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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1235-1242
Fas Ligand Is Present in Human Erythroid Colony-Forming Cells and
Interacts With Fas Induced by Interferon  to Produce Erythroid Cell
Apoptosis
By
Chun-Hua Dai,
James O. Price,
Thomas Brunner, and
Sanford B. Krantz
From the Hematology Division, Department of Medicine and Department
of Pathology, Department of Veterans Affairs Medical Center (DVAMC) and
Vanderbilt University School of Medicine, Nashville, TN; and the
Division of Cellular Immunology, La Jolla Institute for Allergy and
Immunology, San Diego, CA.
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ABSTRACT |
Interferon  (IFN) inhibits the growth and differentiation of
highly purified human erythroid colony-forming cells (ECFCs) and
induces erythroblast apoptosis. These effects are dose- and time-dependent. Because the cell surface receptor known as Fas (APO-1;
CD95) triggers programmed cell death after activation by its ligand and
because incubation of human ECFCs with IFN produces apoptosis, we
have investigated the expression and function of Fas and Fas ligand
(FasL) in highly purified human ECFCs before and after incubation with
IFN in vitro. Only a small percentage of normal human ECFCs express
Fas and this is present at a low level as detected by Northern blotting
for the Fas mRNA and flow cytometric analysis of Fas protein using a
specific mouse monoclonal antibody. The addition of IFN markedly
increased the percentage of cells expressing Fas on the surface of the
ECFCs as well as the intensity of Fas expression. Fas mRNA was
increased by 6 hours, whereas Fas antigen on the cell surface increased
by 24 hours, with a plateau at 72 hours. This increase correlated with
the inhibitory effect of IFN on ECFC proliferation. CH-11 anti-Fas antibody, which mimics the action of the natural FasL, greatly enhanced
IFN-mediated suppression of cell growth and production of apoptosis,
indicating that Fas is functional. Expression of FasL was also
demonstrated in normal ECFCs by reverse transcriptase-polymerase chain
reaction and flow cytometric analysis with specific monoclonal antibody. FasL was constitutively expressed among erythroid progenitors as they matured from day 5 to day 8 and IFN treatment did not change
this expression. Apoptosis induced by IFN was greatly reduced by the
NOK-2 antihuman FasL antibody and an engineered soluble FasL receptor,
Fas-Fc, suggesting that Fas-FasL interactions among the ECFCs produce
the erythroid inhibitory effects and apoptosis initiated by IFN.
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INTRODUCTION |
HUMAN INTERFERON (IFN) induces
activation of mononuclear phagocytes and lymphocytes and also
suppresses normal hematopoiesis in vitro under a variety of cell
culture conditions. The inhibitory effects of IFN on murine and
human granulocyte-macrophage colony-forming units, burst-forming
units-erythroid (BFU-E), and colony-forming units-erythroid (CFU-E) in
vitro have been reported by many investigators.1-9 Previous
experiments performed in this laboratory have shown that intermediate,
day-3 to day-6 mature human BFU-E are most sensitive to IFN, whereas
early BFU-E and more mature erythroid colony-forming cells (ECFCs) are
much less sensitive.8
Additional investigations have shown that IFN inhibited cell
proliferation and produced apoptosis of stromal cells, murine pre-B-cell lines, and also human CD4+ or CD8+
thymocytes that had been treated with cyclosporin.10,11
Apoptosis of erythroid cells after incubation with IFN has been
demonstrated in our laboratory by both nuclear condensation and
fragmentation plus flow cytometry with in situ
end-labeling.8 Whereas reduced cell proliferation was noted
by 72 hours, apoptosis was only demonstrated at a later time and was,
therefore, quite different from the apoptosis produced by
erythropoietin (EP) deprivation, which has been shown by 17 hours with
human erythroid cells.12
The process of apoptosis involves many metabolic changes, leading to
the final degradation of genomic DNA into nucleosomal fragments, and
the regulation of this process involves a large number of genes. Both
in vivo and vitro studies have demonstrated that the Fas/Fas ligand
(FasL) system performs a critical function in producing apoptosis in
several different organ systems after triggering of Fas by
FasL.9,13,14 Fas is widely expressed in many cell types,
but the FasL has been demonstrated mainly in activated T cells and in a
few tissues such as the cornea and testis, plus some tumors. However,
Fas/FasL expression on normal erythroid progenitor cells and their role
during hematopoietic development has not been clearly defined. No CD95
mRNA expression was detected by reverse transcriptase-polymerase chain
reaction (RT-PCR) in CD34+ cells freshly isolated from
human bone marrow15,16 and few CD34+ cells from
normal bone marrow expressed Fas antigen, but this was markedly
increased on bone marrow CD34+ cells from patients with
aplastic anemia.17 Immature primitive hematopoietic
progenitors from human fetal liver did express CD95, whereas the more
mature progenitors showed low CD95 expression.18 Both
IFN and TNF induced Fas expression on purified human
CD34+ cells.9,15 FasL expression in purified
hematopoietic progenitors has not been identified.
In this study, we further characterized the inhibitory effect of IFN
on ECFC proliferation and apoptosis induction. Because binding of FasL,
or agonistic anti-Fas antibody, to Fas receptor triggers apoptosis and
because ECFCs underwent apoptosis after incubation with IFN, we have
investigated the expression of Fas and FasL in human ECFCs after
incubation with IFN and the role of Fas and FasL in IFN-induced
apoptotic cell death.
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MATERIALS AND METHODS |
Purification and expansion of human blood ECFCs.
Four hundred milliliters of blood was obtained from normal donors after
informed consent approved by the Vanderbilt University and Department
of Veterans Affairs Medical Centers (Nashville, TN) Institutional
Review Boards. BFU-E were purified by sequential density gradient
centrifugation; depletion of platelets, lymphocytes, and adherent
cells; and further negative selection of contaminant cells with CD2,
CD11b, CD16, and CD45 monoclonal antibodies (MoAbs), as previously
described.8,19 The BFU-E were suspended in 20 mL Iscove's
modified Dulbecco's medium (IMDM) containing 20% heat-inactivated fetal calf serum, 5% heat-inactivated, pooled, human AB serum, 1%
deionized bovine serum albumin (BSA; Intergen Co, Purchase, NY), 5 × 10 5 mol/L 2-mercaptoethanol, 10 µg/mL
insulin, 2 U/mL recombinant human (rh) EP, 50 U/mL interleukin-3,
penicillin at 500 U/mL, and streptomycin at 40 µg/mL in 50-mL
polystyrene flasks to generate ECFCs.19 After incubation at
37°C in 5% CO2/95% humidified air for 4 or 5 days
(day-5 or day-6 cells), the cells were collected, further enriched by
centrifugation through 10% BSA and over Ficoll-Hypaque, and aliquoted
for Northern and flow cytometric analyses plus plasma clot assays for
ECFCs. In some experiments, incubation was continued in liquid medium
with or without rhIFN (4.75 × 107 U/mg; Genzyme
Corp, Cambridge, MA) for additional times to observe later effects of
rhIFN.
Plasma clot assay for ECFCs.
Cells were plated at a concentration of 103/mL in an IMDM
mixture containing 20% fetal calf serum, 5% human AB serum, 1%
deionized BSA, 10 µg/mL insulin, 2 U/mL rhEP, penicillin plus
streptomycin, 2 mg/mL fibrinogen (Sigma, St Louis, MO), and 0.2 U/mL
bovine thrombin (Parke-Davis, Morris Plains, NJ) in 48-well
flat-bottomed tissue culture plates with 0.2 mL/well. In some
experiments, rhIFN; anti-Fas antibodies, CH-11 (Immunotech Inc,
Westbrook, ME), and/or ZB4 (MBL, Watertown, MA); anti-FasL
antibody, NOK-2 (kindly provided by Hideo Yagita, Juntendo University,
Tokyo, Japan); Fas-Fc, a fusion protein composed of human Fas and a
human Ig constant region prepared as previously
described20; control MoAb isotype IgGI (Becton Dickinson,
San Jose, CA); or control human IgG Fc fragment (Accurate Chemical and
Scientific Corp, Westbury, NY) were added at the indicated
concentrations. The clots were fixed on day 15 and stained
with 3,3 dimethoxybenzidine and hematoxylin. Colonies of 2 or
more hemoglobinized cells were scored as ECFCs. The ECFC purity was
determined by the plating efficiency and data were expressed as the
means ± SD with significance calculated by the t-test.
RNA preparation and Northern analysis.
Total RNA was prepared from day-5 to day-8 cells treated or not treated
with rhIFN using ULTRASPEC or RNAzol (BIOTECX Laboratories, Inc,
Houston, TX). Quantification of RNA, formaldehyde gel electrophoresis, blotting onto nylon membranes, and hybridization with
32P-labeled DNA probes for FAS and  -actin were performed
as previously described.21 Rehybridization of the blots
with a similarly labeled probe for actin mRNA was also performed to
correct for variation in loading. Actin mRNA is maintained at constant
levels in ECFCs in the presence of rhEP during the periods
analyzed.21 Fas mRNA expression was quantitated with a
laser scanning densitometer and normalized to the amount of total RNA
present in the lane by comparison with the 18S band as well as the
actin signal. The human -actin probe was purchased from
Calbiochem-Novobiochem International (La Jolla, CA) and
the human Fas probe was generated by RT-PCR.
RT-PCR.
Total RNA was extracted as described above. Using a Gene Amp RNA PCR
kit (Perkin Elmer, Cetus, Norwalk, CT), single-stranded cDNA was
synthesized and RT-PCR was performed. Fas and FasL specific primers
were designed according to sequence data published
previously.22,23 We used antisense primer 5-T ACT
CAA GTC AAC-3 (bases no. 873-885), for generation of Fas cDNA,
and sense primer 5 ATG CTG GGC ATC TGG ACC CTC CTA-3
(bases no. 195-218) plus antisense primer 5-TGG AGA TTC ATG AGA
ACC TTG GTT-3 (bases no. 810-833) for amplification of Fas cDNA.
We used random primers to generate FasL cDNA and sense primer
5-C AAG TCC AAC TCA AGG TCC ATG CC-3 (bases no. 517-540)
plus antisense primer 5-CAG AGA GAG CTC AGA TAC GTT GAC-3
(bases no. 839-862) for amplification of FasL cDNA. Thirty-five cycles
of PCR, with denaturation at 94°C for 30 seconds, annealing at
60°C for 30 seconds (Fas) or 60 seconds (FasL), and extension at
72°C for 1 minute, were performed on a programmed-temperature system (Hybaid OmniGene; Midwest Scientific, St Louis, MO). The amplified PCR product was electrophoresed in a 1% agarose gel and
ethidium bromide-stained. The gel containing an expected single band
was sliced out and DNA was purified using a QIAquick Gel Extraction kit
(QIAGEN, Santa Clarita, CA). Both PCR products were verified by
sequencing.
Detection of Fas and FasL by flow cytometric analysis.
Data acquisition was performed on a Becton Dickinson FACScan flow
cytometer with data saved as listmode files. All incubations were
performed on ice. The ECFCs cultured with or without rhIFN were
collected and washed three times in phosphate-buffered saline (PBS)
with 1% BSA. Cell-surface expression of Fas was assayed by direct
immunofluoresence flow cytometry. Briefly, 1 × 106
cell aliquots in 0.1 mL PBS with 1% BSA from each group were incubated
with either fluorescein isothiocyanate (FITC)-murine anti-Fas MoAb
(UB2) at 10 µg/mL (Immunotech) or FITC-murine IgG1 (Becton Dickinson)
as a control for 30 minutes. The cells were then fixed in PBS with 1%
formaldehyde before analysis.
FasL was shown by indirect immunoflourescence. ECFCs were fixed and
permeabilized using a commercial Fix & Perm Cell Permeabilization kit
(Caltag Laboratories, South San Francisco, CA) before staining. Primary
incubations were performed with 106 cells and murine
antihuman FasL MoAb at 10 µg/mL (clone G 247-4; PharMingen, San
Diego, CA) or murine IgG1 at the same concentration for 30 minutes.
These cells were then washed once with PBS/1% BSA. Secondary
incubations were performed with FITC-goat antimouse IgG1 at 10 µg/mL
(Southern Biotechnology Assoc Inc, Birmingham, AL) for another 30 minutes.
To ensure that the FasL+ cells were ECFCs, dual staining
was performed in some experiments combining indirect immunofluorescence for FasL with direct staining using phycoerythrin (PE)-MoAb (5 µg/mL)
to CD71 (transferrin receptor; Caltag). After indirect staining as
described above, unbound goat antimouse binding sites were blocked with
5 µg/mL normal mouse IgG in PBS for 20 minutes. Direct-labeled,
antigen-specific PE-MoAb to CD71 or PE-murine IgG1 (Becton Dickinson)
was then incubated with the cells for an additional 30 minutes. The
cells were then fixed and analyzed by flow cytometry. Cells were
incubated with murine IgG1, indirectly stained with FITC-goat antimouse
IgG1, and counterstained with direct-labeled, nonspecific PE-murine
IgG1 to show nonspecific fluorescence on both axes. For specific
comparison of murine antihuman FasL MoAb on ECFCs, we used indirect
murine IgG1 stained with FITC-goat antimouse IgG1 and counterstained
with specific PE-MoAb to CD71.
To detect soluble FasL in the cell medium, the sFas Ligand Elisa Kit
purchased from MBL was used.
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RESULTS |
Inhibition of ECFC growth by IFN.
To study the effect of IFN on Fas and FasL, we first defined the
optimum dose of rhIFN and the appropriate duration of incubation with day-5 cells to produce its inhibitory effects. Inhibition of ECFC
growth was produced by an rhIFN concentration as low as 50 U/mL
(Fig 1A) and was clearly evident after 72 hours of incubation with rhIFN (Fig 1B). Cytospin preparations of
these cells after 96 hours of incubation with 1,000 U/mL of rhIFN
showed cells with morphologic changes characteristic of apoptosis, such as nuclear condensation, fragmentation, and reduced size, which have
correlated with DNA fragmentation demonstrated by in situ end-labeling
in ECFCs.8 A marked reduction of the number and size of
erythroid colonies and hemoglobin formation was also induced by
rhIFN, and programmed cell death within the erythroid colonies was
enhanced as the concentration of rhIFN was increased
(Fig 2). Thirty percent of erythroid
colonies contained apoptotic cells after incubation with 50 U/mL of
rhIFN and 50% of erythroid colonies were greatly reduced in size
and had an enhanced number of cells with the characteristic morphologic
changes of apoptosis at the highest concentration of rhIFN. Similar
results were obtained in day-6 cells (data not shown).
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| Fig 1.
Dose- and time-dependent inhibition of day-5 ECFCs by
IFN. Day-5 ECFCs were incubated in liquid medium with rhIFN from
0 to 1,000 U/mL. After 96 hours of incubation, the cells were collected and the number of cells was counted (A), or the cells were cultured with or without 1,000 U/mL rhIFN and harvested at the indicated times. (B) The number of total cells in each control without rhIFN was taken as 100% and was compared with that of the rhIFN groups. Each graph represents the mean from two experiments. The purity of the
day-5 ECFCs, determined by plasma clot assay, was 54% ± 6% (A) and
56% ± 9% (B).
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| Fig 2.
IFN inhibits colony formation and produces apoptosis
of erythroid cells. Day-5 cells at 200 cells/well were plated in 0.2-mL plasma clots with or without rhIFN. The clots were fixed at day 15 and stained with benzidine-hematoxylin. The size of the colonies was
estimated as follows: large colonies, greater than 500 cells/colony; medium colonies, 50 to 500 cells/colony; and small colonies, 2 to 49 cells/colony. The colonies that contained 10% or more cells with the
morphologic changes of apoptosis were counted as apoptotic colonies,
and the percentage of apoptotic colonies is shown in the top panel.
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Expression of Fas on ECFCs.
We next determined whether IFN treatment induced Fas mRNA
expression. Day-5 ECFCs were cultured with rhIFN at a variety of
increasing concentrations and durations of incubation. The cells were
then harvested and Northern analysis for Fas was performed (Fig 3). A very low level of Fas mRNA was
detected among the original cells without incubation. After incubation
with rhIFN, expression of Fas mRNA was markedly enhanced. A greater
than fivefold enhancement of Fas mRNA by laser scanning densitometry
was evident at an rhIFN concentration of 500 U/mL or higher. This
induction of Fas mRNA expression by rhIFN was clearly increased
by 6 hours after incubation with rhIFN, and Fas mRNA
continued to increase as the time of treatment was extended.
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| Fig 3.
Dose- and time-dependent induction of Fas mRNA in ECFCs
treated with rhIFN. Day-5 cells were incubated with IFN at 0 to 2,000 U/mL for 72 hours or with 2,000 U/mL for 0 to 72 hours, and then
the total RNA was prepared. Twenty micrograms of total RNA was loaded
in each lane. The blots were hybridized with a labeled probe for Fas
and rehybridized with a probe for actin after stripping the initial
probe. The ECFC purity at day 5 was 51% ± 9% and 56% ± 8%, respectively, and by day 8 it was 89% ± 6% and 91% ± 8%.
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Fas antigen expression on the cell surface was measured by direct
immuno-fluorescence with specific FITC-MoAb and flow cytometry. Only a
small percentage of Fas+ cells was present among the
original cells before incubation, but a very large increase of
Fas+ cells was demonstrated after 48 hours of incubation
with increasing concentrations of rhIFN
(Fig 4A). An enhanced expression of Fas antigen on the cells, including both the number of positive cells and
the intensity per cell, was clearly apparent at 24 hours and reached a
plateau after 48 to 72 hours of incubation with rhIFN (Fig 4B). No
effect was evident at 6 hours (data not shown). An enhanced
concentration of Fas on the surface may be required for inhibition of
cell proliferation and induction of apoptosis, because a significant
reduction in cell number occurred at 72 hours when the cells had a
higher Fas distribution on their surface.
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| Fig 4.
Expression of Fas on ECFCs after incubation with
rhIFN. Day-5 cells were cultured in liquid medium in the presence of
rhIFN at 0 to 1,000 U/mL for 48 hours (A) or at 1,000 U/mL for 24 to 96 hours (B). At the indicated times, the cells were incubated with
FITC-MoAb to Fas (CD95) or FITC-murine IgG1. Flow cytometric analysis
was then performed. The open histogram shows the CD95 fluorescence of
the cells incubated without rhIFN and the solid histogram shows the
CD95 fluorescence of the rhIFN-treated cells. The purity of the
day-7 cells was 89% ± 9% (A) and 81% ± 7% (B).
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FasL expression in ECFCs.
Experiments were performed first to demonstrate FasL mRNA by RT-PCR.
These studies showed that FasL mRNA was present in cells treated or not
treated with rhIFN (Fig 5). To
investigate the expression of FasL protein in ECFCs, day-5 cells and
their descendent day-8 cells, derived from the day-5 cells by
incubation with or without rhIFN for 72 hours, were fixed and
permeabilized for identification of FasL and CD71 with specific MoAb.
Although all proliferating cells including activated T and B cells and
macrophages also bear CD71, the number of transferrin receptors in
erythroid progenitor cells is 15-fold higher than the concentrations
associated with other cells.24 The high-intensity
CD71+ cells were also larger cells than those with
low-intensity CD71+ and ECFCs are large cells. Therefore,
when flow cytometric analysis for FasL was performed, large cells with
high intensity CD71+ were gated. This group of cells
represented more than 80% of the total population. To exclude
interference due to staining with two fluorescence markers, not only
isotype, normal mouse FITC-IgG with mouse PE-IgG was set as a control,
but also mouse FITC-IgG with PE-MoAb to CD71 was used as a second
control.
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| Fig 5.
Detection of FasL mRNA by RT-PCR in human ECFCs. Day-5
cells from three donors were cultured in control (C) medium or with 1,000 U/mL rhIFN () for 3 days and total RNA was prepared
followed by RT-PCR for FasL. The PCR product corresponding to FasL (346 bp) was purified after electrophoresis and verified by sequencing (marker,  x174 DNA-Hae III digest).
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Approximately 50% of the gated CD71+ cells expressed FasL
by two-color flow cytometric analysis (Fig
6). Figure 6 compares FasL expression on all day-5 CD71+
cells (Fig 6A) and high-intensity CD71+ cells (Fig 6B). All
CD71+ cells were 47% FasL+, whereas
CD71+ bright cells were 64% FasL+. Plasma clot
assay showed that at least 65% ± 6% of the cells were ECFCs. This
indicated that most of the FasL+ cells were ECFCs. FasL was
constitutively expressed in the erythroid progenitors from day 5 to day
8 (Table 1) and was not altered in day-8
cells after 72 hours of incubation with 1,000 U/mL of rhIFN (data
not shown). When nonpermeabilized cells were used in these experiments,
FasL was present on the cell surface in 13% ± 16% of the ECFCs.
Day-8 cell culture media from five separate experiments showed an
increase in soluble FasL of 83 ± 41 pg/mL compared with
undetectable levels in day-8 media incubated without cells. This
concentration of soluble FasL is significantly above that of the normal
serum level of 58 ± 35 pg/mL (P < .05).
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| Fig 6.
FasL expression in day-5 cells. Day-5 cells were analyzed
for FasL by indirect immunofluorescence using a specific murine antihuman FasL MoAb or an isotype control added after permeabilization and were stained with FITC-goat antimouse IgG1. After blocking with
mouse IgG in PBS, the cells were incubated with PE-MoAb to human CD71
to identify ECFCs. The histogram shown represents three separate
staining procedures with superimposed data: (1) population in lower
left quadrant represents fluorescence produced by presence of indirect
isotype control plus FITC-goat antimouse IgG1 and murine PE-IgG1; (2)
gray represents the fluorescence produced by the presence of indirect
isotype control plus FITC-goat antimouse IgG1 and specific PE-MoAb to
human CD71; (3) dark black represents fluorescence produced by indirect
MoAb to FasL stained with FITC-goat antimouse IgG1 and PE-MoAb to CD71.
(A) CD71+ large cell population, representing 84% of all
cells; 99% of the cells were CD71+ and 47% were
FasL+. (B) Large cells bearing high intensity
CD71+ from the same experiment, representing 79% of all
cells. Among these cells, 100% were CD71+ and 64% were
FasL+. The purity of the ECFCs was 65% ± 6%.
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Demonstration of functional Fas and FasL.
To determine whether IFN inhibition of cell growth was produced by
the interaction between Fas (induced by IFN) and FasL (already
present in the cells), the results of ligation of murine antihuman Fas
MoAb to ECFCs was studied. Two specific MoAbs to Fas (CH-11 [IgM],
which is known to mimic the action of FasL, and ZB-4 [IgG], which
blocks the effect of CH-11) were tested.25 Dose-response
curves for CH-11 and ZB-4 to determine the optimum concentrations for
our experiments were performed and these were compared with the
isotype-matched murine Igs to ensure that the effects of the antibody
were specific and not related to IgM or IgG addition. When CH-11 or
ZB-4 were added to day-5 ECFCs, no effect on colony size, colony
number, or hemoglobin concentration was apparent (data not shown).
Day-5 ECFCs were then cultured in liquid medium in the presence of a
concentration of rhIFN known to result in a large increase of Fas,
and approximately 50% inhibition of the cell number plus an additional
40% inhibition of viability were noted
(Fig 7A). The addition of anti-Fas MoAb, CH-11, to the rhIFN-treated ECFCs greatly potentiated the reduction of cell number by rhIFN, whereas ZB-4 had little effect. When CH-11
and ZB-4 were added together, the effect of CH-11 was blocked by ZB-4.
Fas MoAb-mediated inhibition of colony formation was also observed (Fig
7B). CH-11 strongly increased the rhIFN inhibitory effect on colony
size and number. More than 90% of erythroid colonies were suppressed
with the addition of CH-11, even when the concentration of rhIFN was
quite low (50 U/mL) and ZB-4 neutralized the CH-11 reduction of colony
number as well. These data clearly indicated that Fas induced by IFN
was functional and could be activated.
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| Fig 7.
Effects of anti-Fas MoAbs, CH-11 and ZB4, on IFN
inhibition of cell growth and erythroid colony formation by day-5
cells. Replicate aliquots of day-5 cells were cultured in liquid medium in the presence of CH-11 (100 ng/mL) and/or ZB4 (500 ng/mL)
with or without 50 U/mL rhIFN (A). After incubation for 96 hours, the cells were harvested and trypan blue stains were performed. Aliquots of day-5 cells from the same experiment were plated in 0.2-mL
plasma clots at 200 cells/well with same concentrations of rhIFN and
antibodies (B). At day 15, the clots were fixed and stained. ( )
Large colonies, containing more than 500 cells per colony; ( ) medium
colonies, containing 50 to 500 cells per colony; ( ) small colonies,
containing 2 to 49 cells per colony.
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NOK-2, a specific neutralizing MoAb to human FasL,26 was
used to confirm the functional activity of FasL in these cells. The
results from two experiments are shown in
Table 2. In the presence of rhIFN at 250 U/mL, the first experiment showed that more than two thirds of the
colonies had morphologic apoptotic changes and less than one third
looked normal. Erythroid colony size and colony number were greatly
reduced. The addition of NOK-2 almost completely restored the number of
colonies, although colony size remained small. Apoptosis was not
evident in the presence of NOK-2. In the second experiment, the donor
cells were more sensitive to rhIFN and normal colony formation was
almost completely inhibited with rhIFN. More than 70% of the
colony-forming cells were rescued and had normal morphologic
characteristics in the presence of NOK-2. These experimental results
were confirmed by similar findings with the addition of
Fas-Fc,20 although the latter was not quite as potent
(Table 2).
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DISCUSSION |
The inhibitory effect of IFN on the ECFCs was observed both in
liquid and plasma clot cultures and inhibition of cell proliferation occurred at a concentration of rhIFN as low as 50 U/mL. Evidence that this inhibition is a direct effect of rhIFN is provided by the
observations that the degree of inhibition did not vary significantly
as the ECFCs were purified from 19.8% to 52.6%,27 that
ECFCs with a purity of 80% were still inhibited,8 and that
ECFCs have rhIFN receptors.28 This finding is consistent with past work demonstrating the production of DNA fragmentation in
day-6 ECFCs by in situ end-labeling and flow cytometry.8
The Fas/FasL system is a very important cellular pathway responsible
for the initiation of apoptosis. Fas is a 45-kD membrane glycoprotein
belonging to the tumor necrosis factor (TNF) receptor family. Fas
contains a 70 amino acid cytoplasmic domain that is necessary and
sufficient for transduction of the apoptotic signal. Fas mRNA is widely
expressed in lymphocytes, thymocytes, monocytes, epithelial and
endothelial cells, eosinophils, leukemia cells, and many tissues, such
as bone marrow, heart, kidney, liver, and ovary.17,22,25,26,29-33 FasL is a 40-kD, type II protein
member of the TNF family.23,34 In contrast to Fas, FasL was
thought to be relatively restricted in its cell and tissue
distribution. It was initially found to be expressed in activated T
cells and has a major role in T-cell cytoxicity.23 However,
FasL expression in other tissues and cells, such as stroma cells of the
eye, Sertoli cells of the testis, thyrocytes, and large granular
lymphocytic leukemia cells, as well as in various nonlymphoid carcinoma
cells, including colon plus hepatocellular carcinomas and melanoma
cells has been recently reported.35-41 This work indicated
that expression of FasL was important for maintaining immune privilege
and that it appears to have a role in enabling tumor cells to escape
from immune attack.
To determine if Fas/FasL expression correlated with IFN induction of
ECFC apoptosis, we studied the presence of both of these molecules in
highly purified ECFCs in the presence or absence of IFN. Northern
and flow cytometric analyses showed that only a small percentage of
normal human ECFCs expressed Fas and that this was at a very low level.
Addition of rhIFN potently induced Fas expression. Fas mRNA clearly
increased by 6 hours after the incubation of ECFCs with IFN and at a
concentration of IFN as low as 50 U/mL. Fas protein on the ECFCs was
clearly enhanced by 24 hours after incubation with IFN and most of
the ECFCs expressed Fas antigen by 48 hours. The amount of Fas on the
ECFC surface gradually increased to a maximum after 72 hours of
incubation in the presence of IFN when the inhibitory effect of
IFN on the ECFCs clearly appeared. Further experiments showed
activation of Fas by the anti-Fas MoAb, CH-11, which is known to
functionally mimic FasL. Ligation of Fas with this antibody markedly
reduced ECFC viability, the generation of ECFCs in liquid culture and erythroid colony formation, while the anti-Fas MoAb ZB4 corrected the
CH-11 inhibition, indicating that Fas expression was not only associated with functional inhibition of the ECFCs, but also, when the
Fas system for producing apoptosis was activated, it produced the
inhibition.
Nevertheless, CH-11 is an artifactual MoAb added in culture. If
apoptosis is mediated via Fas, interaction with an agonistic ligand
must occur. Our search for this ligand using flow cytometric analysis
showed that approximately 50% of the large CD71+ cells,
which were 80% of the total cells, expressed FasL. Because not all the
cells in the population were ECFCs, especially at day 5, and because
erythroid cells have 15-fold more CD71 than other cells, we performed
flow cytometric analysis with the cells gated to include only the
high-intensity, CD71+ ECFC. Our results showed that
approximately 66% of the large size, high-intensity, CD71+
erythroid cells were FasL+ and that FasL was constitutively
expressed on ECFCs from day 5 to day 8. Because day-8 cells were almost
homogenous, with 90% or more of the day 8 cells proven to be ECFCs by
plasma clot assay, most of the FasL+ cells in our cultures
are ECFCs and rhIFN did not appear to alter FasL expression.
Nok-2, an anti-FasL MoAb, has the biological property of neutralizing
FasL.26 When this MoAb was added to the ECFC cultures, the
induction of apoptosis and the inhibition of ECFC proliferation and
colony formation by IFN were partially prevented, indicating that
FasL is functional as an actuator of apoptosis in the system. Comparable results were observed with Fas-Fc. Our observations thus
suggest a mechanism responsible for at least part of the inhibitory
effect of IFN on human ECFCs. Because normal ECFCs express
negligible amounts of Fas, they do not undergo apoptosis. Simultaneous
expression of functional Fas and FasL on ECFCs, the former induced by
IFN, results in programmed cell death.
Similar findings in Hashimoto's thyroiditis have now been
reported.41 FasL is constitutely expressed in normal
thyroid cells, but only the cells of Hashimoto's thyroiditis express
large amounts of Fas on their cell surface, which is thought to be
induced by IL-1. Coexpression of Fas and FasL also has been thought
to contribute to the rapid rate of spontaneous neutrophil
apoptosis.42
Because Fas and FasL were coexpressed in ECFCs after incubation with
IFN, they may commit the cells to an autocrine death as shown with
T-cell hybridoma cells20 and/or soluble FasL may function in a paracrine pathway to mediate cell death.43,44 Further studies are now in progress to delineate the precise mechanism of this interaction. The mechanism by which IFN exerts an inhibitory effect on the cell growth of ECFCs and induces apoptosis may be multifactorial. Our laboratory has found that IFN downregulates stem
cell factor and EP receptors as well as the mRNA for these receptors.45 Nevertheless, the present study shows that the Fas/FasL system strongly contributes to the inhibition of
erythropoiesis by IFN.
| |
FOOTNOTES |
Submitted May 15, 1997;
accepted October 8, 1997.
Supported by a Veterans Health Administration Merit Review Grant and by
Grants No. DK-15555, GM-52735, and 5 T32-DK07186 from the National
Institutes of Health.
Address reprint requests to Sanford B. Krantz, MD, Department of
Medicine/Hematology, Vanderbilt University Medical School MRB II Room
547, Nashville, TN 37232-6305.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
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Abstract
Introduction
Abstract