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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3742-3749
HEMATOPOIESIS
Interferon gamma delays apoptosis of mature erythroid progenitor
cells in the absence of erythropoietin
Ilseung Choi,
Koichiro Muta,
Amittha Wickrema,
Sanford B. Krantz,
Junji Nishimura, and
Hajime Nawata
From the Department of Medicine and Bioregulatory Science, Graduate
School of Medical Science, Kyushu University, Fukuoka, Japan; Stem
Cell/Bone Marrow Processing Laboratory, Hematology/Oncology Section,
University of Illinois; Department of Medicine-Hematology/Oncology,
Vanderbilt University School of Medicine; and Department of Clinical
Immunology, Medical Institute of Bioregulation, Kyushu University,
Beppu, Japan.
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Abstract |
Based on the hypothesis that interferon gamma (IFN- ) may have
stimulating effects on survival of hematopoietic progenitor cells, we
examined the effect of IFN- on apoptosis of mature erythroid
colony-forming cells (ECFCs) derived from human peripheral blood obtained from normal, healthy volunteers. When the cells were
cultured in the presence of IFN-, even without erythropoietin (EPO),
the viability of the cells was maintained for at least 36 hours. When
apoptosis of ECFCs was assessed by flow cytometric analysis', using
annexin V, IFN- reduced the extent of apoptosis of the cells, as
well as EPO. DNA fragmentation of ECFCs was also reduced by IFN-. In
cells cultured with IFN- alone, expression of Bcl-x was detected but
the level of expression decreased gradually during incubation for 36 hours, and the expression level was lower than incubation with EPO. Fas
expression and activation of downstream caspases were assessed by flow
cytometric analysis or fluorometric protease assay. IFN- induced Fas
expression of the cells without the activation of caspase8 or caspase3
during 16 hours of incubation, while deprivation of EPO induced
expression of Fas and the activation of both caspase8 and caspase3. We
propose that IFN- produces a stimulating signal for the survival of
mature erythroid progenitor cells by reducing apoptosis through a
mechanism other than modulating Fas and one related to the expression
of Bcl-x.
(Blood. 2000;95:3742-3749)
© 2000 by The American Society of Hematology.
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Introduction |
Interferon gamma (IFN-), produced by activated T
cells and by natural killer cells,1 is a potent inhibitor
of hematopiesis.2 This cytokine is believed to play a
crucial role in the pathophysiology of hematopoietic disorders
associated with bone marrow failure such as aplastic
anemia3,4 and hemophagocytic syndrome.5 It has
been demonstrated that IFN- inhibits in vitro colony formation by
bone marrow- and blood-derived hematopoietic progenitor
cells,6-12 and the inhibitory effects of IFN- on
hematopoietic cells are apparently due to inhibition of cell cycle
progression10 or induction of apoptosis.2,11
Apoptosis was facilitated by upmodulation of Fas
expression both in CD34+ cells7 and blood-derived immature erythroid colony forming cells (ECFCs).6 It has been
reported that IFN- reduces receptors for stem cell factor (SCF) and
erythropoietin (EPO) in immature ECFCs,8 and these
receptors, when activated, normally prevent apoptosis of
ECFCs.13 This suggests that IFN- inhibits growth of
hematopoietic progenitor cells mainly by enhancing apoptosis.
Although numerous studies have focused on the inhibitory effect of
IFN- on hematopoiesis, IFN- also has been reported to stimulate
growth of hematopoietic cells.14-16 IFN- increased the number of blood CD34+ cells expanded ex vivo when added together with
SCF, interleukin-1 (IL-1), IL-3, IL-6, and EPO,14
and IFN- enhanced the colony formation induced by IL-3 in
purified CD34+ cells.15 These contradictory findings
regarding IFN- action may depend on the stage of maturation of the
hematopoietic progenitors16 and on differences in growth
factors added to the cultures.
EPO, the principal growth factor for erythroid progenitor cells,
maintains the viability of these cells by allowing them to undergo the
process of proliferation and
maturation.13,17-21 It has been demonstrated
that deprivation of EPO-induced apoptosis of ECFCs occurs through
down-regulation of Bcl-x,22 a member of the Bcl-2 family
known as an important regulator of apoptosis in various cell
systems.23-25 The Fas-Fas ligand system also
has a role in apoptosis of erythroid progenitor cells6
through deprivation of EPO-induced activation of
apopain/caspase3,22 a cystein protease which acts as a
downstream signal mediator in the apoptosis induced by Fas
activation.26-28
We examined the effects of IFN- on apoptosis of mature erythroid
progenitor cells (day 7 ECFCs), and found that IFN-, as well as EPO,
can prevent apoptosis of mature ECFCs through mechanisms other than
modulating expression of Fas, and is related to the expression of
Bcl-x.
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Materials and methods |
Reagents
Recombinant human erythropoietin (rhEPO) was kindly provided by
Chugai Pharmaceutical Co Ltd (Tokyo, Japan); recombinant human interleukin3 (rhIL-3) and recombinant human stem cell factor (rhSCF) were kindly provided by Kirin-Brewery Co Ltd (Tokyo, Japan);
recombinant human interferon gamma 1a (rhIFN-) was kindly provided
by Shionogi Co Ltd (Osaka, Japan). rhIFN- inhibits the proliferation
of A-498 cells, which are derived from human renal cell carcinoma, by
50% at a concentration of 2.5 U/mL (data not shown).
Anti-IFN- antibody (catalog number 955000010) and
mouse anti-IgG1 antibody (catalog number 857070000) were purchased from
Life Technologies, Inc (Rockville, MD). Rabbit anti Bcl-x antibody
(B22 630) was purchased from Transduction Laboratories (Lexington,
KY). Rabbit anti-Bax antibody was purchased from Santa Cruz
Biotechnology, Inc (Santa Cruz, CA). Mouse antiactin antibody (N350)
was from Amersham Life Science (Buckinghamshire, England). Horseradish
peroxidase (HRP) conjugated antirabbit (NA934) and mouse (NA931) whole
Ig secondary antibodies were from Amersham.
Generation of ECFCs
ECFCs were prepared using a modified method described by Sawada et
al.17,18,29 Light-density mononuclear cells were obtained from 40 mL of heparinized peripheral blood buffy coat
from healthy Japanese volunteers by density centrifugation using
lymphocyte separation medium (LSM, density 1.0770-1.0800 g/mL; ICN
Biomedicals, Aurora, OH). Red blood cells were lysed by
suspending the mononuclear cell pellet in red cell lysis buffer (0.16 mol/L ammonium chloride, 10 mmol/L potassium bicarbonate,
5 mmol/L EDTA). Platelets were removed by cell centrifugation through
phosphate-buffered saline (PBS) containing 10% human serum albumin
(HSA, kindly provided by the Chemo-Sero-Therapeutic Research Institute,
Kumamoto, Japan). Adherent cells were depleted by a 1-hour incubation
in a polystyrene tissue-culture flask at 4°C. Nonadherent cells
were then collected and 2 cycles of negative selection were performed
using anti-CD3, -CD11b, -CD15 and -CD45RA antibodies and
immunomagnetic beads with Vario-Macs columns (Miltenyi Biotech, Auburn,
CA). The remaining cells were then cultured in Iscove modified Dulbecco
medium (IMDM; GIBCO BRL, Grand Island, NY) containing 15%
heat-inactivated fetal calf serum (FCS; Commonwealth Serum
Laboratories, Melbourne, Australia), 15% pooled human AB serum, 2 U/mL
EPO, 20 ng/mL SCF, 10 ng/mL IL-3, 100 U/mL penicillin, and 100 µg/mL
streptomycin (GIBCO) at 37°C in a high-humidity, 5%
CO2, 95% air incubator (day 0). On day 3, the cells,
referred to as day 3 ECFCs, were centrifuged over LSM, then collected
and incubated under the same conditions, but without IL-3. The cultured
cells were collected on day 7, referred to as day 7 ECFCs, and used in
the following experiments. The purity of the day 7 ECFCs, with
proerythroblastlike features, was 95% ± 3%, as determined in
cytospin preparations. Cell purity was assessed in each experiment.
Serum-free liquid cultures of ECFC
The cells (day 7 ECFCs, 2 × 105 cells/mL) were
incubated in serum-free liquid medium containing 50% IMDM/50% F-12
medium (Sigma Chemical Co, St Louis, MO) with 1% detoxified bovine
serum albumin (BSA; Stem Cell Technologies Inc, Vancouver, BC), 300 µg/mL iron-saturated transferrin (652202; Boehringer Mannheim,
Mannheim, Germany), lipid suspension (oleic acid, 2.8 µg/mL; L- -phosphatidylcholine, 4.0 µg/mL; cholesterol, 3.9 µg/mL, Sigma), and prepared as described30 with 100 U/mL
penicillin and 100 µg/mL streptomycin at 37°C in a high-humidity,
5% CO2, 95% air incubator. We added rhEPO
and rhIFN- as indicated.
Determination of cell viability
Viability of the cells was determined by trypan blue exclusion using
a hemocytometer.
Plasma clot assay
Erythroid colony-forming capacity of ECFC was determined by the
plasma clot method.13 A total of 1 mL medium consisting of
IMDM, 20% FCS, 1% BSA, 10 ng/mL SCF, rhEPO as indicated, and 10%
pooled citrated human AB plasma containing 600 cells were plated on 3 35-mm culture dishes and incubated at 37°C in a high-humidity, 5%
CO2, 95% air incubator for 7 days. The clots were then
fixed and stained with 3,3' dimethoxybenzidine. The colonies of 8 or more hemoglobinized cells were defined as colony-forming
unit-erythroid (CFU-E), and aggregates consisting of 2 to 7 hemoglobinized cells were defined as small erythroid clusters. The
colonies consisting of 8 to 19 hemoglobinized cells were referred as to
medium erythroid colonies, and those consisting of 20 to 49 hemoglobinized cells were referred to as large erythroid
colonies.17
Apoptosis assay
Apoptosis was assessed by measuring membrane redistribution of
phosphatidilserine31 using an annexin
V-fluorescein-5-isothiocyanate (FITC) apoptosis
detection kit (Immunotech, Marseille, France) according to the
manufacturer's protocol. Briefly, cells were washed twice with PBS and
incubated for 30 minutes on ice in 500 µL binding buffer containing
FITC-conjugated annexin V antibody (5 µL) and propidium iodide (PI, 5 µL of 250-µg/mL stock). Cells were analyzed on the Epics Elite ESP
flow cytometer (Coulter Co, Miami, FL). The annexin V-positive
fraction was detected as apoptotic.
Analysis of DNA breakdown
To quantitate breakdown of cellular DNA during apoptosis, the amount
of fragmented DNA was measured by a modified method, as previously
described.13 Day 7 ECFCs were preincubated in IMDM
containing 30% FCS and 0.25% HSA at 37°C for 30 minutes. [3H]thymidine
(1.85 × 10-2 MBq/mL; 0.2479 MBq/mmol;
New England Nuclear Corp, Boston, MA) was added to 106
cells/mL and further incubation at 37°C was carried out for more than 30 minutes. The cells were collected, resuspended in 2 mL IMDM,
and carefully layered over 2 mL PBS containing 10% BSA for centrifugation at 1000g, 4° C for 5 minutes. Replicated
cells (5 × 105) were then transferred to 1 mL of
serum-free medium, as described above. To block further incorporation
of [3H]thymidine, thymidine and
deoxycytidine (20 µ mol/L; Sigma) were added. IFN- was added as
indicated. After culture in serum-free medium for 16 hours, the cell
replicates were collected and lysed in 1 mL of 50 mmol/L
Tris-HCl, pH 8.0, containing 10 mmol/L sodium chloride
(NaCl), 20 mmol/L EDTA, 0.5% sodium dodecyl sulfate
(SDS), and proteinase K (200 µ g/mL; GIBCO), followed
by incubation overnight at 37°C. DNA was extracted with
phenol:Chloroform (1:1 vol/vol), precipitated in ethanol,
and dissolved in 30 µL of 10 mmol/L Tris-HCl, pH 8.0, containing 1 mmol/L EDTA. The DNA samples were electrophoretically separated on alkaline pH, 0.6% agarose gels. Each lane was cut into
sixteen 5 mm fractions, and the radioactivity of each fraction was
expressed as a percent of the total radioactivity in each lane. The sum
of the radioactivity in fractions 5 through 16 was considered as the
amount of fragmented DNA, and was expressed as a percentage
of the total radioactivity.
Protein sample preparation and Western blotting
The cells (1 × 106) were lysed in buffer
containing 62.5 mmol/L Tris-HCl (pH 6.8), 100 mmol/L
dithiothreitol, 2% (wt/vol) SDS, and 10%
Glycerol.32 Cellular proteins (80 µg) were
separated by 12.5% sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS PAGE) and transferred to polyvinylidene difluoride
membranes (Immobilon, IPVH00010; Millipore, Bedford, MA).
The membranes were blocked in PBS-containing 0.1% Tween 20 (PBST) with
5% skim milk. The membranes were washed 3 times with PBST, then were
incubated in PBST containing 5% skim milk with anti-Bcl-x or
Bax antibody as a primary antibody at room temperature
for 1 hour. The membranes were then washed with PBST and incubated with
HRP conjugated secondary antibody (rabbit Ig). Specific signals were
detected on X-ray films using an enhanced chemiluminescence detection
system (ECL, PRN2106; Amersham). To remove the antibodies, the
membranes were incubated in 0.0625 mol/L Tris-HCl (pH
6.8) and 2% SDS at 50°C for 30 minutes and were reblotted first
with mouse antiactin antibody and then with HRP mouse Ig as the
secondary antibody. Specific signals were detected as described above.
K562 cells served as positive controls.
Detection of Fas by flow cytometry
The cells (1 × 106) were washed twice with PBS
and suspended in 0.1 mL PBS, then incubated with either FITC-conjugated
murine anti-Fas monoclonal antibody (UB2, 1506; Immunotech) at 0.2 µL or FITC-conjugated murine IgG1 (349041 Becton Dickinson, San Jose, CA) on ice for 30 minutes as a control. PBS
(400 µL) was added and analysis was performed on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA).
Evaluation of caspase3 activity
Activation of caspase3 was assessed using the PhiPhiLux G1D2
caspase3 activity detection kit (AK304R1G; Oncoimmunin Inc, College Park, MD), according to the manufacturer's instructions.
Briefly, 1 × 106 cells were washed twice with PBS,
and 50 µL of substrate solution (10 µmol/L; GDEVDGI)
was added, followed by incubation for 60 minutes in a 5%
CO2, 95% air incubator at 37°C. Five hundred
microliters of cold-flow cytometry solution were added to each sample,
followed by analysis using a FACScan flow cytometer at 488 nm FL1 channel.
Evaluation of capase8 activity
Activation of caspase8 was evaluated using a FLICE/caspase8
fluorometric protease assay kit (BV-K112; Medical and Biological Laboratories Co, Ltd, Nagoya, Japan) according to the
manufacturer's instructions. Briefly, lysates from
1 × 106 cells were incubated with fluorogenic
substrate IETDAFC for 60 minutes at 37°C in buffer
containing 5 mmol/L dithiothreitol. Samples were then
analyzed using an ARVO multilabel counter (Wallac Oy, Turku, Finland)
at 535 nm.
Statistical analysis
The t test was used to determine significant differences
between the groups.
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Results |
Effects of IFN- on viability of ECFCs
To determine the effect of EPO and IFN- on survival of ECFCs, we
examined the viability of these cells in serum-free liquid culture in
the presence or absence of either rhEPO or rhIFN-, or
both (Figure 1A). While the
viability of ECFCs rapidly decreased without rhEPO and rhIFN-
(22.9% ± 7.0%, mean ± SD of triplicates at 16 hours), in
the presence of rhEPO (10 U/mL), the viability was maintained
(61.8% ± 2.1% at 72 hours). When the cells were incubated with
rhIFN- (1000 U/mL) alone, viability of the cells was significantly
greater than that seen without rhEPO for at least 36 hours of culture
(45.9% ± 10.8% vs 7.4% ± 1.0%, P < .01 at
36 hours). In the cultures with rhEPO plus rhIFN-, viability of the
cells was similar to that seen with rhEPO alone (59.5% ± 9.0%
vs 61.8% ± 2.1%, P = .70 at 72 hours).
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| Fig 1.
Effect of IFN- on day 7 ECFCs.
(A) Day 7 ECFCs were cultured with or without rhEPO (10 U/mL) and/or
rhIFN- (1000 U/mL) for the indicated time. Viability of the cells
was determined by trypan blue exclusion. Each point indicates the
mean ± SD of triplicates. (B) Erythroid colony-forming capacity
after liquid culture for 16 hours with or without rhEPO (10 U/mL) or
rhIFN- (1000 U/mL), or both, was determined by plasma
clot assay. (C) Colony-forming capacity of day 7 ECFCs. Cells were
cultured in plasma clots, in the presence or absence of rhEPO (10 U/mL)
or rhIFN- (1000 U/mL), or both. Large erythroid
colonies include more than 20 hemoglobinized cells per colony; medium
erythroid colonies include 8 to 19 cells per colony; and small
erythroid clusters include 2 to 7 hemoglobinized cells per aggregate,
(B) and (C). Each point indicates the mean of triplicate
studies.
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IFN- maintains colony-forming capacity of ECFCs
To determine if IFN- alone could maintain colony-forming capacity
during liquid culture of ECFCs, plasma-clot assays were performed.
After 16 hours of incubation, with or without rhIFN- or rhEPO or
both, in serum-free liquid medium, the cells were transferred to plasma-clot cultures containing rhEPO (2 U/mL), and incubations were carried out for another 7 days (Figure 1B). Addition of rhIFN-
alone resulted in maintenance of a greater colony-forming capacity than
that seen without additives. However, the number of large- and
medium-sized colonies formed from the cells incubated with rhIFN-
alone was significantly smaller than that seen in the culture with
rhEPO alone (35.0 ± 10.8 and 76.7 ± 14.6,
P < .05, mean colony number from 200 cells ± SD of
triplicates, respectively). Addition of rhIFN- plus rhEPO resulted
in a significant decrease of large- and medium-sized colony-forming
capacity (36.6 ± 9.2, P < .05) compared to that seen
with rhEPO alone. Instead, addition of rhIFN- resulted in a greater
increase in the number of small erythroid clusters, both in the
presence and absence of rhEPO, than in those without
rhIFN-. Therefore, no significant difference was
present in the sum of the number of large- and medium-sized
colonies plus small erythroid clusters among cultures with or without rhIFN-, both in the presence and absence of rhEPO.
To determine if IFN- could substitute for EPO during erythroid
proliferation and maturation, rhIFN- was added to plasma clots at
the beginning of the cultures of day 7 ECFCs. As can be seen from
Figure 1C, addition of rhIFN- alone could not support formation of
medium- and large-sized erythroid colonies. However, a larger number of
small erythroid clusters did form with rhIFN- alone than was seen
with no additives (no additives, 7.0 ± 10.4; rhIFN- alone,
28.3 ± 17.0, P = 0.14). Addition of rhIFN- together with rhEPO resulted in a reduced number of large- and medium-sized colonies, but the sum of the number of clusters and colonies was similar to that seen with rhEPO alone (116 ± 9.3 of rhEPO alone and 106 ± 12.8 of both additives, P = 0.31), since the
number of small erythroid clusters increased in the presence of
rhIFN-.
IFN--reduced apoptosis of ECFCs
EPO maintains viability of these cells by reducing apoptosis. Since
IFN- maintained viability of the cells for up to 36 hours, as did
EPO, we wondered if IFN- would reduce
the apoptosis of ECFCs. Experiments were conducted using serum-free
medium to exclude the effect of unknown factors in serum, and apoptosis
was measured by flow cytometry using annexin V as described.
Annexin V binds the membrane phospholipid phosphatidylserine (PS),
which is externalized from the inner to the outer leaflet of plasma
membrane in the early stage of apoptosis. When membrane integrity is
lost, as seen in the latter stage of cell death resulting from either the apoptotic or necrotic processes, propidium iodide (PI)
staining becomes positive. According to the results of our time-course
study, annexin V- and PI-double-positive cells gradually increased
during incubation of ECFCs without rhEPO and rhIFN- (data not
shown). When we determined the ratio of the later stage of apoptosis to
the earlier stage of apoptosis, there were no significant differences
among various treatments (data not shown). Therefore, it was evident
that annexin V-positive and PI-negative cells (earlier
stage of apoptosis) and positive cells (later stage of apoptosis) were
apoptotic cells.
As shown in Figure 2A, when the cells were
incubated for 16 hours without rhEPO (10 U/mL) and rhIFN- (1000 U/mL), annexin V-positive cells were 66.9% ± 13.2%
(mean ± SD of 12 replicates from 4 independent experiments).
Addition of rhIFN- alone significantly reduced the number of annexin
V-positive cells (34.0% ± 8.8%, P < .01), compared
to that seen without rhEPO and rhIFN-. Addition of rhEPO alone
significantly reduced the number of annexin V-positive cells
(20.8% ± 4.5%, P < .01), and addition
of rhIFN- together with rhEPO further significantly reduced the
number of annexin V-positive cells (16.7% ± 4.9%,
P < .05), compared to that seen with rhEPO alone.
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| Fig 2.
Effect of IFN- on apoptosis of ECFCs.
(A) Day 7 ECFCs were incubated in serum-free medium without rhEPO and
rhIFN- (upper left), with rhEPO (10 U/mL) (upper right), with
rhIFN- (1000 U/mL) (lower left), and both (lower right). After
incubation for 16 hours, apoptosis was measured with PI and annexin V,
using a flow cytometer. Data shows typical results of 12 replicates
from 4 experiments. In each panel, the right lower quadrant (annexin
V-positive and PI negative) indicates early apoptosis, and the right
upper quadrant (annexin V- and PI positive) indicates late apoptosis.
Both annexin V-positive fractions were assessed as apoptotic cells.
(B) The day 7 ECFCs were labeled with
[3H]thymidine and cultured in serum-free
medium without rhIFN- (left), or with rhIFN- (1000 U/mL) (right),
for 16 hours. Cellular DNA was isolated and analyzed by alkaline pH,
0.6% agarose gel electrophoreses. The sum of the radioactivity of
fractionations 1-4 is designated as uncleaved DNA and is expressed as a
percentage of the total radioactivity.
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Apoptosis was qualitatively confirmed by agarose gel electrophoreses of
cellular DNA using [3H]thymidine. As shown
in Figure 2B, when ECFCs were cultured for 16 hours without rhEPO, high
molecular weight DNA was cleaved into small fragments. When cells were
cultured with rhIFN-, the amount of DNA fragmentation was greatly
reduced (71% of uncleaved DNA, sum of fraction 1 to 4, with rhIFN- and 19% with no additives).
To confirm whether IFN- maintains viability of mature ECFCs and
prevents them from apoptosis, neutralizing experiments were performed
using anti-IFN- antibody (Table 1). When
neutralizing antibody was added together with IFN-, the protective
effect of IFN- on apoptosis of ECFCs was nil, and both the viability and the number of apoptotic cells were similar to the experiment without IFN-.
When different concentrations of rhIFN- were added to the cultures,
a dose-dependent reduction of apoptosis was evident, and significant
suppression of apoptosis was obtained with a concentration of 10 U/mL
(P < .01, Figure 3A).
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| Fig 3.
Effect of IFN- on ECFCs with various concentrations of
rhIFN- and rhEPO.
(A) Dose-dependent effects of IFN- on reducing apoptosis of day 7 ECFCs. Cells were incubated for 16 hours with the indicated
concentrations of rhIFN-, without rhEPO. (B) Cells were incubated
with the indicated concentrations of rhEPO or rhIFN- (1000 U/mL), or
both. Each point shows the mean ± SD of
triplicates. Apoptosis was evaluated by flow cytometry after staining
cells with annexin V and PI.
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To determine the effect of IFN- under more physiologic conditions,
we examined the effect of IFN- in the presence of low concentrations
of EPO. As shown in Figure 4B, when cells were incubated with rhIFN-
(1000 U/mL) plus various concentrations of rhEPO, suppression of
apoptosis of ECFCs was evident in cultures with a lower concentration
of rhEPO.
Effects of IFN- on expression of Bcl-2-family proteins
To determine the mechanism by which IFN- influences apoptosis of
ECFCs, we examined the extent of expression of Bcl-2-family proteins
(Bcl-x, Bax, and Bcl-2) using Western blotting (Figure 4A). When ECFCs were incubated without
rhEPO and rhIFN- for the indicated times, Bcl-x was not detected.
When the cells were incubated with rhIFN- alone, Bcl-x was detected
but the level decreased gradually during incubation for 36 hours. As
can be seen from Figure 4B, the level of Bcl-x in the cells cultured
with rhIFN- was lower than that of cells cultured with rhEPO alone
or rhEPO and IFN-. When cells were cultured with rhEPO alone,
expression of Bcl-x was maintained, and addition of rhIFN- together
with rhEPO did not affect the expression of Bcl-x (Figure
4A, right). The expression levels of Bax
showed no difference among variables (Figure 4A), and
Bcl-2 was not detected in the erythroid progenitors (data not shown).
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| Fig 4.
Effect of IFN- on expression of Bcl-x and Bax.
Expression of Bcl-x and Bax was evaluated by Western blot
analysis. (A) Cells were incubated with or without rhEPO (10 U/mL) or rhIFN- (1000 U/mL), or both, for the
indicated time, and the same amount of whole cell lysates was loaded.
The upper panel shows the expression of Bcl-x, and the lower
panel shows the expression of Bax. (B) Lysates from cells incubated for
16 hours with or without rhEPO or IFN-, or both, were
loaded.
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Effect of IFN- on expression of Fas and activation of
caspases
Since the Fas-Fas ligand system mediates the apoptotic signal from
various stimuli, we examined the effect of IFN- on Fas expression of
ECFCs by flow cytometry. When the cells were incubated for 16 hours
without rhEPO and rhIFN-, Fas was expressed on 20.2% of the cells
(Figure 5B), a value exceeding that seen
with rhEPO alone (10.8%, Figure 5C). When
rhIFN- was added to the cultures, the percentage of cells that
expressed Fas increased further both in the absence or presence of EPO
(29.7% and 23.2% respectively, Figures 5D
and E). The activities of caspase3 and caspase8, downstream mediators
of apoptotic signaling induced by activation of Fas, were determined by
flow cytometry and fluorometric protease assay, respectively (Figures
6 and 7). When the cells were incubated with rhEPO alone, the activity of caspase3 was less than that seen
without rhEPO and rhIFN- (Figure 6A and
B). Addition of rhIFN- alone for 16 hours reduced the
activity of caspase3, as did rhEPO alone(Figure
6C), and addition of rhIFN- together
with rhEPO further reduced the activity of caspase3 at a greater rate than that seen with each additive alone. The
response of caspase8 to rhIFN- was similar to that seen in caspase3
(Figure 7). IFN- as well as EPO reduced
the activity of caspase8 at a greater rate than that seen without these
additives. Addition of rhIFN- plus rhEPO resulted in
less caspase8 activity than was seen using each additive
alone.
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| Fig 5.
Effect of IFN- on expression of Fas.
Data show typical results of 3 independent experiments. Panel (A) shows
the baseline expression of Fas on day 7 ECFCs, prior to serum-free
liquid culture. Cells were incubated with or without rhEPO (10 U/mL) or
rhIFN- (1000 U/mL), or both, for 16 hours. Cells were
then incubated on ice with FITC-Fas or FITC-murine IgG1
as control for 30 minutes, and then flow cytometric analysis was
performed. The open histogram indicates murine IgG1 as control, and the
gray histogram indicates Fas. Each panel shows data on cells incubated
without rhEPO and rhIFN- (B), with rhEPO alone (C), with rhIFN-
alone (D), and together with rhEPO plus rhIFN- (E).
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| Fig 6.
Effect of IFN- on activation of caspase3.
Data show typical results of 3 independent experiments. Cells were
incubated with or without rhEPO (10 U/mL) or rhIFN- (1000 U/mL), or
both, in serum-free medium for 16 hours. Cells were then
incubated with caspase3 substrate solution GDEVDGI for 1 hour at
37°C, and flow cytometric analysis was performed. The open
histogram shows the fluorescence of day 7 ECFCs as control. Each panel
shows data on cells incubated without rhEPO and rhIFN- (A), with
rhEPO alone (B), with rhIFN- alone (C), and together with rhEPO plus
rhIFN- (D).
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| Fig 7.
Effect of IFN- and EPO on activation of caspase8.
Cells were incubated under the indicated conditions in serum-free
medium for 16 hours. Cells were then incubated with fluorogenic
caspase8 substrate IETDAFC for 1 hour at 37°C. The fluorescence
intensity was then measured using a multilabel counter. The mean of
triplicate studies is shown.
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Discussion |
It has been reported that IFN- produces apoptosis of
hematopoietic progenitor cells, and thus inhibits the
growth of hematopoietic cells in vitro and in vivo.2-12
However, when we measured the effect produced by IFN- during 16 hours of cell culture, this cytokine also significantly reduced
apoptosis of day 7 ECFCs, especially in the absence of or with lower
concentrations of EPO. This protective effect on the survival of ECFCs
was also evident in the erythroid colony assay, though the size of the
colonies was reduced by exposure to IFN-, thereby indicating the
inhibition of cellular proliferation, or production of a later
apoptosis, of ECFCs by this cytokine. This early contradictory effect
of IFN- on apoptosis of erythroid progenitor cells can be reasonably explained as follows: Inhibition of expansion of human erythroid progenitor cells by IFN- was described in detail by Dai et
al,6,11,33 who used cells purified according to similar
methods, but the cells were in an earlier stage of maturation and
underwent longer incubation times. In their report, Dai
et al clearly indicated that IFN- induced apoptosis of erythroid
progenitor cells at a relatively restricted stage of maturation through
96 hours of incubation. Mature BFU-E was obtained on
days 3-6 in culture, earlier than what was achieved with the progenitor
cells that we used in this study. Dai et al
did not evaluate the effect of IFN- on apoptosis in
the absence of EPO, but we did see an apoptotic effect in this
report. Therefore, it is not contradictory to see a
suppressive effect of IFN- on apoptosis instead of apoptotic induction, since cells were in a different stage of maturation and were
examined at an earlier time, and thus may show a
distinctly different response to the same cytokine.
The number of receptors for IFN- gradually decreases during
maturation of erythroid progenitor cells, as shown by Taniguchi et
al.34 While the amount of expression of the receptor may be
altered, intracellular components that mediate the signal induced by
IFN- could also be reorganized during erythroid maturation and hence
would show a different response from that seen in immature cells.
While day 7 ECFCs immediately underwent apoptosis when EPO was
excluded from the culture medium, cells cultured for
another 3 days (day 10 cells, which are at the
poly-ortho chromatic erythroblast stage)
are relatively resistant to EPO deprivation (data not shown). This
means that erythroid cells probably lose the intracellular "death" mechanism that mediates apoptosis at the terminal stage of erythroid maturation. Again, the report by Dai et
al11 showed that 2 to 4 days of contact with IFN- is
necessary to induce apoptosis of erythroid progenitors. Hence, when
IFN- was added to day 7 ECFCs, these cells may have undergone the
terminal stages of erythroid maturation before IFN- initiated the
process by which the cells undergo apoptosis. Since day 7 ECFCs are
less sensitive to the "death signal" induced by IFN-, possibly
for the reason mentioned above, this might explain why we
observed the enhancing effect of IFN- on survival of mature ECFCs,
which was not evident during the process of apoptosis as it was in the case of immature erythroid progenitor cells.
Alternatively, by looking at cells after 16 hours of
incubation without EPO, we may have detected the effects
of an early set of genes activated by IFN-, the effects of which are
not seen during the process of apoptosis.
A stimulating effect of IFN- on hematopoietic progenitor cells has
also been reported. Brugger et al14 found that IFN- increased peripheral blood CD34+ progenitor cell
expansion when added to a cocktail of growth factors. Shiohara et
al16 reported that the addition of IFN- to cultures
containing SCF resulted in a synergistic effect on the development of
murine hematopoietic progenitors. These data may support the notion
that IFN- does not always function as a strictly inhibitory factor
on hematopoietic progenitor cells. Recently, Baxter et al35
reported that tumor necrosis factor-, which is also an inhibitory
cytokine, can stimulate proliferation of mitotically quiescent
cells, while it induces apoptosis of mitotically active
cells. Interferon alpha (IFN-) enhances survival of B cells by
reducing apoptosis,36 while IFN- induces
apoptosis of malignant plasma cells.37 This
observation also supports the notion that an
"inhibitory" cytokine can reduce apoptosis of hematopoietic cells
under certain conditions and at certain stages of development.
While IFN- increased Fas expression of mature ECFCs, this expression
did not directly induce apoptosis in these cells. Deprivation of EPO
induced Fas expression plus activation of caspase8 and caspase3, which
are known to be activated during Fas-mediated apoptosis prior to DNA
fragmentation.26,28,38 Since the Fas ligand (FasL) is
present on the surface of ECFCs,6 Fas-FasL interaction may
play an important role in the apoptosis of ECFCs induced by EPO
deprivation. The Fas-FasL system appears to play an
important role in erythroid homeostasis through its induction of
apoptosis in immature erythroblasts.39 In contrast, while IFN- also induced Fas expression on mature ECFCs, activation of
caspase8 and caspase3 was not increased over 16 hours, and apoptosis of
the cells was reduced despite the absence of EPO. These data indicate
that IFN- induces expression of Fas throughout different stages of
erythroid maturation, but influences downstream caspases in a
differential manner according to the stage of erythroid maturation and
the time of incubation. IFN- induces Fas expression but might block
the Fas-mediated apoptosis of mature ECFCs through intracellular
components such as FLIPs (FLICE- [Fas-associated death-domain-like
IL-1-converting enzyme] inhibitory protein)40 that
block activation of caspase8 and downstream caspases in the T cells
resistant to Fas-FasL-induced apoptosis or other mechanisms.
Bcl-x plays an important role in protecting cells from
apoptosis by blocking the release of cytochrome c from mitochondria, a
critical step in the activation of the caspase protease
cascade.41 Gregoli and Bondurant22 demonstrated
that deprivation of EPO reduced expression of Bcl-x to induce apoptosis
of ECFCs via activation of caspase3. They also showed that expression
of Bcl-x was highly EPO-dependent and that it increased greatly during
the terminal differentiation of ECFCs. When mature ECFCs were cultured
in the presence of IFN- without EPO, an increased amount of Bcl-x
was detected compared to findings in cells without EPO and IFN-. However, the level of expression was less than in
cultures with EPO. This suggests that IFN- may at least
partially protect ECFCs from apoptosis through a pathway
independent of expression of Bcl-x, which still plays an important role
in the maintenance and survival of the cells in the
presence of IFN-.
The complete significance of suppression of apoptosis shown in this
report is not fully understood. IFN- may allow mature erythroid
progenitor cells to maintain erythropoiesis, while immature progenitors
are impaired during inflammatory stress.
To clarify the mechanism by which IFN- suppresses apoptosis of
mature erythroid progenitors, further investigation on
intracellular components that mediate cytokine stimuli, such as
MAP kinase,42 Jaks, STATs,43 and
phosphatidylinositol 3-kinase,44 is needed.
| |
Acknowledgments |
We express our deep appreciation to S. Aoki and S. Isewaki for
excellent technical assistance.
| |
Footnotes |
Submitted July 8, 1999; accepted February 7, 2000.
Supported in part by National Institutes of Health grant DK-15 555 of
S.B.K.).
Reprints: Koichiro Muta, Department of Medicine and
Bioregulatory Science, Graduate School of Medical Science,
Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; e-mail: mmmmm{at}intmed3.med.kyushu-u.ac.jp.
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.
| |
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Abstract
Introduction