Human Subjects Committee of the University of Kansas Medical Center (Kansas City, KS). The tissues were manually dissected and fixed in 4% paraformaldehyde-PBS overnight at 4°C, then blocked into paraffin at low temperature. The human trophoblast-derived choriocarcinoma cell lines, Jar and JEG-3, and the U937 myelomonocytic cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The Wilkinson Laboratory for Cancer Research at the University of Kansas Medical Center kindly provided THP-1 monocyte/macrophage cells. The cell lines were cultured at 37°C in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA) and antibiotics (Sigma-Aldrich) (growth medium).
Immunoblot analysis
Human term placental protein (Human Placenta Protein Medley; Clontech Laboratories, Palo Alto, CA) was fractionated by standard 10% SDS-PAGE (33) using a MiniPROTEAN II Dual Slab Cell (Bio-Rad Laboratories, Hercules, CA). The gels were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) using the Bio-Rad MiniTrans-Blot Cell. TRAIL was identified according to the manufacturer’s instructions using goat anti-human TRAIL (K-18) Ab (1 µg/ml), which recognizes amino acids 233–250 in the C-terminal region of TRAIL, and donkey anti-goat IgG-HRP secondary Ab, both from Santa Cruz Biotechnology (Santa Cruz, CA). Goat IgG from Sigma-Aldrich served as a control for nonspecific binding. The enhanced chemiluminescence substrate was SuperSignal (Pierce, Rockford, IL); detection was by exposure to Hyperfilm MP (Amersham Life Science, Arlington Heights, IL).
Immunohistochemistry
The paraformaldehyde-fixed tissues were embedded in paraffin at low temperature, and two 5-micron sections were taken onto glass slides for analysis by immunohistochemistry, as previously described (7), with the following modifications. Following blocking with normal horse serum, the goat IgG anti-TRAIL primary Ab K-18 or control goat IgG (both at 15 µg/ml) was incubated with the tissues overnight at 4°C. Biotinylated horse anti-goat IgG (15 µg/ml; Vector Laboratories, Burlingame, CA) was incubated with the samples for 30 min at room temperature, and endogenous peroxidase was blocked after this step. For peptide inhibitions, primary Ab (10 µg/ml) was incubated with a 19-fold weight excess of peptide K-18 (Santa Cruz Biotechnology) for 1 h at room temperature. The mixtures were centrifuged and tissue sections were incubated with the supernatant as a replacement for the primary Ab. Following incubation with substrate, the tissues were lightly counterstained with hematoxylin, dried, and coverslipped for light microscopy.
Probes
cDNAs encoding TRAIL, DR4, DR5, DcR1, and DcR2 were excised from
their respective plasmid vectors using appropriate restriction enzymes.
The linear fragments were resolved on 1% agarose gels and visualized
by UV transillumination, and appropriately sized bands were eluted from
the gel by centrifugation on GenElute Agarose Spin Columns
(Sigma-Aldrich). Eluted linear ds cDNA was ethanol precipitated and
dissolved in 10 mM Tris-HCl, pH 8, 1 mM EDTA. Probes from interior
regions of the cDNAs for TRAIL (
550 bp encoding the extracellular
soluble region (19)), DR4 (bp 739–952, 214 bp), DcR1 (bp 217–566, 350
bp), and DcR2 (bp 573–787, 215 bp) were generated by PCR and agarose
gel purified, as described above.
RNA isolation and Northern blot analysis
For Northern blot analysis, total RNA was prepared from
untreated cultured cell lines (1–2 x 108 cells per
preparation) using TRIzol (Life Technologies, Gaithersburg, MD) or TRI
reagent (Sigma-Aldrich), according to the manufacturer’s
protocol. Total RNA from human term placenta was purchased
from Ambion (Austin, TX). RNA samples (8–10 µg/lane) were resolved
on 1% agarose/2 M formaldehyde gels. The gels were examined by UV
transillumination and immediately blotted to Nytran using a
TurboBlotter, as directed (Schleicher & Schuell). UV cross-linking was
performed with a UV Stratalinker 1800 (Stratagene Cloning Systems, La
Jolla, CA). Dry blots were either used immediately or sealed in
heat-seal bags and stored at 4°C. Twenty-five nanograms of each cDNA
or PCR-generated probe were random-prime labeled with
[
-32P]dCTP (3000 Ci/mmol; ICN Pharmaceuticals, Costa
Mesa, CA) using the Random Primers DNA Labeling System (Life
Technologies), according to the manufacturer’s instructions.
Unincorporated nucleotides were removed from labeled cDNA probes by
centrifuging through Micro Bio-Spin 6, SSC Chromatography Columns
(Bio-Rad Laboratories). Specific activity of labeled cDNA probes was
determined by liquid scintillation, and 5 x 106
cpm/ml (4 ml total hybridization solution) was used for each
hybridization. Hybridization was performed in a Hybaid oven using
QuikHyb (Stratagene) hybridization solution essentially as recommended
by the manufacturer: prehybridization for at least 20 min at 68°C,
hybridization with labeled probe for 1 h at 68°C, two 15-min low
stringency washes (2x SSC, 0.1% SDS) at room temperature, one 30-min
high stringency wash (0.1x SSC, 0.1% SDS) at 60°C, and a second
15-min high stringency wash at the same conditions. The blots were
briefly rinsed in 2x SSC at room temperature before being sealed in
heat-seal bags (KAPAK, Minneapolis, MN) and exposed to Hyperfilm MP
(Amersham Life Science) in the presence of intensifying screens.
IFN- treatment of cell lines
Four 100-mm tissue culture dishes for each cell line were seeded
and incubated at 37°C, 5% CO2 in humid air: the
trophoblastic cell lines Jar and JEG-3 at 2 x 106
cells in 10 ml of growth medium 1 day before treatment and the
monocytic cell lines U937 and THP-1 at 3 x 106 cells
in 10 ml of growth medium 2–4 h before treatment. A total of 100 µl
of 1 x 104 U/ml of human rIFN- (rhuIFN-;
Genzyme, Cambridge, MA) was added to two of the dishes for each line,
and 100 µl of medium was added to the remaining two dishes
(controls). Incubation was resumed for 24 h, at which time one
medium control and one rhuIFN--treated culture dish for each cell
line were removed for total RNA preparation using TRIzol, as described
above. Total RNA was prepared from the remaining cultures after an
additional 24 h (48 h total incubation time). Northern blot
analysis was performed as described above. Gels were transilluminated
and photographed with Polaroid 665 black and white film to obtain
ethidium bromide-stained 28S and 18S bands (loading controls). Northern
blot films and the Polaroid negatives were quantitatively analyzed
using a Molecular Dynamics (Sunnyvale, CA) Personal
Densitometer and ImageQuaNT software.
Cytotoxicity assays
JEG-3, Jar, U937, THP-1, and HeLa human endometrial adenocarcinoma cells from ATCC were plated into 96-well microplates (0.1 ml/well, replicates of three wells) in medium containing 10% FBS. U937 and THP-1 cells were plated at 5 x 104 cells/well, and all other cells were plated at 1 x 104 cells/well. After overnight culture at 37°C, rTRAIL (20) was added to a final concentration of 0, 10, or 1000 ng/ml, and cultures were continued for 20 h. Mitochondrial enzyme activity was evaluated using an MTT kit from Promega (Madison, WI), following the manufacturer’s directions. Color intensity was determined spectrophotometrically at A570. Duplicate plates were established, and cytotoxicity was assessed by exclusion of the vital dye, trypan blue.
Nuclear fragmentation assay
To determine whether the rTRAIL used in cytotoxicity assays killed by apoptosis, HeLa cells were seeded into eight-chamber Lab-Tek Tissue Culture Chamber Slides (Nunc International, Naperville, IL) at 3 x 105 cells/well in 0.3 ml of culture medium and cultured overnight at 37°C. On the following day, 30 µl of culture medium (controls) or culture medium containing 1000 ng/ml of rTRAIL was added to each well for a final concentration of 100 ng/ml. The cells were incubated for 2 h at 37°C, then the slides were rinsed briefly in PBS, air dried, and fixed for 30 min in 1% paraformaldehyde at 4°C. The slides were washed in PBS and incubated in the dark at room temperature for 15 min with 0.4 µg/ml DAPI (4', 6-diamidino-2-phenylindole; Sigma) in PBS. The slides were rinsed briefly in PBS and mounted using Slow Fade Light Antifade glycerol mounting medium (Molecular Probes, Eugene, OR) and examined for nuclear fluorescence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have reported that human placentas contain TRAIL
mRNA (9, 10). To determine whether the messages were translated and to
identify the cells containing TRAIL protein, immunohistochemical
experiments were performed. Specificity of a commercially available Ab
to a human TRAIL-specific amino acid sequence was first verified by
immunoblotting. As shown in Fig. 1
,
left panel, the goat anti-human TRAIL Ab detected a
polypeptide of Mr 33,000–34,000 (major
band) as well as less prominent bands at Mr =
42,000, Mr = 32,000, and
Mr = 31,000. These results were generally
consistent with previous reports on TRAIL protein (17). This same Ab
was then used for immunohistochemical experiments.
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, right panel, shows that TRAIL in first trimester
placentas was prominent in syncytiotrophoblast, where it was localized
primarily to the apical brush border. Immunoreactive TRAIL was detected
in villous stroma and stromal cells, particularly the round, highly
vacuolated cells known as Hofbauer cells (placental macrophages), but
was low to absent in fibroblastic mesenchymal cells and endothelial
cells (Fig. 1A). Normal goat IgG did not bind to any cells
in first trimester placentas (Fig. 1B), and these controls
were also negative with all other tissues. Fig. 1C shows
that large maternal decidual cells in first trimester tissues were
TRAIL protein positive. Leukocytic aggregates identified as "L" in
Fig. 1C and endothelial cells were TRAIL negative. Staining
was essentially abolished by preincubating the anti-TRAIL reagent
with specific peptide (Fig. 1D).
In term placentas, immunoreactivity with anti-TRAIL was less
intense. Positive signals were detected in both syncytiotrophoblast and
macrophage-like mesenchymal cells (data not shown). In term
extraplacental membranes, TRAIL protein was clearly evident in the
amnion membrane (Fig. 1E) as well as in a few
macrophage-like stromal cells located between the amnion and chorion
membranes and maternal decidual cells. Chorionic cytotrophoblasts
contained little or no TRAIL. Staining in the extraplacental membranes
was completely abrogated by preincubating the primary Ab with specific
peptide (Fig. 1F).
These results confirmed translation of TRAIL messages in placentas and indicated that TRAIL is differentially expressed at the maternal-fetal interface, with synthesis probable in syncytiotrophoblast cells, placental macrophages, amnion epithelial cells, and maternal decidual cells.
Detection of TRAIL mRNA in human placentas and cell lines
In accordance with previous reports (9, 10), Northern blots of
term placental RNA demonstrated TRAIL transcripts migrating to a
position on the gels corresponding to 2 kb (Fig. 2). We then used the same experimental
method to test cell lines representing two of the major TRAIL
protein-positive cell types identified by immunohistochemistry in human
placentas, trophoblasts (Jar, JEG-3 cells), and monocytes/macrophages
(U937, THP-1 cells).
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shows that TRAIL transcripts were present in all of the cell
lines. Transcripts were prominent in Jar and JEG-3 trophoblastic cells,
but steady state levels of TRAIL mRNA were reduced in
monocytes/macrophages, with lower expression in U937 cells than in
THP-1 cells. This and other TRAIL Northern blots used a PCR-generated
(approximately 550-bp) DNA probe that identified sequences encoding the
external soluble portion of the TRAIL protein. In all Northern
analyses, equal loading of lanes and quality of RNA were verified by
examining 18S and 28S rRNA content by ethidium bromide staining (Fig. 2, lower panel). These experiments supplied experimental support for the idea that both trophoblasts and placental macrophages transcribe the TRAIL gene.
rhuIFN- enhances TRAIL mRNA in trophoblast and macrophage cell
lines
Activation with Con A (T cells) and LPS (B cells) increases TRAIL
expression in T and B lymphocytes, respectively (17). Macrophages are
activated by IFN-, and trophoblasts have some macrophage-like
characteristics (34). Therefore, we tested rhuIFN- for effects on
TRAIL mRNA in the cell lines. Jar, JEG-3, U937, and THP-1 cells were
exposed to 100 U/ml of rhuIFN-. RNA was harvested at 24 and 48
h, and Northern blot hybridization was used to analyze TRAIL mRNA.
Fig. 3 shows that IFN- efficiently
enhanced TRAIL mRNA in all four cell lines, although the kinetics of
enhancement varied. Equal loading was verified by examining 28S and 18S
bands on ethidium bromide-stained agarose gels. Table I shows the means and SDs of three
independent experiments, in which the results were analyzed
quantitatively using scanning densitometry. Mean increases in steady
state levels of TRAIL mRNA compared with 28S RNA varied from 2–11-fold
at 24 h, and 2–3-fold at 48 h. Exposure for 24 h was
maximal for Jar, JEG-3, and THP-1 cells, and levels declined at 48
h, whereas 3-fold increases were observed at both 24 and 48 h in
U937 cells. No cell lineage-specific patterns were observed in these
experiments.
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on steady state levels
Potential target cells for placental TRAIL were then investigated
by using Northern blot hybridization to detect receptor mRNA.
Initially, full-length cDNAs encoding each of the four receptors were
used to generate probes. Because the DNA sequences for the four
receptors contain sizable regions of identity and similarity, there was
some cross-detection, particularly of DR5 by the full-length probes for
DR4, DcR1, and DcR2. (The full-length cDNA probe for DR5 did not detect
transcripts for the other three receptors, however.) To reduce this
cross-detection, PCR was used to generate smaller probes for DR4, DcR1,
and DcR2 from regions of less DNA sequence similarity. Identical
specific transcript bands were detected by full-length cDNA and the
PCR-generated probes. Cross-detection of DR5 transcript by probes for
the other three receptors was greatly reduced or eliminated when the
PCR-generated probes were used, as shown in Fig. 4. Approximate sizes of the transcripts
detected were 5 and 3 kb for DR4; 4.5 kb for DR5; 5, 3, and 1.5 kb for
DcR1; and 4 kb for DcR2. All are consistent with published reports
(19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32).
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A illustrates mRNAs for the apoptosis-inducing DR4 and
DR5 receptors, and Fig. 4B illustrates mRNAs for the decoy
receptors, DcR1 and DcR2. Placentas contained specific mRNA encoding
each of the four TRAIL-R, although signals were generally weak in
comparison with cell lines. This may have been due to the mixed cell
types present in tissue and to the comparatively lower quality of the
placental RNA available from commercial sources. In the cell lines,
transcripts encoding the apoptosis-inducing DR4 receptor were detected
only in the THP-1 cells, whereas specific mRNA encoding the second
apoptosis-inducing receptor, DR5, were present in all four of the cell
lines. The THP-1 cells, which contained mRNA encoding the DR4 receptor,
had the least mRNA for the DR5 receptor, and the reverse was true for
JEG-3, Jar, and U937 cells.
As illustrated in Fig. 4B, only the two cell lines derived
from trophoblastic cells, JEG-3 and Jar, contained DcR1 mRNA; specific
transcripts were undetectable in the two tumor-derived
monocyte/macrophage cell lines, U937 and THP-1. In striking contrast,
DcR2 decoy receptor transcripts were present only in macrophage cell
lines and not in the trophoblastic cell lines. Matching ethidium
bromide-stained agarose gels are shown for each receptor Northern blot
to verify equal loading of the lanes.
Table I shows the mean fold change in receptor mRNA induced by treating
the cell lines with 100 U/ml of rhuIFN- for 24 or 48 h.
Cytokine treatment had essentially no effect on receptor mRNA levels in
Jar, JEG-3, and U937 cells. By contrast, DR4 message was doubled in the
only cell line that transcribed this gene, the THP-1 cells.
Cytotoxicity of TRAIL for trophoblast and macrophage cell lines
The results described above regarding expression of specific TRAIL receptors predicted that both trophoblast and macrophage cell lines would be protected against killing by TRAIL, the first by DcR1 and the second by DcR2. To evaluate this postulate, trophoblast-derived and macrophage cell lines were incubated with rTRAIL and tested for mitochondrial enzyme activity 20 h later using a commercially available MTT assay.
Table II shows that trophoblasts were
fully protected against TRAIL cytotoxicity. By contrast, U937 and THP-1
tumor macrophages as well as HeLa endometrial adenocarcinoma cells were
sensitive to killing by TRAIL and the effects were dose dependent. The
MTT results were verified in trypan blue assays for cell viability
(results not shown).
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shows nuclear changes
induced by exposing HeLa cells to 100 ng/ml of rTRAIL for 2 h. The
nuclei of untreated control cells were of fairly uniform size and
shape, contained distinct nucleoli, and exhibited comparatively
homogenous staining, whereas the nuclei of TRAIL-treated cells varied
in size and shape and exhibited areas of intense staining as well as
fragmentation characteristic of apoptotic changes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. We verified transcription of all four TRAIL-R genes in
human placentas, and in this study provide evidence that the array of
receptor messages in cell types comprising placentas is also cell type
specific. The results of a final series of experiments implied that
trophoblast is well equipped to participate in TRAIL-mediated killing
without sustaining injury, and that this may be due to expression of
DcR1. TRAIL is likely to play a major role in maintaining placental immune privilege. Syncytiotrophoblast, which is continuously bathed in maternal blood containing immune cells, was the major cell type containing immunoreactive TRAIL. It is now well established that TRAIL cooperates with FasL in limiting lymphocyte proliferation (17, 35), a process termed activated cell death as opposed to programmed cell death (36). FasL was the first of the apoptosis-inducing TNF family members to be implicated in activated cell death, and the early demise of natural mutant mice deficient in FasL or its receptor, Fas, as a consequence of uncontrolled lymphoproliferation illustrates its biologic importance. Through this ability to kill activated lymphocytes, FasL has been implicated in immune privilege in the testis, eye, and pregnant uterus (8, 37, 38). Our identification of immunoreactive TRAIL in a placental cell layer that is already known to produce FasL strongly suggests cooperativity in this setting, with both TRAIL and FasL potentially involved in killing activated lymphocytes to confer immune privilege to the placenta.
Activated lymphocytes express increased TRAIL, and in this study we
report that activation of trophoblasts with IFN- has the same
result: steady state levels of TRAIL mRNA are efficiently enhanced.
This is probably of importance in vivo; IFN- is present in placentas
and trophoblast has an abundance of IFN- receptors (39, 40, 41). In the
only reported experiments similar to these, Snell et al. identified
IFN- as an enhancer of TRAIL mRNA in PBMC, but the RT-PCR results
were inconsistent among preparations from different individuals (14).
TRAIL-mediated self-destruction as well as destruction by paracrine
TRAIL may be prevented in trophoblast by the production of the decoy
receptor, DcR1. This idea is offered cautiously; whether DcR1
expression, which was prominent in choriocarcinoma cells (this study),
is also a feature of normal trophoblast in situ is unknown. Specific Ab
to DcR1 that could be used for protein identification has not been
reported. However, trophoblast is the main cell type in placentas, and
DcR1 is exceptionally high in this organ, as noted recently by Marsters
et al. (30). Trophoblast-derived choriocarcinoma cells containing DcR1
mRNA were entirely resistant to killing by rTRAIL. Although our
presumption is that DcR1 provided total protection, it is equally
possible that trophoblast cell DR5 apoptosis-inducing receptors are
either not produced or are nonfunctional. TRAIL protection is provided
differently from both FasL and TNF-, which are characterized by
limited expression of the ligand (Fas/FasL) and soluble receptors that
interfere with membrane binding and signal transduction (TNF-). With
its array of four different receptors, TRAIL may have significantly
greater flexibility in determining cell life or death than other
apoptosis-inducing TNF-related cytokines.
Macrophages comprised a second placental cell type that contained TRAIL
protein in situ. Further studies on cell lines showed that this lineage
expresses the TRAIL gene and contains higher levels of mRNA following
activation with IFN-. This finding was of considerable interest;
modulation of TRAIL expression had not been demonstrated in either
trophoblasts or macrophages before this study. The functions of TRAIL
in villous macrophages are unknown, but it does not seem unreasonable
to suggest that these migratory cells, which are phagocytic, produce
cytokines, and protect against transport of unwanted substances into
the fetus (42), might use their TRAIL for placental modeling and/or
killing of neoplastic cells. The studies on macrophage cell lines
suggested that at least in this lineage, DR5 delivers a death signal
more effectively than DR4. The U937 cells, which were highly
susceptible to TRAIL-induced apoptosis, expressed more DR5 and less DR4
mRNA than THP-1 cells, which were less sensitive to killing by TRAIL.
Interestingly, the DR4 message was elevated by treating THP-1 cells
with IFN-, whereas a slight decrease in DcR2 mRNA following cytokine
treatment was observed, suggesting a reciprocal relationship between an
apoptosis-inducing and a protective receptor deserving of further
investigation. The inability of extremely high concentrations of rTRAIL
to achieve total lysis of macrophage cell lines (Table II) is of
interest and implies that, as suggested in other contexts (43),
mechanisms other than decoy receptors may be important in protecting
cells from destruction by TRAIL.
A third type of fetal cell that contained TRAIL protein was the amnion epithelial cell, but no experiments were done to determine whether this lineage produces TRAIL or simply endocytoses the protein, and its potential functions are unclear.
Maternal decidual cells in the placental bed residing side by side with migrating trophoblasts were TRAIL positive. It seems unlikely that these TRAIL-expressing cells kill the trophoblast cells; there is little evidence for trophoblast cell death in the placental bed. There has been considerable interest and speculation regarding the role of TRAIL in killing tumor cells (14, 44), suggesting the possibility that destroying neoplastic cells that arise in this dynamic situation of cell growth and proliferation may be a major role for decidual cell TRAIL.
In summary, the results of this study strongly suggest that high expression of TRAIL and the decoy receptor, DcR1, in trophoblast cells exposed to maternal blood may contribute importantly to immune tolerance during pregnancy. Moreover, the observations reported in this work are consistent with the postulate that TRAIL has other roles at the maternal-fetal interface that could include protecting against the development of tumors and contributing to placental modeling and homeostatis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joan S. Hunt, Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160-7400. E-mail address:
3 Abbreviations used in this paper: FasL, Fas ligand; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; hu, human; TRAIL, TNF-related apoptosis-inducing ligand.
Received for publication September 10, 1998. Accepted for publication February 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA and protein are present in human placental and uterine cells at early and late stages of gestation. Am. J. Pathol. 139:327.[Abstract]
B. Immunity 7:831.[Medline]
B pathway. Immunity 7:821.[Medline]
B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7:813.[Medline]
receptor in first trimester placenta to trophoblasts but lack of stimulation of HLA-DRA, -DRB, or invariant chain mRNA expression by IFN-. J. Immunol. 149:2675.[Abstract]
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 T. Yamamoto, H. Nagano, M. Sakon, H. Wada, H. Eguchi, M. Kondo, B. Damdinsuren, H. Ota, M. Nakamura, H. Wada, et al. Partial Contribution of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)/TRAIL Receptor Pathway to Antitumor Effects of Interferon-{alpha}/5-Fluorouracil against Hepatocellular Carcinoma Clin. Cancer Res., December 1, 2004; 10(23): 7884 - 7895. [Abstract] [Full Text] [PDF]
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 J. K. Riley, J. M. Heeley, A. H. Wyman, E. L. Schlichting, and K. H. Moley TRAIL and KILLER Are Expressed and Induce Apoptosis in the Murine Preimplantation Embryo Biol Reprod, September 1, 2004; 71(3): 871 - 877. [Abstract] [Full Text] [PDF]
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R. M. Gill and J. S. Hunt Soluble Receptor (DcR3) and Cellular Inhibitor of Apoptosis-2 (cIAP-2) Protect Human Cytotrophoblast Cells Against LIGHT-Mediated Apoptosis Am. J. Pathol., July 1, 2004; 165(1): 309 - 317. [Abstract] [Full Text] [PDF]
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 X. Y. Zhang, X. D. Zhang, J. M. Borrow, T. Nguyen, and P. Hersey Translational Control of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Death Receptor Expression in Melanoma Cells J. Biol. Chem., March 12, 2004; 279(11): 10606 - 10614. [Abstract] [Full Text] [PDF]
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 K. E. Naylor, A. Rogers, R. B. Fraser, V. Hall, R. Eastell, and A. Blumsohn Serum Osteoprotegerin as a Determinant of Bone Metabolism in a Longitudinal Study of Human Pregnancy and Lactation J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5361 - 5365. [Abstract] [Full Text] [PDF]
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M. Lonergan, D. Aponso, K. W. Marvin, R. J. A. Helliwell, T. A. Sato, M. D. Mitchell, T. Chaiwaropongsa, R. Romero, and J. A. Keelan Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), TRAIL Receptors, and the Soluble Receptor Osteoprotegerin in Human Gestational Membranes and Amniotic Fluid during Pregnancy and Labor at Term and Preterm J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3835 - 3844. [Abstract] [Full Text] [PDF]
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H. Ka and J. S. Hunt Temporal and Spatial Patterns of Expression of Inhibitors of Apoptosis in Human Placentas Am. J. Pathol., August 1, 2003; 163(2): 413 - 422. [Abstract] [Full Text] [PDF]
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 K. Ichikawa, W. Liu, M. Fleck, H. Zhang, L. Zhao, T. Ohtsuka, Z. Wang, D. Liu, J. D. Mountz, M. Ohtsuki, et al. TRAIL-R2 (DR5) Mediates Apoptosis of Synovial Fibroblasts in Rheumatoid Arthritis J. Immunol., July 15, 2003; 171(2): 1061 - 1069. [Abstract] [Full Text] [PDF]
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 T. A. Phillips, J. Ni, and J. S. Hunt Cell-specific expression of B lymphocyte (APRIL, BLyS)- and Th2 (CD30L/CD153)-promoting tumor necrosis factor superfamily ligands in human placentas J. Leukoc. Biol., July 1, 2003; 74(1): 81 - 87. [Abstract] [Full Text] [PDF]
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M. G. Petroff, L. Chen, T. A. Phillips, D. Azzola, P. Sedlmayr, and J. S. Hunt B7 Family Molecules Are Favorably Positioned at the Human Maternal-Fetal Interface Biol Reprod, May 1, 2003; 68(5): 1496 - 1504. [Abstract] [Full Text] [PDF]
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R. M. Gill, J. Ni, and J. S. Hunt Differential Expression of LIGHT and Its Receptors in Human Placental Villi and Amniochorion Membranes Am. J. Pathol., December 1, 2002; 161(6): 2011 - 2017. [Abstract] [Full Text] [PDF]
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H.-o. Lee, J. M. Herndon, R. Barreiro, T. S. Griffith, and T. A. Ferguson TRAIL: A Mechanism of Tumor Surveillance in an Immune Privileged Site J. Immunol., November 1, 2002; 169(9): 4739 - 4744. [Abstract] [Full Text] [PDF]
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T. S. Soderstrom, M. Poukkula, T. H. Holmstrom, K. M. Heiskanen, and J. E. Eriksson Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling in Activated T Cells Abrogates TRAIL-Induced Apoptosis Upstream of the Mitochondrial Amplification Loop and Caspase-8 J. Immunol., September 15, 2002; 169(6): 2851 - 2860. [Abstract] [Full Text] [PDF]
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H. S. Kim, J. H. Cho, H. W. Park, H. Yoon, M. S. Kim, and S. C. Kim Endotoxin-Neutralizing Antimicrobial Proteins of the Human Placenta J. Immunol., March 1, 2002; 168(5): 2356 - 2364. [Abstract] [Full Text] [PDF]
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X. D. Zhang, X. Y. Zhang, C. P. Gray, T. Nguyen, and P. Hersey Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis of Human Melanoma Is Regulated by Smac/DIABLO Release from Mitochondria Cancer Res., October 1, 2001; 61(19): 7339 - 7348. [Abstract] [Full Text] [PDF]
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D. Ait-Azzouzene, S. Caucheteux, F. Tchang, J. Wantyghem, R. Moutier, A. Langkopf, M.-C. Gendron, and C. Kanellopoulos-Langevin Transgenic Major Histocompatibility Complex Class I Antigen Expressed in Mouse Trophoblast Affects Maternal Immature B Cells Biol Reprod, August 1, 2001; 65(2): 337 - 344. [Abstract] [Full Text] [PDF]
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H. Behbahani, E. Popek, P. Garcia, J. Andersson, A.-L. Spetz, A. Landay, Z. Flener, and B. K. Patterson Up-Regulation of CCR5 Expression in the Placenta Is Associated with Human Immunodeficiency Virus-1 Vertical Transmission Am. J. Pathol., December 1, 2000; 157(6): 1811 - 1818. [Abstract] [Full Text] [PDF]
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W. D. Thomas, X. D. Zhang, A. V. Franco, T. Nguyen, and P. Hersey TNF-Related Apoptosis-Inducing Ligand-Induced Apoptosis of Melanoma Is Associated with Changes in Mitochondrial Membrane Potential and Perinuclear Clustering of Mitochondria J. Immunol., November 15, 2000; 165(10): 5612 - 5620. [Abstract] [Full Text] [PDF]
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 L. Zamai, P. Secchiero, S. Pierpaoli, A. Bassini, S. Papa, E. S. Alnemri, L. Guidotti, M. Vitale, and G. Zauli TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis Blood, June 15, 2000; 95(12): 3716 - 3724. [Abstract] [Full Text] [PDF]
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N. Hanna, I. Hanna, M. Hleb, E. Wagner, J. Dougherty, D. Balkundi, J. Padbury, and S. Sharma Gestational Age-Dependent Expression of IL-10 and Its Receptor in Human Placental Tissues and Isolated Cytotrophoblasts J. Immunol., June 1, 2000; 164(11): 5721 - 5728. [Abstract] [Full Text] [PDF]
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