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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3716-3724
HEMATOPOIESIS
TNF-related apoptosis-inducing ligand (TRAIL) as a
negative regulator of normal human erythropoiesis
Loris Zamai,
Paola Secchiero,
Sabina Pierpaoli,
Alessandra Bassini,
Stefano Papa,
Emad S. Alnemri,
Lia Guidotti,
Marco Vitale, and
Giorgio Zauli
From the Institute of Morphological Sciences, University of Urbino,
Urbino, Italy; Department of Morphology and Embryology, Human Anatomy
Section, University of Ferrara, Ferrara, Italy; Institute of Histology
and General Embryology, University of Bologna, Bologna, Italy; Kimmel
Cancer Institute, Jefferson Medical College, Philadelphia,
Pennsylvania; Department of Biomedical Sciences and Biotechnologies,
Human Anatomy Section, University of Brescia, Brescia, Italy; Institute
of Cytomorphology NP CNR, c/o "Codivilla-Putti" Research
Institute, Bologna, Italy; and the Institute of Human Morphology,
"G. D'Annunzio," University of Chieti, Chieti, Italy.
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Abstract |
The impact of tumor necrosis factor (TNF)-related apoptosis-inducing
ligand (TRAIL) on normal hematopoietic development was investigated
using adult peripheral blood CD34+ hematopoietic
progenitor cells, induced to differentiate along the erythroid,
megakaryocytic, granulocytic, and monocytic lineages by the addition of
specific cytokine cocktails. TRAIL selectively reduced the number of
erythroblasts, showing intermediate levels of glycophorin A
(glycophorin Ainterm) surface expression, which appeared in
liquid cultures supplemented with stem cell factor + interleukin
3 + erythropoietin at days 7-10. However, neither immature (day 4)
glycophorin Adim erythroid cells nor mature (day 14)
glycophorin Abright erythroblasts were sensitive to
TRAIL-mediated apoptosis. Moreover, pre-exposure to TRAIL significantly
decreased the number and size of erythroid colonies in semisolid
assays. These adverse effects of TRAIL were selective for
erythropoiesis, as TRAIL did not significantly influence the survival
of cells differentiating along the megakaryocytic, granulocytic, or
monocytic lineages. Furthermore, TRAIL was detected by Western blot
analysis in lysates obtained from normal bone marrow mononuclear cells.
These findings indicate that TRAIL acts in a lineage- and stage of
differentiation-specific manner, as a negative regulator of normal erythropoiesis.
(Blood. 2000;95:3716-3724)
© 2000 by The American Society of Hematology.
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Introduction |
The maintenance of organ and tissue homeostasis and
regulation of their size are controlled by finely tuned signals of
proliferation and programmed cell death, an active form of death also
known as apoptosis.1 In particular, adult hematopoiesis is
maintained by a balanced production of cytokines, which mainly act to
stimulate or to prevent survival and proliferation of hematopoietic
progenitor and precursor cells.2
Several hematopoietic growth factors, such as interleukin (IL)-3, have
been shown to deliver survival/proliferation signals to hematopoietic
cells. However, it is becoming increasingly clear that hematopoietic
progenitor cells die not only as a consequence of growth factor
withdrawal but also in response to apoptosis inducers, such as
transforming growth factor  1, tumor necrosis factor (TNF)- ,
interferon (IFN)- , and Fas (CD95) ligand (FasL/CD95L) (reviewed in
Park2). These negative regulators of hematopoiesis are
likely involved in both the physiological control of hematopoiesis as
well as the induction of pathological conditions. In this respect, it
has been shown that TNF-- and IFN--treated hematopoietic progenitors, when exposed to an agonist CD95 monoclonal antibody (mAb),
display a poor colony formation in semisolid cultures and undergo
apoptosis.3,4 Moreover, mouse embryos, bearing a defective
expression of FADD, a death adaptor molecule downstream to various death receptors, including CD95 and TNF-R1, die within 12 days of gestation because of erythroid accumulation and impaired heart
muscle development (reviewed in Ashkenazi and Dixit5). Altogether, these findings suggest that the CD95/CD95L system is
involved in the negative regulation of hematopoiesis and, in particular, of normal erythroid development (reviewed in Niho and
Asano6). Moreover, an up-regulation of the CD95/CD95L
system has been proposed to play a prominent role also in the
pathogenesis of aplastic anemia.7-10
TNF-related apoptosis-inducing ligand (TRAIL) is a recently described
member of the TNF-related proteins, which shows structural and
functional similarities with CD95L,11-18 including the use of FADD as adaptor molecule.19 The unique feature of TRAIL
with respect to CD95L and TNF- is considered its ability to induce apoptosis of various cell lines and of primary tumor cells, including several of hematopoietic origin12 without displaying toxic
effects on normal cells and tissues. So far, the only primary cells
susceptible to TRAIL cytotoxic activity are cultured
astrocytes.20 Thus, a role for this cytokine in
physiological conditions has not been envisioned yet.
The aim of this study was to investigate whether TRAIL might play a
role in the homeostatic control of hematopoiesis. For this purpose,
TRAIL was tested on freshly isolated adult peripheral blood (PB)
CD34+ hematopoietic progenitors, as well as on erythroid
(glycophorin A+), megakaryocytic (CD61+),
granulocytic (CD15+), and monocytic (CD14+)
precursor cells generated in vitro in liquid suspension and semisolid
cultures. Moreover, the expression of TRAIL was investigated on lysates
obtained from human bone marrow (BM) mononuclear cells.
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Materials and methods |
Reagents
Both rHis6-tagged TRAIL and rHis6-tag control
peptides were produced in bacteria and purified by affinity
chromatography on Ni2+ affinity resin, as
previously described.21 The functional activity of each
TRAIL preparation used in this study was tested on the TRAIL-sensitive
Jurkat cell line (J32 clone), as previously
described.22 In preliminary dose- and time-course
experiments performed in Jurkat cells, increasing apoptosis was evident
by about 4-6 hours, it was complete by 20 hours, and reached a plateau
at the concentrations of 0.1-1 µg/mL. In contrast, equimolar
concentrations of rHis6-tag alone did not show any significant
toxicity on Jurkat cells. Therefore, a 20-hour incubation time and the
concentration of 1 µg/mL of TRAIL were chosen to perform the
experiments in primary hematopoietic cells.
Agonist anti-CD95 immunoglobulin (Ig)M mAb (CH11, UBI, Lake Placid, NY)
or isotype-matched control IgM was used at the concentration of 0.2 µg/mL.
Purification of the cells
Bone marrow aspirates were taken from the posterior iliac crest of 3 normal donors, whereas leukapheresis units were obtained from PB of 21 normal donors. All individuals gave their informed consent to the study
according to the Helsinki declaration of 1975. Mononuclear cells from
either BM or PB samples were isolated by density gradient
(Ficoll/Histopaque-1077, Sigma Chemical Co, St Louis, MO). Whereas BM
mononuclear cells were immediately lysed for Western blot analysis, PB
mononuclear cells were let to adhere to plastic for 1 hour at 37°C.
After removal of adherent cells, PB CD34+ cells were
isolated with the use of a magnetic cell sorting program Mini-MACS and
the CD34 isolation kit (Miltenyi Biotech, Auburn, CA) in accordance
with the manufacturer's instructions and as previously
described.23 The purity of CD34-selected cells was determined for each isolation by FACStar Plus (Lysis II program, Becton
Dickinson, San Jose, CA), using a mAb that recognizes a separate
epitope of the CD34 molecule (HPCA-2, Becton Dickinson) directly
conjugated to fluorescein isothiocyanate (FITC). CD34+
cells ranged about 85%-98%.
Unilineage liquid cultures of hematopoietic progenitor cells and
phenotypic evaluation
In most experiments, purified CD34+ cells were cultured
in Ex-vivo (Biowittaker, Walkersville, MA) serum-free medium,
supplemented with nucleosides (10 µg/mL each), 0.5% bovine serum
albumin (BSA, Chon fraction V), 10-4 mol/L BSA-adsorbed
cholesterol, 10 µg/mL insulin, 200 µg/mL iron-saturated transferrin, 5 × 10-5mol/L
2--mercaptoethanol (all purchased from Sigma). Cells were adjusted
to an optimal cell density of 5 × 104/mL and seeded
in culture in the presence of stem cell factor (SCF; 50 ng/mL) + IL-3
(10 ng/mL) + erythropoietin (EPO; 4 U/mL) to induce erythroid
differentiation; IL-3 (10 ng/mL) + thrombopoietin (TPO; 100 ng/mL) to
induce megakaryocytic differentiation; SCF (50 ng/mL) + IL-3 (10 ng/mL) + granulocyte-colony stimulating factor (G-CSF; 10 ng/mL) to
induce granulocytic differentiation; SCF (50 ng/mL) + IL-3 (10 ng/mL) + macrophage-CSF (M-CSF; 10 ng/mL) + 10% fetal calf serum
(Gibco, Grand Island, NY) to induce monocytic differentiation. All
cytokines were purchased from Genzyme (Cambridge, MA). Every 3-4 days,
cultures were demi-populated by removing half volume of the medium,
which was substituted with fresh medium supplemented or not with
lineage-specific cytokines (EPO, TPO, G-CSF, or M-CSF). At these time
points, the cells removed were counted, stained, and analyzed by flow
cytometry, whereas the cell density was readjusted to
1 × 105/mL. TRAIL (1 µg/mL), anti-CD95 CH11 mAb
(0.2 µg/mL), or isotype-matched mAb (0.2 µg/mL) + rHis6-tag (0.15 µg/mL) were added either
immediately after purification of CD34+ cells (day 0) or at
various culture times as specified in the text.
The surface phenotype of CD34-derived cells was analyzed by FACStar
Plus. Staining was performed at 4°C for 30 minutes on 2 × 105 cells in 200 µL of phosphate-buffered
saline (PBS) containing 1% BSA, 5% human plasma, 0.1% sodium azide,
and the following mAbs: FITC- or phycoerythrin (PE)-conjugated
anti-CD34, PE-anti-CD71, PE-anti-CD33 (Becton Dickinson); FITC- or
PE-anti-CD14, FITC-anti-CD15, FITC- or PE-anti-CD61 (Cymbus, LTD, Tema
ricerca srl, Bologna, Italy); PE-anti-glycophorin A and PE-anti-Fas
(CD95) mAb (Pharmingen, San Diego, CA); FITC-anti-CD42b (Southern
Biotechnology Associates Inc, Birmingham, AL); FITC-anti-glycophorin A
(DAKO, Copenhagen, Denmark); PE-anti-CD41 (Coulter-Immunotech,
Marseille Cedex, France); PE-anti-CD11b (BIO-RAD SPD, Segrate, Italy);
and FITC-anti-CD16 (Caltag, San Francisco, CA).
In some experiments, erythroid cells obtained in liquid culture were
spun on coverslips, fixed in 4% paraformaldehyde in PBS for 10 minutes
at room temperature, washed twice with PBS, stained with
May-Grunwald-Giemsa, and observed at light microscopy with an Axyophot
Zeiss microscope.
Measurement of apoptosis
Cell death was evaluated by FACStar Plus, using 3 distinct
procedures: (1) FITC-conjugated annexin V staining (Bender, Wien, Austria) that detects phosphatidyl-serine exposed on the outer cell
membrane following caspase activation, (2) propidium iodide (PI;
Calbiochem, CA) that goes into membrane-damaged dead cells, (3)
forward-side-scatter analysis that detects a strong decrease of the
forward scatter signal (FSC) in the presence of a still stable side
scatter signal (SSC) due to profound morphological changes (shrinkage)
of apoptotic cells.24 The staining methods were chosen in
each experiment depending on the fluorochrome conjugated to the mAb
used for the surface antigen detection. When performed in parallel
experiments, all 3 methods gave basically overlapping results.
In vitro colony forming assays
Erythroid (BFU-E), megakaryocytic (CFU-meg), and
granulocytic/macrophagic (CFU-GM) colonies were assayed in plasmaclot
cultures, as previously described.25 Briefly,
5 × 103 CD34+ cells were cultured in 1 mL of IMDM (Gibco), containing 10% detoxified BSA, 10%
heat-inactivated pooled human AB sera, 10% citrated bovine plasma
(Gibco), 20 mg of L-asparagine (Sigma), 3.4 mg/mL
CaCl2. The standard sources of growth factors were the same
as used in liquid cultures: SCF (50 ng/mL) + IL-3 (10 ng/mL) + EPO
(4 U/mL) for the growth of BFU-E; IL-3 (10 ng/mL) + TPO (100 ng/mL)
for the growth of CFU-meg; SCF (50 ng/mL) + IL-3 (10 ng/mL) + G-CSF (10 ng/mL) for the growth of CFU-GM. All semisolid cultures were supplemented with TRAIL (1 µg/mL), CH11 anti-CD95 mAb (0.2 µg/mL), or isotype-matched mAb (0.2 µg/mL) + rHis6-tag (0.15 µg/mL). In selected experiments, CD34+ cells were
pre-incubated for 2 days with the cytokine combinations illustrated
above in liquid cultures and then treated for an additional 20 hours
with TRAIL, CH11 anti-CD95 mAb, or IgM irrelevant
mAb + rHis6-tag irrelevant peptide before being seeded in
semisolid assays. At day 14, for BFU-E and CFU-GM identification, the
clots were fixed in situ with methanol-acetone 1:3 for 20 minutes and stained with 3,3' dimethoxybenzidine and hematoxylin. Colonies of
> 50 red hemoglobin-containing cells were scored as BFU-E, whereas
colonies of > 50 nonhemoglobinized cells were scored as CFU-GM.
CFU-meg was identified after immunofluorescence staining with anti-CD41
mAb directly conjugated to FITC (Pharmingen), as aggregates of 3 or
more fluorescent cells.
Western blotting
For the analysis of TRAIL protein expression, Western blot was
performed on approximately 4 × 106 BM mononuclear
cells. Cells were harvested in lysis buffer containing 1% Triton
X-100, sonicated, and processed by Western blot. Protein determination
was performed by Bradford assay (Bio-Rad, Richmond, CA). Human Jurkat
cell line and murine Friend cell line were used as positive and
negative controls of TRAIL expression, respectively (not shown). For
each sample, 100 µg of proteins was migrated in 12% acrylamide gels
and blotted onto nitrocellulose filters. Blotted filters were blocked
for 30 minutes in a 3% suspension of dried skimmed milk in PBS and
incubated overnight at 4°C with 1:100 dilution of anti-TRAIL mAb
(clone B35-1 from Pharmingen). Filters were washed and further
incubated for 1 hour at room temperature with 1:1000 dilution of
peroxidase-conjugated anti-mouse IgG (Sigma Chemicals) in 0.1% BSA.
Specific reactions were revealed with the ECL Western blotting
detection reagent (Amersham Corp, Arlington Heights, IL).
Statistical analysis
Data were analyzed with the use of the 2-tailed, 2-sample t
test (Minitab statistical analysis software, State College, PA). Values
of P < .05 were considered significant.
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Results |
Lack of TRAIL toxicity on freshly isolated CD34+ cells
In the first set of experiments, we have investigated the
sensitivity of PB CD34+ hematopoietic progenitor cells to
TRAIL and anti-CD95-triggering (CH11) mAb by both FSC/SSC and PI
staining analyses, which gave similar results in terms of percentage of
dead cells (Figure 1). Although the
addition of hematopoietic growth factors (SCF + IL-3) significantly
(P < .05) reduced the percentage of freshly isolated CD34+ cells undergoing apoptosis after 20 hours of culture,
TRAIL was unable to significantly modify the degree of
CD34+ cell death either in the absence or in the presence
of SCF + IL-3 (Figure 1 and Table 1). As
expected, because of the absence of surface CD95-antigen expression in
CD34+ cells (not shown),3,4,7,10 anti-CD95
agonist mAb was unable to significantly alter the percentage of
CD34+ cell death, irrespective of the presence of cytokines
(Figure 1 and Table 1).
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| Fig 1.
Evaluation of TRAIL activity on freshly isolated PB
CD34+ cells.
The percentage of cell death was evaluated in CD34+ cells
treated with irrelevant IgM mAb and rHis6-tag peptide (CONT),
TRAIL, or anti-CD95 (CH 11) IgM mAb for 20 hours in medium supplemented
with (CYTOK, right panels) or without SCF + IL-3 (left panels).
CD34+ cell death was detected by either FSC/SSC (upper 2 panels) or PI staining (lower 6 panels) analyses. Percentages of dead
cells are indicated in each panel. Representative FSC/SSC analyses are
shown only for control (CONT) cultures, since similar profiles were
observed in TRAIL- and anti-CD95-treated cells (not shown). In the
lower 6 panels, quadrants were set based on negative controls stained
with isotype-matched irrelevant mAb (not shown). X-axis: relative CD34
expression; Y-axis: propidium iodide staining.
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Table 1.
Percentages of dead CD34+ cells incubated
soon after purification (CD34+ cells >90%) for 20 hours with the indicated treatments
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Because CD34+ cells represent an heterogeneous population
of hematopoietic stem and progenitor cells,26 we next
sought to test TRAIL and anti-CD95 CH11 mAb on hematopoietic
progenitors committed toward the erythroid, megakaryocytic,
granulocytic and monocytic lineages. For this purpose, unilineage
serum-free liquid cultures were set by the addition of pluripotent
hematopoietic growth factors (SCF + IL-3) plus lineage-specific
cytokines (EPO, TPO, G-CSF, or M-CSF).
Selective TRAIL-mediated induction of apoptosis in erythroid cells
at intermediate stages of differentiation in unilineage liquid culture
Erythroid differentiation, which strictly requires the presence of
EPO, is characterized by initial expression of CD71, followed by the
appearance of glycophorin A antigen.27 At day 4 of culture, most of the CD34+-derived cells cultured with
SCF + IL-3 + EPO expressed CD71, whereas approximately 10% of
cells showed a low expression of glycophorin A (glycophorin
Adim) (Figure 2A). About half
of the CD34+-derived hematopoietic progenitor cells
cultured for 4 days with IL-3 + TPO expressed CD61 (gpIIIa) (Figure
2A), which represents an early marker expressed during human
megakaryocytic development.28 When CD34+
hematopoietic progenitor cells were cultured for 4 days with SCF + IL-3, regardless of the presence of G-CSF or M-CSF, most of the
cells expressed CD33 at low density (Figure 2A).
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| Fig 2.
Evaluation of TRAIL effects on unilineage committed
hematopoietic progenitors at early stages of differentiation (day 4 of
culture).
(A) The percentage of cell death was evaluated by annexin V staining in
unilineage erythroid (SCF + IL-3 + EPO), megakaryocytic
(IL-3 + TPO), and myeloid (SCF + IL-3) cells at day 4. Cultures
were supplemented with irrelevant IgM mAb and rHis6-tag peptide
(CONT), TRAIL, or anti-CD95 (CH 11) IgM mAb for 20 hours before annexin
V staining. Quadrants were set based on negative controls stained with
isotype-matched irrelevant mAbs. X-axis: annexin V relative expression.
Y-axis: glycophorin A, CD71, CD61, CD33 relative expression. (B) shows
CD95 surface expression (thick lane histograms) evaluated at day 4 of
unilineage cultures. Isotype-matched negative controls are represented
by the thin lane histograms. X-axis: CD95 relative expression. Y-axis:
relative cell number.
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In all unilineage cultures, the percentage of dead cells was somewhat
higher in 20-hour TRAIL-treated samples than in control samples, but
these differences were small (Figure 2A) and did not result in a
significant reduction of viable cells committed toward the different
lineages (Table 2). Similarly, 20-hour
incubation with anti-CD95 CH11 mAb did not result in a significant
increase of cell death (Figure 2A and Table 2), despite that, at this time point, most of the cells expressed detectable levels of surface CD95 molecule (Figure 2B).
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Table 2.
Percentages of viable cells expressing lineage specific
markers and dead cells (reported only for the erythroid lineage at days
7-10) on unilineage cultures at different time points after 20 hours of
incubation with the indicated treatments
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We next analyzed TRAIL- and CD95-mediated sensitivity of
hematopoietic precursors at intermediate stages of development. At day
7, most of the cells generated in cultures supplemented with EPO
expressed CD71 and about 30%-40% of these were
CD71bright/glycophorin A+ (Figure
3A). Most CD34+-derived
hematopoietic cells cultured for 7 days with TPO expressed CD61 (and
CD41 at low density) early markers, whereas CD42b antigen, which
appears later during megakaryocyte development,28 was virtually undetectable (Figure 3A). Of note, a 20-hour incubation with
TRAIL induced a statistically significant (P < .05)
decrease of viable glycophorin A+ cells concomitant with a
parallel increase (P < .05) of cell death at day 7 of
culture (Table 2), as shown by both annexin V staining followed by flow
cytometry (Figure 3A) and by morphological analysis of cultured
erythroblasts (Figure 3B). In agreement with a previous
report,9 we found that erythroblasts obtained at day 7 of
liquid culture could be induced to die by apoptosis also by anti-CD95
CH11 mAb (Figure 3A-B, Table 2). At light microscopy examination, many
cells treated with either TRAIL or anti-CD95 CH11 mAb showed several
features characteristic of apoptosis, such as membrane blabbing and
chromatin condensation (Figure 3B). These effects were specific for the
erythroid lineage, as demonstrated by the absence at the same time
point of significant reduction of the percentages of cells belonging to
the megakaryocytic (CD61+) lineage (Figure 3A), which is
closely related to the erythroid lineage.28
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| Fig 3.
TRAIL-mediated induction of apoptosis in erythroid
precursors at intermediate stages of differentiation.
Seven-day unilineage erythroid (SCF + IL-3 + EPO) (A and B) and
megakaryocytic (IL-3 + TPO) (A) cultures were incubated with
irrelevant IgM mAb and rHis6-tag peptide (CONT), TRAIL, or
anti-CD95 (CH 11) IgM mAb for 20 hours. (A) Erythroid and
megakaryocytic cells were phenotypically characterized by double
staining with anti-CD71 + anti-glycophorin A or
anti-CD41 + anti-CD42b, respectively (upper 2 panels). Percentages of
glycophorin A+ and CD41+ cells are indicated in
each panel. Apoptotic erythroid cells were detected by staining with
annexin V combined to glycophorin A, whereas apoptotic megakaryocytes
were detected by propidium iodide staining combined to CD61 (lower 6 panels). Percentages of cells in the respective quadrants are
indicated. Quadrants were set based on negative controls stained with
isotype-matched irrelevant mAbs (not shown). Panel (B) shows the
morphological analysis of erythroblasts stained with
May-Grunwald-Giemsa. Apoptotic cells are indicated by arrowheads.
Original magnification 600×.
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In the next experiments, we have investigated whether the
known anti-apoptotic activity of EPO29 was
able to interfere with TRAIL ability to kill erythroid cells. In this
respect, it should be noticed that, in the experiments illustrated
above, TRAIL was typically added in culture 3-4 days after the last
addition of EPO, which occurred at the time of the first
demi-population (day 4 of culture). Although EPO is consumed by
cultured erythroid cells,30 we have not assayed the EPO
concentrations at various culture times. Thus, to precisely
characterize whether EPO showed any protective effect, erythroid cells
were washed at day 8 to eliminate residual EPO and then seeded in
culture in the absence or presence of fresh EPO (4 U/mL) for 12 hours
before TRAIL treatment. Within the heterogeneous erythroid cell
population, TRAIL specifically inhibited erythroblasts showing an
intermediate glycophorin A expression (glycophorin
Ainterm), appearing in culture between days 7 and 10 (Figure 4). Fresh EPO
counteracted the ability of 20-hour TRAIL treatment to deplete the
glycophorin Ainterm cells (Figure 4). This effect was
accompanied by a significant (P < .05) reduction in the
number of dead cells in TRAIL-treated samples from 33% ± 5% in
the absence of EPO to 15% ± 7% in the presence of EPO (means ± SD of 3 separate experiments). The addition of fresh EPO showed a
similar protective effect also against CD95-mediated cell death (data
not shown).
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| Fig 4.
EPO prevention of the TRAIL-mediated depletion of
glycophorin Ainterm erythroblasts.
The expression of glycophorin A was evaluated on viable cells
electronically gated on the basis of FSC/SSC pattern (see caption of
Figure 1) at day 10 of culture with the indicated cytokines. Cultures
were supplemented (bottom 2 panels) or not with fresh EPO for 12 hours
and then with TRAIL or rHis6-tag peptide (CONT) for the last 20 hours of culture before glycophorin A staining. X-axis: glycophorin A
relative expression. Y-axis: relative cell number. Percentages of
glycophorin A+ cells are indicated.
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In parallel experiments, we found that 7- to 10-day granulocytic and
monocytic unilineage cultures were minimally affected by both TRAIL and
anti-CD95 CH11 mAb (Figure 5 and Table 2).
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| Fig 5.
Lack of toxicity of TRAIL on unilineage committed
myelomonocytic precursors at intermediate stages of differentiation.
Ten-day unilineage granulocytic (SCF + IL-3 + G-CSF) and monocytic
(SCF + IL-3 + M-CSF) cultures were incubated with irrelevant IgM
mAb and rHis6-tag peptide (CONT), TRAIL, or anti-CD95 (CH 11)
IgM mAb for 20 hours. Apoptotic granulocytic cells were detected by
staining with propidium iodide combined to CD15, whereas apoptotic
monocytic cells were detected by annexin V staining combined to CD14.
Percentages of cells in the respective quadrants are indicated.
Quadrants were set based on negative controls treated with
isotype-matched irrelevant mAbs (not shown).
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At later culture times (days 14-15), maturation along the different
lineages reached the maximal levels before senescence of the culture
(Figure 6). In fact, most cells
differentiating along the erythroid lineage were
CD71bright/glycophorin Abright, whereas cells
differentiating toward the megakaryocytic lineage were characterized by
an up-regulation of both early CD61 and late CD42b megakaryocytic
surface antigens (Figure 6). Full maturation along the granulocytic
lineage was accompanied by the up-regulation of CD15, followed by
appearance of CD11b,31 whereas cells induced to
differentiate along the monocytic lineage were characterized by the
up-regulation of CD14, concomitant with the appearance of CD16 antigen
(Figure 6). At this time point, although mature erythroid cells have a
decreased size with respect to immature erythroblasts, they were still
clearly distinguishable from apoptotic cells by either scatter
characteristics or PI staining (Figure 7A).
At variance with glycophorin Ainterm cells, mature
glycophorin Abright erythroid cells were not significantly
affected by 20-hour treatment with TRAIL (Figure 7B and Table 2).
Similarly, anti-CD95 CH11 mAb failed to induce apoptosis in mature
glycophorin Abright erythroid cells, in spite of a clearly
detectable expression of surface CD95 antigen, which reached levels
comparable to that previously observed in immature glycophorin
Adim erythroid cells (Figure 2A).
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| Fig 6.
Surface phenotype of unilineage hematopoietic cells at
day 14 of culture.
At day 14, cells obtained in unilineage cultures with the indicated
cytokines were phenotypically characterized by double staining with
anti-CD71 + anti-glycophorin A (erythroid antigens),
anti-CD61 + anti-CD42b (megakaryocytic antigens),
anti-CD15 + anti-CD11b (granulocytic antigens), or
anti-CD16 + anti-CD14 (monocytic antigens). Percentages of cells in
the respective quadrants are indicated. CD14+ cells were
65%. Quadrants were set based on negative controls treated with
isotype-matched irrelevant mAbs (not shown).
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| Fig 7.
Loss of sensitivity to TRAIL of erythroid cells at late
stages of differentiation.
(A) 14-day viable erythroid cells were detected by either FSC/SSC or PI
staining analyses. Representative FSC/SSC (left and right panels) and
PI (central panel) staining analyses are shown only for control
cultures, since similar profiles were obtained in TRAIL- and
anti-CD95-treated cells. The same cluster of dead cells was clearly
detectable by both FSC/SSC and PI staining analyses. Viable cells,
gated in R1 (region 1), were examined for glycophorin A expression, as
shown in (B). (B) Cultures were supplemented with irrelevant IgM mAb
and rHis6-tag peptide (CONT), TRAIL, or anti-CD95 (CH 11) IgM
mAb for the last 20 hours before glycophorin A staining. X-axis:
glycophorin A relative expression. Y-axis: relative cell number.
Percentages of glycophorin A+ cells are indicated.
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At these late culture times (days 14-15), megakaryocytic, granulocytic,
and monocytic unilineage cultures were still virtually unaffected by
both apoptosis-inducing stimuli (Table 2).
TRAIL-induced inhibition of pre-stimulated BFU-E in semisolid
cultures
To establish whether TRAIL- and anti-CD95 CH11 mAb-treated
hematopoietic progenitors maintained the ability to form colonies, CD34+ cells were seeded in semisolid cultures either
immediately after purification or when they were first induced to
proliferate for 2 days with hematopoietic growth factors in liquid
culture and then treated for an additional 20 hours with TRAIL or
anti-CD95 CH11 mAb before performing semisolid cultures. As shown in
Figure 8A, the plating efficiency of
freshly isolated CD34+ cells was modestly affected by
either TRAIL or anti-CD95 mAb. Both TRAIL and anti-CD95 CH11
significantly (P < .05) decreased the number (Figure 8A)
and the size (Figure 8B) of BFU-E in pre-stimulated cultures. Moreover,
CFU-meg and CFU-GM were minimally affected by either TRAIL or anti-CD95
CH11 mAb in both experimental conditions (Figure 8A). These results
obtained in semisolid cultures show an apparent discrepancy with the
data illustrated in Figure 2A, in which we have found a lack of
cytotoxic effect of TRAIL on early erythroid progenitors in liquid
cultures. These differences can be explained by the fact that the
clonogenic assay, which occurs over 12-14 days of culture, is a more
sensitive indicator of early TRAIL-mediated cytotoxic effects than the
annexin assay. An alternative, not mutually exclusive, explanation is
that TRAIL, which is administered in semisolid cultures for 14 days
instead of 20 hours, shows a combination of apoptotic and
anti-differentiative effects, as previously shown for
anti-CD95,32 which are preferentially reveled by the
semisolid assay.
View larger version (2072K):
[in this window]
[in a new window]
| Fig 8.
TRAIL-induced inhibition on pre-stimulated clonogenic
erythroid progenitors (BFU-E) in semisolid cultures.
(A) Erythroid (BFU-E), megakaryocytic (CFU-meg), and
granulocytic-macrophagic (CFU-GM) colonies were scored after 14 days of
semisolid cultures. CD34+ cells were either seeded in
semisolid cultures immediately after purification (white columns) or
pre-stimulated in liquid cultures with specific cytokine cocktails for
2 days (black columns). Before seeding, cells were treated for 20 hours
with irrelevant IgM mAb and rHis6-tag peptide (CONT), TRAIL, or
anti-CD95 (CH 11) IgM mAb. Data are expressed as means ± SD of 4 separate experiments performed in duplicate. (B) Representative BFU-E
are shown to document the difference in terms of colony size between
TRAIL, CH11, and control cultures. Original magnification (400×).
|
|
Expression of TRAIL in human BM
To investigate the expression of TRAIL in hematopoietic tissues, we
next performed an immunoblot analysis on BM mononuclear cells, obtained
from 3 different donors, using an anti-TRAIL mAb. In whole cell lysates
obtained from 3 human BM cells, the anti-TRAIL mAb recognized a protein
with an apparent molecular mass of 32-33 kDa, which corresponds to the
membrane-bound form of TRAIL protein.13,14 Results are
shown in Figure 9.
View larger version (93K):
[in this window]
[in a new window]
| Fig 9.
Immunoblot analysis of TRAIL protein expression in human
BM mononuclear cells.
Whole cell lysates of human BM mononuclear cells from 3 different
donors were tested with anti-TRAIL antibody (clone B35-1). Molecular
mass markers are indicated (kDa).
|
|
| |
Discussion |
The most important cytokine controlling erythrocyte
production in vivo is EPO that functions mainly to increase the
survival of erythroid progenitor cells.29 Previous
studies,3,6-10,32 however, have implicated the CD95/CD95L
system in the negative regulation of erythroid differentiation in both
normal and pathophysiological conditions. In particular, the presence
of relatively immature apoptotic erythroblasts, surrounded by
macrophages phagocytosing apoptotic cells, has been described in vivo
under physiological conditions in erythroblastic islands of the
BM.9 It has been proposed that mature CD95L+
erythroblasts kill, through a negative feedback regulatory mechanism, immature CD95+ erythroblasts and that macrophages are
responsible for clearance of dead erythroid cells.9
However, lpr-mutant mice bearing a defective Fas
(CD95) gene do not show gross abnormalities in erythroid
development.33 Moreover, inhibition of
IFN--induced erythroid apoptosis was only partially
restored (notably large colonies have never been rescued) after
blockage of the CD95 pathway.8 Taken together, these
findings suggest that apoptotic pathways, other than the CD95/CD95L,
are involved in the control of erythropoiesis.
In the present report, we have demonstrated that TRAIL can
act, alternatively to CD95L, as a negative regulator of erythropoiesis. An interesting similarity between anti-CD95 mAb and TRAIL is that both
specifically affect glycophorin Ainterm but neither
immature glycophorindim nor mature glycophorin
Abright erythroblasts. Furthermore, neither CD95 agonists
nor TRAIL significantly affected cells differentiating along the
megakaryocytic, granulocytic, or monocytic pathways. Finally, the
addition of fresh EPO efficiently counteracted both the TRAIL- and
anti-CD95-mediated apoptosis of erythroid cells.
The importance of TRAIL as a physiological inhibitor of erythropoiesis
is underlined by the demonstration that TRAIL protein is expressed in
normal human BM. Both CD95L and TRAIL exist as full-length
membrane-bound molecules and as a shorter soluble form.13
At variance with CD95L that contains a long intracellular region of 81 amino acids, TRAIL has a very short intracellular tail of 17 amino
acids and appears regulated at the cell surface of different cell types
by a proteolytic event sensitive to cysteine protease
inhibitors.13,14
Although the source of TRAIL in the BM microenvironment remains to be
investigated, it has been previously shown that PB
monocytes/macrophages express and deploy CD95L34 and
produce TRAIL in response to IFN- or IFN-.35 Thus,
resident monocytes/macrophages are likely candidates for the production
of TRAIL at the BM levels, and it is conceivable that they play a
central role in erythroid differentiation, altering the balance between
survival factors (EPO) and apoptotic inducers (TRAIL and CD95L).
Moreover, because mature erythroid cells obtained in EPO-containing
liquid cultures after 10-14 days express TRAIL protein (data not
shown), it is possible that new BM erythroblasts also contribute to the
regulation of erythropoiesis through a negative feedback loop.
TRAIL has been involved in a variety of pathological conditions, mainly
as a mediator of inflammatory cytokines or
chemotherapy.18,35-45 In particular, much attention has
been focused on the ability of TRAIL to selectively kill cancer
cells.12,13,16,39-45 In this respect, the ability of TRAIL
to adversely affect normal erythropoiesis should be taken in
consideration in the prospective to employ TRAIL for anticancer
treatments in vivo,44,45 as proposed on the basis of the
supposed preferential sensitivity of cancer cells with respect to
normal cells to TRAIL-induced apoptosis. In this respect, in agreement
with our present data, Ashkenazi et al44 reported the
presence of mild anemia after in vivo TRAIL administration to nonhuman
primates. However, Walczak et al45 did not observe any
significant alteration in red and white blood cells in mice treated for
14 days with human TRAIL. This finding may be due to
the short-term observation time (14 days) that did not allow time to
detect a TRAIL-induced anemia, which, if inducible in vivo, should take
at least 1 month (life span of mouse erythrocytes). It is also possible
that mouse erythroblasts are more resistant than those of nonhuman
primates to human TRAIL.
Although we have not addressed the molecular mechanisms underlining the
sensitivity of glycophorin Ainterm erythroblasts to TRAIL,
previous studies (reviewed in Ashkenazi and Dixit5) have
demonstrated an extreme complexity of the expression and function of
TRAIL receptors in various cell types. In fact, at least 5 TRAIL
receptors belonging to the apoptosis-inducing TNF-receptor (R) family
have been described so far. TRAIL-R1 (DR4) and TRAIL-R2
(DR5)21,46 transduce apoptotic signals on binding of
TRAIL,11 whereas TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) as
well as osteoprotegerin (reviewed in Ashkenazi and Dixit5)
are homologous to DR4 and DR5 in their cysteine-rich extracellular
domain but lack intracellular death domain and apoptosis-inducing
capability. At first, TRAIL-R3 and TRAIL-R4 have been proposed to
function as decoy receptors, protecting normal cells from
apoptosis.40,41 More recently, it has been shown that
expression of TRAIL-R1 and/or TRAIL-R2 is necessary but not always
sufficient to mediate TRAIL-induced apoptosis, whereas expression of
TRAIL-R3 and/or TRAIL-R4 does not appear to be a significant factor in
determining the resistance or sensitivity of tumor target cells to the
effects of TRAIL.47,48 Similarly, the expression of CD95,
which contains a conserved intracytoplasmic "death
domain"49 indirectly responsible for activating the
caspases enzymatic cascade,50 does not always correlate
with its ability to transduce an apoptotic signal (this study).9,10 The inability of both TRAIL and CD95L to kill cells expressing TRAIL-R1 and/or TRAIL-R2 and CD95, respectively, may
be due to a high level of expression of Bcl-2 protein,51 or
other anti-apoptotic pathways, which are known to modulate the
sensitivity to apoptotic agonists.
In conclusion, our study provides new insights into the mechanisms
involved in the control of erythroid development, showing the presence
of redundancy in the TNF super-family of proteins in the negative
regulation of erythropoiesis. Like CD95L, TRAIL acts in a stage of
development-specific manner. Moreover, our findings predict that the
administration of erythroid protective cytokines, such as EPO, would be
beneficial as a strategy for long-term anticancer therapy, based on the
in vivo administration of TRAIL.
| |
Acknowledgment |
We are grateful to Kristi Bemis for excellent technical assistance.
| |
Footnotes |
Submitted November 12, 1999; accepted January 31, 2000.
Supported by Telethon funds (P.S.), by CNR grant 96.03134. CT04 (S.P.),
by NIH grant CA78890 (E.S.A.), and by local funds and MURST cofin of
the Universities of Urbino, Ferrara, Chieti, Bologna, and Brescia.
Reprints: Loris Zamai, Institute of Morphological Sciences,
University of Urbino, Campus Scientifico Località Crocicchia, 61029 Urbino, Italy; e-mail: zamai1{at}biocfarm.unibo.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Earnshaw WC.
Apoptosis: a cellular poison cupboard.
Nature.
1999;397:387[Medline]
[Order article via Infotrieve].
2.
Park JR.
Cytokine regulation of apoptosis in hematopoietic precursor cells.
Curr Opin Hematol.
1996;3:191[Medline]
[Order article via Infotrieve].
3.
Maciejewski J, Selleri C, Anderson S, Young NS.
Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro.
Blood.
1995;85:3183[Abstract/Free Full Text].
4.
Nagafuji K, Takenaka K, Shibuya T, Harada M, Niho Y.
Fas antigen (CD95) and hematopoietic progenitor cells.
Leuk Lymphoma.
1996;24:43[Medline]
[Order article via Infotrieve].
5.
Ashkenazi A, Dixit VM.
Apoptosis control by death and decoy receptors.
Curr Opin Cell Biol.
1999;11:255[Medline]
[Order article via Infotrieve].
6.
Niho Y, Asano Y.
Fas/Fas ligand and hematopoietic progenitor cells.
Curr Opin Hematol.
1998;5:163[Medline]
[Order article via Infotrieve].
7.
Maciejewski JP, Selleri C, Sato T, Anderson S, Young NS.
Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia.
Br J Haematol.
1995;91:245[Medline]
[Order article via Infotrieve].
8.
Dai CH, Price JO, Brunner T, Krantz SB.
Fas ligand is present in human erythroid colony-forming cells and interacts with Fas induced by interferon gamma to produce erythroid cell apoptosis.
Blood.
1998;91:1235[Abstract/Free Full Text].
9.
De Maria R, Testa U, Luchetti L, et al.
Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis.
Blood.
1999;93:796[Abstract/Free Full Text].
10.
Takenaka K, Nagafuji K, Harada M, et al.
In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95).
Blood.
1996;88:2871[Abstract/Free Full Text].
11.
Wiley SR, Schooley K, Smolak PJ, et al.
Identification and characterization of a new member of the TNF family that induces apoptosis.
Immunity.
1995;3:673[Medline]
[Order article via Infotrieve].
12.
Snell V, Clodi K, Zhao S, et al.
Activity of TNF-related apoptosis-inducing ligand (TRAIL) in haematological malignancies.
Br J Haematol.
1997;99:618[Medline]
[Order article via Infotrieve].
13.
Mariani SM, Matiba B, Armandola EA, Krammer PH.
Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells.
J Cell Biol.
1997;137:221[Abstract/Free Full Text].
14.
Mariani SM, Krammer PH.
Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage.
Eur J Immunol.
1998;28:973[Medline]
[Order article via Infotrieve].
15.
Jeremias I, Herr I, Boehler T, Debatin KM.
TRAIL/Apo-2-ligand-induced apoptosis in human T cells.
Eur J Immunol.
1998;28:143[Medline]
[Order article via Infotrieve].
16.
Herr I, Wilhelm D, Bohler T, Angel P, Debatin KM.
JNK/SAPK activity is not sufficient for anticancer therapy-induced apoptosis involving CD95-L, TRAIL and TNF-alpha.
Int J Cancer.
1999;80:417[Medline]
[Order article via Infotrieve].
17.
Martinez-Lorenzo MJ, Anel A, Gamen S, et al.
Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.
J Immunol.
1999;163:1274[Abstract/Free Full Text].
18.
Bretz JD, Rymaszewski M, Arscott PL, et al.
TRAIL death pathway expression and induction in thyroid follicular cells.
J Biol Chem.
1999;274:23627[Abstract/Free Full Text].
19.
Walczack H, Degli Esposti MA, Johnson RS, et al.
TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.
EMBO J.
1997;16:5386[Medline]
[Order article via Infotrieve].
20.
Rieger J, Ohgaki H, Kleihues P, Weller P.
Human astrocytic brain tumors express AP02L/TRAIL.
Acta Neuropathol.
1999;97:1[Medline]
[Order article via Infotrieve].
21.
MacFarlane M, Manzoor A, Srinivasula SM, Fernandes-Alnemri T, Cohen GM, Alnemri ES.
Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL.
J Biol Chem.
1997;41:25417.
22.
Zamai L, Ahmad M, Bennett IM, Azzoni L, Alnemri ES, Perussia B.
Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells.
J Exp Med.
1998;188:2375[Abstract/Free Full Text].
23.
Zauli G, Vitale M, Falcieri E, et al.
In vitro senescence and apoptotic cell death of human megakaryocytes.
Blood.
1997;90:2234[Abstract/Free Full Text].
24.
Zamai L, Mariani AR, Zauli G, et al.
Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry.
Cytometry.
1998;32:280[Medline]
[Order article via Infotrieve].
25.
Zauli G, Furlini G, Vitale M, et al.
A subset of human CD34+ hematopoietic progenitors express low levels of CD4, the high affinity receptor for human immunodeficiency virus-type 1 (HIV-1).
Blood.
1994;84:1896[Abstract/Free Full Text].
26.
Strauss LC, Bowley SD, La Russa VF, Sharkis SS, Stuart RK, Civin CI.
Antigen analysis of hematopoiesis: characterization of My-10 antigen expression by normal lymphohematopoietic progenitor cells.
Exp Hematol.
1986;14:878[Medline]
[Order article via Infotrieve].
27.
Loken MR, Shan VO, Dattilio KL, Civin CI.
Flow cytometric analysis of human bone marrow. I. Normal erythroid development.
Blood.
1987;69:255[Abstract/Free Full Text].
28.
Zauli G, Catani L.
Human megakaryocyte biology and pathophysiology.
Crit Rev Oncol Hematol.
1995;21:135[Medline]
[Order article via Infotrieve].
29.
Koury MJ.
Programmed cell death (apoptosis) in hematopoiesis.
Exp Hematol.
1992;20:391[Medline]
[Order article via Infotrieve].
30.
Koller MR, Bradley MS, Palsson BO.
Growth factor consumption and production in perfusion cultures of human bone marrow correlate with specific cell production.
Exp Hematol.
1995;23:1275[Medline]
[Order article via Infotrieve].
31.
Terstappen LW, Buescher S, Nguyen M, Reading C.
Differentiation and maturation of growth factor expanded human hematopoietic progenitors assessed by multidimensional flow cytometry.
Leukemia.
1992;6:1001[Medline]
[Order article via Infotrieve].
32.
De Maria R, Zeuner A, Eramo A, et al.
Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1.
Nature.
1999;401:489[Medline]
[Order article via Infotrieve].
33.
Perkins DL, Michaelson J, Marshak-Rothstein A.
The lpr gene is associated with resistance to engraftment by lymphoid but not erythroid stem cells from normal mice.
J Immunol.
1987;138:466[Abstract].
34.
Oyaizu N, Adachi Y, Hashimoto F, et al.
Monocytes express Fas ligand upon CD4 cross-linking and induce CD4 T cell apoptosis.
J Immunol.
1997;158:2456[Abstract].
35.
Griffith TS, Wiley SR, Kubin MZ, Sedger LM, Maliszewski CR, Fanger NA.
Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL.
J Exp Med.
1999;189:1343[Abstract/Free Full Text].
36.
Katsikis PD, Garcia-Ojeda ME, Torres-Roca JF, et al.
Interleukin-1 beta converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection: TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection.
J Exp Med.
1997;186:1365[Abstract/Free Full Text].
37.
Sedger LM, Shows DM, Blanton RA, et al.
IFN-gamma mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression.
J Immunol.
1999;163:920[Abstract/Free Full Text].
38.
Phillips TA, Ni J, Pan G, et al.
TRAIL (Apo-2L) and TRAIL receptors in human placentas: implications for immune privilege.
J Immunol.
1999;162:6053[Abstract/Free Full Text].
39.
Gura T.
How TRAIL kills cancer cells, but not normal cells.
Science.
1997;277:768[Free Full Text].
40.
Pan G, O'Rourke K, Chinnaiyan AM, et al.
The receptor for the cytotoxic ligand TRAIL.
Science.
1997;276:111[Abstract/Free Full Text].
41.
Sheridan JP, Marsters SA, Pitti RM, et al.
Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.
Science.
1997;277:818[Abstract/Free Full Text].
42.
Sheikh MS, Burns TF, Huang Y, et al.
p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha.
Cancer Res.
1998;58:1593[Abstract/Free Full Text].
43.
Keane MM, Ettenberg SA, Nau MM, Russell EK, Lipkowitz S.
Chemotherapy augments TRAIL-induced apoptosis in breast cell lines.
Cancer Res.
1999;59:734[Abstract/Free Full Text].
44.
Ashkenazi A, Pai RC, Fong S, et al.
Safety and antitumor activity of recombinant soluble Apo2 ligand.
J Clin Invest.
1999;104:155[Medline]
[Order article via Infotrieve].
45.
Walczak H, Miller RE, Ariail K, et al.
Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.
Nat Med.
1999;5:157[Medline]
[Order article via Infotrieve].
46.
Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L.
Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate NF-kappaB.
Immunity.
1997;7:821[Medline]
[Order article via Infotrieve].
47.
Zhang XD, Franco A, Myers K, Gray C, Nguyen T, Hersey P.
Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma.
Cancer Res.
1999;59:2747[Abstract/Free Full Text].
48.
Griffith TS, Rauch CT, Smolak PJ, et al.
Functional analysis of TRAIL receptors using monoclonal antibodies.
J Immunol.
1999;162:2597[Abstract/Free Full Text].
49.
Nagata S, Golstein P.
The Fas death factor.
Science.
1995;5:1449.
50.
Cohen GM.
Caspases: the executioners of apoptosis.
Biochem J.
1997;326:1.
51.
Iwai K, Miyawaki T, Takizawa T, et al.
Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils.
Blood.
1994;84:1201[Abstract/Free Full Text].
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|
U. Schmidt, E. van den Akker, M. Parren-van Amelsvoort, G. Litos, M. de Bruijn, L. Gutierrez, R. W. Hendriks, W. Ellmeier, B. Lowenberg, H. Beug, et al.
Btk Is Required for an Efficient Response to Erythropoietin and for SCF-controlled Protection against TRAIL in Erythroid Progenitors
J. Exp. Med.,
March 15, 2004;
199(6):
785 - 795.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Secchiero, E. Melloni, M. Heikinheimo, S. Mannisto, R. Di Pietro, A. Iacone, and G. Zauli
TRAIL regulates normal erythroid maturation through an ERK-dependent pathway
Blood,
January 15, 2004;
103(2):
517 - 522.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Greil, G. Anether, K. Johrer, and I. Tinhofer
Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools
J. Leukoc. Biol.,
September 1, 2003;
74(3):
311 - 330.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Secchiero, D. Milani, A. Gonelli, E. Melloni, D. Campioni, D. Gibellini, S. Capitani, and G. Zauli
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-{alpha} promote the NF-{kappa}B-dependent maturation of normal and leukemic myeloid cells
J. Leukoc. Biol.,
August 1, 2003;
74(2):
223 - 232.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. H. Li, N. C. Kirkiles-Smith, J. M. McNiff, and J. S. Pober
TRAIL Induces Apoptosis and Inflammatory Gene Expression in Human Endothelial Cells
J. Immunol.,
August 1, 2003;
171(3):
1526 - 1533.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. J. Sayers, A. D. Brooks, C. Y. Koh, W. Ma, N. Seki, A. Raziuddin, B. R. Blazar, X. Zhang, P. J. Elliott, and W. J. Murphy
The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP
Blood,
July 1, 2003;
102(1):
303 - 310.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Komatsu, M. Mammolenti, M. Jones, R. Jurecic, T. J. Sayers, and R. B. Levy
Antigen-primed CD8+ T cells can mediate resistance, preventing allogeneic marrow engraftment in the simultaneous absence of perforin-, CD95L-, TNFR1-, and TRAIL-dependent killing
Blood,
May 15, 2003;
101(10):
3991 - 3999.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Hietakangas, M. Poukkula, K. M. Heiskanen, J. T. Karvinen, L. Sistonen, and J. E. Eriksson
Erythroid Differentiation Sensitizes K562 Leukemia Cells to TRAIL-Induced Apoptosis by Downregulation of c-FLIP
Mol. Cell. Biol.,
February 15, 2003;
23(4):
1278 - 1291.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Secchiero, A. Gonelli, G. Ciabattoni, E. Melloni, V. Grill, B. Rocca, G. Delbello, and G. Zauli
TNF-related apoptosis-inducing ligand (TRAIL) up-regulates cyclooxygenase (COX)-1 activity and PGE2 production in cells of the myeloid lineage
J. Leukoc. Biol.,
November 1, 2002;
72(5):
986 - 994.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Secchiero, A. Gonelli, P. Mirandola, E. Melloni, L. Zamai, C. Celeghini, D. Milani, and G. Zauli
Tumor necrosis factor-related apoptosis-inducing ligand induces monocytic maturation of leukemic and normal myeloid precursors through a caspase-dependent pathway
Blood,
September 18, 2002;
100(7):
2421 - 2429.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. A. Papadaki, H. D. Kritikos, V. Valatas, D. T. Boumpas, and G. D. Eliopoulos
Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-alpha antibody therapy
Blood,
June 28, 2002;
100(2):
474 - 482.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Y. Zang, R. G. Goodwin, M. R. Loken, E. Bryant, and H. J. Deeg
Expression of tumor necrosis factor-related apoptosis-inducing ligand, Apo2L, and its receptors in myelodysplastic syndrome: effects on in vitro hemopoiesis
Blood,
November 15, 2001;
98(10):
3058 - 3065.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. T. Jones, K. Ganeshaguru, A. E. Virchis, N. I. Folarin, M. W. Lowdell, A. B. Mehta, H. G. Prentice, A. V. Hoffbrand, and R. G. Wickremasinghe
Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation
Blood,
November 1, 2001;
98(9):
2800 - 2807.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Secchiero, P. Mirandola, D. Zella, C. Celeghini, A. Gonelli, M. Vitale, S. Capitani, and G. Zauli
Human herpesvirus 7 induces the functional up-regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) coupled to TRAIL-R1 down-modulation in CD4+ T cells
Blood,
October 15, 2001;
98(8):
2474 - 2481.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Secchiero, A. Gonelli, C. Celeghini, P. Mirandola, L. Guidotti, G. Visani, S. Capitani, and G. Zauli
Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies
Blood,
October 1, 2001;
98(7):
2220 - 2228.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Matsushima, M. Nakashima, K. Oshima, Y. Abe, J. Nishimura, H. Nawata, T. Watanabe, and K. Muta
Receptor binding cancer antigen expressed on SiSo cells, a novel regulator of apoptosis of erythroid progenitor cells
Blood,
July 15, 2001;
98(2):
313 - 321.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Di Pietro, P. Secchiero, R. Rana, D. Gibellini, G. Visani, K. Bemis, L. Zamai, S. Miscia, and G. Zauli
Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by selective up-regulation of TRAIL-R1
Blood,
May 1, 2001;
97(9):
2596 - 2603.
[Abstract]
[Full Text]
[PDF]
|
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Top
Abstract
Introduction