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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2847-2854
HEMATOPOIESIS
Monocytes express high amounts of Notch and undergo cytokine
specific apoptosis following interaction with the Notch ligand, Delta-1
Kohshi Ohishi,
Barbara Varnum-Finney,
David Flowers,
Claudio Anasetti,
David Myerson, and
Irwin D. Bernstein
From the Clinical Research Division, Fred Hutchinson Cancer Research
Center, and the Departments of Pediatrics, Medicine, and Pathology, the
University of Washington, Seattle, WA.
 |
Abstract |
Notch signaling has been shown to play a key role in cell fate
decisions in numerous developmental systems. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we reported the
expression of human Notch-1 in CD34+ progenitors. In this study, we
evaluated the expression of human Notch-1 and Notch-2 protein by
hematopoietic cells. In immunofluoresence study, we detected low
amounts of Notch-1 and Notch-2 protein in both CD34+ and
CD34+Lin cells, high amounts in CD14+ monocytes as well as B and
T cells, but no expression in CD15+ granulocytes. We further found
that an immobilized truncated form of the Notch ligand, Delta-1,
induced apoptosis in monocytes in the presence of macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage colony-stimulating factor (GM-CSF). The widespread expressions of Notch
proteins suggest multiple functions for this receptor during
hematopoiesis. These studies further indicate a novel role for Notch in
regulating monocyte survival.
(Blood. 2000;95:2847-2854)
© 2000 by The American Society of Hematology.
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Introduction |
In the hierarchical development of the hematopoietic
system, the progeny of stem cells progressively lose self-renewal and proliferative capacity while committing to differentiate along distinct
pathways.1 The complexity of lineage determination during
hematopoiesis has led to the conflicting concepts of instructive and
stochastic models. In instructive models, cytokines have generally been
thought to determine lineage. However, current evidence indicates that
the lineage specificity of cytokine activity results from restricted
expression of their receptors rather than determination properties of
the actual cytokine receptor.2 In purely stochastic models,
lineage fate determination has been thought to result from a
probabilistic event(s), followed by cytokine-mediated expansion of cell
numbers within a particular lineage.3,4 Although this
represents a workable hypothesis, in other developmental systems the
potential of the progeny of multipotent precursor cells to adopt a
particular fate is often modified as a result of cell-cell
interactions, eg, those mediated via the Notch receptor.5,6
The evolutionary conserved Notch transmembrane receptor has been found
to be a fundamental cell-fate controlling mechanism that involves
signals between neighboring precursor cells.5-7 In general,
a precursor cell progresses to a more differentiated state in response
to specific signals. Notch is not associated with the transmission of
these signals, but appears to control fates by modulating a precursor
cell's ability to respond to signals. Although not confined to a
particular precursor type, Notch signaling is generally capable of
influencing the ability of a cell to progress from a less to a more
differentiated state. For example, during neurogenesis, a single cell
within a cluster of neuroepithelial precursor cells upon commitment to
neural development, initiates Notch-mediated signaling in adjacent
cells, inhibiting their neural potential, while allowing their
development along the epithelial lineage.8
Four paralogs of the Notch gene, Notch-1,-2,-3, and
-4,9-13 and 5 Notch ligands, including Jagged-1,
and -2, and Delta-1,-2, and -3,14-21 have been identified
in vertebrates. Notch interactions have been shown to affect cell-fate
decisions in multiple systems in both invertebrates and vertebrates.
Because successive cell-fate choices mediated by cell-cell interactions
may underlie hematopoietic processes, Notch signaling could potentially
play an important role.22 In support of this, we have
previously reported expression of Notch-1 messenger RNA (mRNA) in bone
marrow cells, including the CD34+ subset,23 and have
recently identified expression of Notch-1 and Notch-2 protein by
purified murine hematopoietic precursors.24 We and others
further demonstrated the expression of the Notch-1 ligand, Jagged-1, by
hematopoietic stroma cells.24-26 It has also been shown
that the abnormal activation of Notch signaling in either the murine or
human hematopoietic system, by expressing mutant, constitutively
activated forms of the receptor, profoundly affects hematopoietic
lineages.27-30 Most importantly, we have shown
that incubation of hematopoietic precursor cells with purified or
cell-bound Jagged-1 generated an increase in the formation of a
primitive precursor cell population, HPP-mix.24 Taken
together, these results provide ample evidence for a role of Notch
signaling in influencing cell-fate decisions during hematopoietic development.
To gain further insight into the specific cell-fate decision points
where Notch signaling may influence hematopoietic decisions, we
determined the protein expression of 2 members of this receptor family,
Notch-1 and Notch-2, in cells from marrow and
peripheral blood. We detected relatively high amounts of these
receptors in monocytes as well as in T and B lymphocytes, lower levels
in developing precursor cells, and no expression of Notch-1 or Notch-2 in granulocytes. Because high levels of Notch receptors were found in
monocytes, we further investigated the potential role of Notch signaling in the development of these cells. To activate Notch receptors in monocytes, we used a truncated form of the Notch ligand,
Delta-1, consisting of the extracellular domain without the
transmembrane and cytoplasmic domains. We have previously found this
ligand, when immobilized on a plastic surface, inhibits the
differentiation of C2 myoblast cells, indicative of Notch activation
(Varnum-Finney et al, submitted). We found that incubation of monocytes
with the plastic-immobilized Notch ligand in the presence of macrophage
colony-stimulating factor (M-CSF) led to apoptotic cell death, but did
not in the presence of granulocyte-macrophage colony-stimulating factor
(GM-CSF). These studies therefore demonstrate widespread expression of
Notch receptors during hematopoiesis, suggesting multiple roles for
Notch signaling, and further demonstrated a novel role for Notch in the
cytokine specific regulation of monocyte survival.
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Materials and methods |
Separation of marrow cells and peripheral blood
Human marrow samples were obtained after informed consent. Cells
were separated by Ficoll-Hypaque density gradient centrifugation (density 1.077), washed, and resuspended in phosphate-buffered saline
(PBS) with 2% human-type AB serum. Peripheral blood cells were
obtained from healthy volunteers and separated over a 2-layer density
gradient (density 1.090 and 1.108) by mixing Ficoll-Hypaque (density
1.077) and Hypaque (density 1.3) to separate monocytes and
granulocytes. The monocyte-rich layer was harvested and resuspended in
PBS with 2% human-type AB serum. The cells were further purified by
depleting T, B, and NK cells using antibodies 9.6 (anti-CD2), HD37
(anti-CD19), B1 (anti-CD20), CD56 (Immunotech, Marseilles, France), and
R10 (antiglycophorin A), followed by goat antimouse IgG (Dynabeads
M-450, Oslo, Norway), and then immunomagnetic bead depletion according
to the manufacture's instructions. Monocytes were further purified
using fluorescence-activated cell sorting by selecting cells with
forward and side-scatter properties characteristic of monocytes and by
excluding propidium iodide (Sigma, St Louis, MO) positive dead cells.
The proportion of monocytes in the isolated cell populations were
verified by staining with anti-CD14 antibody, with more than 95% of
sorted cells showing positive staining in all experiments.
Antibodies and immunofluoresence studies
Cells were incubated in 96-well v-bottom plates with antibodies
against hematopoietic differentiation antigens, including phycoerythrin
(PE)-labeled IgG antibodies against CD2, CD4, CD8, CD20, and CD33 (all
from Becton Dickinson, Sunnyvale, CA), and PE-labeled antiglycophorin A
(Pharmingen, San Diego, CA). Three color studies were performed using
the PE-Cy5 tandem dye directly conjugated to either CD14 or CD34 (both
from Immunotech, Westbrook, ME). For studies of granulocytes, the
unlabeled mouse IgM anti-CD15 antibody, 1G10, was used. PE-labeled
affinity purified antimouse IgM (Amac, Westbrook, ME) was used to
detect CD15 positive cells. Briefly, for 2-color or 3-color analysis,
cells were incubated at room temperature with PE-Cy5 antibodies and/or
with 1G10. After this, cells were washed twice and the 1G10-labeled
cells were incubated at room temperature with the PE antimouse IgM
antibody. The antihuman myc antibody F(ab')2 was
prepared from the 9E10 hybridoma in our laboratory.31 The
F(ab')2 fragment of the 9E10 antibody was prepared by
incubation of purified 9E10 (2 mg/mL in 0.1 mol/L sodium citrate pH
3.5) with Pepsin (Sigma Chemical Co, St Louis, MO; ratio of enzyme to
antibody was 1:200). The mixture was incubated at 37°C for 4 hours,
neutralized with 3 mol/L Tris pH 8.0 and chromatographed on HiTrap
Protein A Column (Amersham Pharmacia Biotech AB, Uppsala, Sweden).
For Notch-1 or -2 expression, cells were then washed twice and made
permeable by incubation with PermeaFix (Ortho, Raritan, NJ), washed
twice, and then incubated at room temperature with the rat antihuman
Notch-1 antibody 18G or antihuman Notch-2 antibody Bhn6 (kindly
provided by S. Artavanis and P. Zagouras, Massachusetts General
Hospital, Boston, MA). The preparation and characterization of these
antibodies that detect the intracellular domain of Notch-1 and -2 has
been described previously.32 Cells were again washed twice,
incubated at 4°C with mouse-adsorbed rabbit antirat antiserum, conjugated to biotin (Vector Laboratories, Burlingame, CA), washed twice, and then incubated at 4°C with streptavidin-fluorescein (Biosouce/Tago, Camarillo, CA.) Control staining was measured using
isotype-matched directly labeled or unlabeled antibody of irrelevant
specificity, except for the IgG2a CD14-PE-Cy5 antibody in which an
IgG1-PE-Cy5 control was used instead. Cells were analyzed and sorted
using a Vantage flow cytometer or FACScan (Becton Dickinson Co,
Mountain View, CA).
Immunohistochemical staining for Notch
Immunohistochemistry was performed on formalin-fixed, formic
acid-ethylenediamine tetraacetic acid (EDTA) decalcified,
paraffin-embedded tissues using the TechMate automated histostainer
(Ventana Medical Systems, Tucson, AZ). Sections 4 µm thick were cut
onto slides and allowed to dry for at least 1 hour in a
57°C oven. After deparaffinization and rehydration to
water, tissue sections were heated in 0.01 mol/L citric acid buffer, pH
6.0, for 20 minutes in a vegetable steamer and cooled for 5 minutes at
room temperature. Tissues were pretreated with 5% normal goat serum,
2% bovine serum albumen (BSA), 0.1% Tween-20 for 10 minutes. Primary
rat anti-Notch-1 or control antibody was diluted in PBS, 1% BSA, and
0.03% Tween-20 and was applied for 30 minutes at room temperature.
After washing, biotinylated rabbit antirat antibody (Dako, Carpinteria,
CA), was applied for 30 minutes, followed by quenching of endogenous peroxidase in 3% H2O2 for 7.5 minutes. The
biotinylated secondary antibody was detected with avidin-biotin complex
(ABC) (Vector, Burlingame, CA), followed by the chromogen
diaminobenzidine 0.5mg/mL, 0.1% NiCl2, 0.01%
H2O2. Sections were counterstained with 0.1% Acridine Orange/ 0.05% Safranin O, dehydrated, and mounted in a
toluene-based mounting media (Polymount, Poly Scientific, Bay Shore, NY).
Double staining for Notch and glycophorin was performed using
simultaneous biotin-streptavidin peroxidase and mouse APAAP techniques.
Briefly, anti-Notch-1 (18G) and mouse antiglycophorin antibodies
(Dako, Carpinteria, CA) were applied together, followed by simultaneous
application of biotinylated rabbit antirat and rabbit antimouse
antibody (Dako Carpinteria, CA). They were detected with pooled
streptavidin peroxidase (Zymed, South San Francisco, CA) and mouse
APAAP (Dako, Carpinteria, CA). Vector Red chromogen (Vector) was
applied after the diaminobenzidine step. Double staining for
myeloperoxidase and Notch was performed by applying rabbit antimyeloperoxidase antibody (Dako, Carpinteria, CA), which was detected with alkaline phosphatase-conjugated goat antirabbit antibody
(Dako, Carpinteria, CA), followed by Vector Red chromogen. After steam
pretreatment in citrate buffer, Notch staining was performed as above.
Extracellular domain of Delta-1 generation
These constructs were engineered to encode the extracellular domain
of human Delta-1(amino acids 1-536, GenBank accession number AF003722,
kindly provided by G. Gray, R. Mann, and S. Artavanis). cDNA sequences
were subcloned into the pcDNA3, adding a Cla-1 site to the 3' end
of the truncated Delta-1. A HindIII and Cla-1 fragment containing the
Delta-1 sequence was subcloned into PCS2 vector that contained
sequences encoding 6 consecutive myc epitopes (kindly provided by David
Turner and Hal Weintraub), so that 6 myc epitopes were expressed from
the carboxy terminus. An Eco-R1 fragment containing the truncated
Delta-1 and the myc tags was then subcloned into the expression vector
pcDNA3.1/amp (Invitogen, San Diego, CA) that added sequences to encode
6 histidines and a stop codon at the carboxy terminus.24
NSO myeloma cells were electroporated with constructs to generate
stable cell lines for protein production. G418-resistant clones were
screened for secretion of the fusion proteins using a quantitative
enzyme-linked immunosorbent assay (ELISA). In brief, wells were coated
with the 9E10 anti-myc antibody, washed, and then blocked with gelatin. Supernatant material generated by transfected cells was then added. After a period of incubation, Deltaext-myc protein was
detected, using a biotinylated anti-myc tag antibody. Clones that
generated the highest amounts of Deltaext-myc were expanded
in roller bottles and the medium collected. The medium was then
concentrated and the truncated ligand purified using methods previously
reported for the purification of a truncated Jagged-1
ligand.24 This procedure involved the binding of
concentrated medium to a nickel column (Ni-NTA Agarose, Qiagen,
Chatsworth, CA) using the His-Bind buffer Kit (Novagen, Madison, WI),
according to the manufacturer's instructions. After elution with
imidizol (5 mmol/L-1 mol/L), fractions that contained
Deltaext-myc were identified with Western blots using the
9E10 antibody. Positive fractions were then pooled, concentrated, and
dialyzed with PBS. The protein concentration was determined by
OD280. The control conditioned medium was collected from
nontransfected NSO and was similarly prepared using all the steps used
for purifying the ligand.
Monocyte cultures
Isolated monocytes were incubated in tissue culture wells containing
Deltaext-myc or control medium. In some cases, a mouse
anti-Myc antibody, 9E10, in the form of an F(ab')2
fragment, or a control murine anti-CD20 antibody
F(ab')2 fragment (kindly provided by D. Maloney, Fred
Hutchinson Cancer Research Center), was adhered to a 96-well plate
(Corning Incorporated, Corning, NY) at the concentration of 10 µg/mL
for 30 minutes at 37°C. After washing, coated wells were blocked
with Iscove's Modified Dulbecco's Medium (IMDM) containing 20% fetal
bovine serum (FBS) (Hyclone, Logan, UT) for 30 minutes at 37°C.
After washing, Deltaext-myc or control medium was applied
to the coated well for 30 minutes at 37°C in a humidified
atmosphere of 5% CO2 in air. Isolated monocytes were then
cultured in these wells in the presence of 10% FCS IMDM, containing 10 ng/mL M-CSF (Peprotech, Rocky Hill, NJ) or 100 ng/mL GM-CSF (Amgen,
Thousand Oaks, CA). Cultured cells were harvested by incubation with
PBS, containing 200 µmol/L EDTA (Gibco, Grand Island, NY) for 15 minutes on ice to enhance the detachment of adherent cells.
Apoptosis assays
Annexin V assays were performed according to the manufacture's
specifications. Briefly, cultured cells were collected, washed with
binding buffer, and incubated in 200 µL of binding buffer containing
5 µL of annexin V-FITC (Pharmingen, San Diego, CA) and 1.25 µg of
propidium iodide at room temperature. Apoptotic cells were analyzed
using a FACScan (Becton Dickinson Co, Mountain View, CA).
Statistical analysis
A Student t test was used to determine statistical significance.
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Results |
Immunocytochemistry of normal bone marrow
We determined the expression of Notch-1 in formalin-fixed biopsy
specimens of normal human bone marrow. As seen in Figure 1A, significant staining was observed, with
stained cells appearing immature, including cells with features of
monocytic and/or primitive erythroid cells. Background staining with an
isotype-matched control antibody was minimal (Figure 1B). To assess
Notch-1 expression in erythroid cells, we double-stained sections with
both anti-Notch-1 and antiglycophorin antibodies. As shown in Figure
1C, a portion of glycophorin-expressing cells also expressed Notch-1.
The remaining portion of Notch negative, glycophorin positive cells had
the appearance of more mature erythroid cells, such as acidophilic normoblasts. To assess Notch-1 expression in granulocyte precursors, we
double-stained sections with both anti-Notch-1 and antimyeloperoxidase antibodies. No overlap of staining was seen, indicating the absence of
Notch-1 expression in granulocytic cells (Figure 1D). These results
therefore demonstrate that substantial numbers of maturing cells
expressed the Notch-1 receptor, and mainly include monocytic and
erythrocytic elements.
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| Fig 1.
Immunocytochemistry of normal bone marrow.
(A) shows normal bone marrow stained with anti-Notch-1 antibody.
Positive staining is brown-black and the counterstain is a light-blue
hematoxylin. Positive staining is seen in the cytoplasm and appears to
be on the cell membrane in both erythroid precursors (seen in a patch
on the left as a narrow cytoplasmic rim in round cells with round
nuclei) and in myeloid precursors (seen in a patch on the right as
cytoplasmic staining of polygonal cells). Background staining with an
isotype-matched antibody of irrelevant specificity was minimal (B). (C)
shows a double stain for Notch-1 and glycophorin, with the latter seen
as red. A portion of the glycophorin-staining cells also contained
Notch-1. This portion contained mainly the larger more immature
glycophorin-staining cells, which the more mature, smaller erythroid
precursors did not stain for Notch 1. A few cells in this field contain
both. It appears that Notch-1 is lost as the cells mature and gain
glycophorin. (D) shows double-staining for Notch-1 and myeloperoxidase,
with the myeloperoxidase staining red. The majority of cells in this
field are of the granulocytic lineage, staining with myeloperoxidase
and sometimes showing polylobulated nuclei characteristic of mature
neutrophils. The Notch-1 stained cells appear not to be myeloperoxidase
positive, even when lightly stained. Some of the Notch-1 stained cells
are clearly small, round erythroid precursors, others have a more
polygonal cytoplasmic shape and sometimes a clefted nucleus consistent
with a monocytic cell. All photomicrographs were taken at magnification
× 60.
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Immunofluoresence analysis of normal bone marrow and peripheral
blood
We next used flow cytometry to study Notch-1 and Notch-2 expression
by marrow cells. In the CD34+ population, there was a small homogenous
shift in the fluorescence intensity curve after staining with either
Notch-1 or Notch-2 antibodies, indicating relatively low expression of
both Notch-1 and Notch-2 by essentially all cells (see representative
examples in Figure 2A). This result was
reproducibly seen in 4 individuals. Similarly, low levels of Notch-1
and Notch-2 were also found on the less differentiated CD34+
CD33Lin subpopulation (excludes cells expressing antigens associated with T [CD2] or B [CD19] cells). To assess Notch
expression in more mature myeloid populations, marrow cells were
double-stained with either Notch-1 or Notch-2 antibodies and either an
anti-CD14 antibody to identify monocytes or an anti-CD15 antibody to
identify granulocytes. Notch-1 and Notch-2 were seen in substantial
levels on a major portion of cells that expressed CD14 (Figure 2B). The fluorescence intensity of Notch-1 staining was greater on the CD14+
cells than on CD34+ cells. In contrast, no Notch-1 or Notch-2 was
detected on CD15+ cells. To assess Notch expression in developing erythroid cells, marrow cells were double-stained with either Notch-1
or Notch-2 antibodies and with antiglycophorin A antibodies. Little or
no Notch-1 and Notch-2 was detected in cells that expressed glycophorin
A (Figure 2B). However, further analysis revealed low levels of Notch-1
in cells expressing low levels of glycophorin A, suggesting that, as in
the immunocytochemistry studies, immature erythroid precursors express
Notch-1 (data not shown). Notch-2 was not detected in cells expressing
either high or low levels of glycophorin A (data not shown).
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| Fig 2.
Expression of Notch-1 and Notch-2 by human bone marrow
cells and peripheral blood.
(A) Fluorescence histograms of human bone marrow cells stained with
anti-Notch-1 and -2 antibodies and CD34, or CD34 and the absence of
other antigens associated with lineage commitment. The x-axis
represents log fluorescence intensity and the y-axis represents cell
number. The solid line represents staining with anti-Notch-1 and -2 antibodies, and the dashed line represents staining with an
isotype-matched control antibody of irrelevant specificity. (B) Notch-1
and -2 expression by human bone marrow cells committed to monocytic
(CD14), granulocytic (CD15), and erythroid (glycophorin A) lineages.
The x-axis represents log fluorescence intensity and the y-axis
represents cell number. The solid line represents staining with
anti-Notch 1-and 2-antibodies, and the dashed line represents staining
with an isotype-matched control antibody. (C) Notch-1 and -2 expression
by human peripheral blood cells. Fluorescence histograms for 2 color
studies gated on cells committed to monocytic (CD14), granulocytic
(CD15), T-cell (CD4, 8), or B-cell (CD20) lineages are shown. The
x-axis represents log fluorescence intensity and the y-axis represents
cell number. The solid line represents staining with anti-Notch-1 and
-2 antibodies, and the dashed line represents staining with an
isotype-matched control antibody.
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To assess Notch expression in peripheral blood, cells were
double-stained with antibodies to either Notch-1 or Notch-2 and antibodies to CD14, CD15, CD4, and CD8 (antigens expressed by subsets
of T lymphocytes) and CD20 (antigen expressed by B cells). As observed
in marrow, both Notch-1 and Notch-2 were detected in CD14+ monocytes,
but not CD15+ granulocytes (Figure 2C). Both Notch-1 and Notch-2 were
also found on CD4+ and CD8+ cells. Moreover, as shown in Figure 2C, a
slightly greater staining intensity was seen with Notch-1 in the CD8+
cells, compared with the CD4+ T cells. This difference was seen in 7 of
10 individuals studied (data not shown). Notch-2 staining, however, was
essentially equivalent in both CD4+ and CD8+ T cells. In addition, both
Notch-1 and Notch-2 were detected in CD20+ B cells.
Effect of Notch ligand on CD14+ monocytes in the presence of
M-CSF
Because monocytes express the highest amounts of Notch-1 and Notch-2
within the myeloid lineages, we further investigated the effect of
Notch activation on isolated monocytes in vitro. Monocytes were
isolated from Ficoll-Hypaque-separated peripheral blood mononuclear
cells, based on forward and right angle light-scatter properties.
Isolated cells were shown to be more than 95% positive for CD14
staining (data not shown). These cells were incubated with a truncated
form of the extracellular domain of the Notch ligand, Delta-1, which
contained the highly conserved DSL domain, thought to be critical for
activation of Notch and all the epidermal growth factor (EGF) repeats.
The truncated Delta-1 was further engineered to contain 6 myc tag
epitopes at the carboxy-terminus, Deltaext-myc (Figure
3A). Previous studies have demonstrated
that the differentiation of C2 myoblasts is inhibited by overexpressing
the constitutively active intracellular domain of Notch-1 or exposure
to cell-membrane-expressed Notch ligand.14,33 More recent
studies in our laboratory revealed that Deltaext-myc
inhibited differentiation of C2 cells, but only if it was first immobilized on the plastic surface of the culture wells (Varnum-Finney, submitted). We therefore tested the effect of Deltaext-myc
in a solution as well as in an immobilized form on monocyte growth and
differentiation. Deltaext-myc was immobilized by attaching
it to plastic via its myc tags, by first binding 9E10 anti-myc
F(ab')2 antibody fragments to the plastic surface. We
used F(ab')2 fragments because the whole antibody appeared to affect monocytes in culture, presumably as a result of the
effects of the Fc domain.
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| Fig 3.
Presentation of Deltaext-myc.
(A) A schematic diagram of the truncated form of the extracellular
domain of Delta-1, Deltaext-myc. The construct contains the
extracellular domain of Delta, including the signal peptide (SP),
conserved DSL domain, EGF repeats, and 6 myc tag epitopes. (B)
A schematic diagram of the presentation of Deltaext-myc.
Each panel represents a mode of presentation of
Deltaext-myc. In Panel A, Deltaext-myc is
presented in immobilized form, bound to 9E10 F(ab')2 antibody
fragments that is bound to the plastic. In Panel B,
Deltaext-myc is presented in the absence of antibody
fragments (see "Materials and methods").
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Increased amounts of Deltaext-myc were added to tissue
culture wells with or without anti-myc F(ab')2
antibody fragments bound to the plastic (Figure 3B). After 30 minutes,
monocytes were incubated in these wells in the presence of M-CSF, a
cytokine known to promote monocyte survival and differentiation into
macrophages.34,35 After 6 days, there was a
substantially lower number of cells in cultures containing
Deltaext-myc that was immobilized by attachment to
plastic-bound antibody fragments in comparison with cultures with only
nonimmobilized Deltaext-myc (Figure
4A). The reduction in cell
numbers in cultures containing immobilized Deltaext-myc was
maximal at 1 µg/mL of ligand, consistent with ELISAs demonstrating maximal saturation of the plastic-bound antibody by this dose of ligand
(data not shown). As additional controls, we evaluated the effect of
medium that was collected from nontransfected NSO cells and purified
identically to Deltaext-myc (control medium), and also
evaluated the effect of Deltaext-myc in wells coated with a
control F(ab')2 fragment. We found no effect of the
control material in the presence of the plastic-bound 9E10 antibody
fragments, compared with Deltaext-myc (Figure 4B). Nor did
we find an effect of Deltaext-myc in the presence of the
control, isotype-matched anti-CD20 F(ab')2 antibody
fragments, compared with the anti-myc antibody fragments, whereas
significant decreases in cell numbers were again seen in wells
containing both Deltaext-myc and the plastic-bound anti-myc
antibody fragments (Figure 4B).
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| Fig 4.
Effect of Deltaext-myc on monocyte survival.
Ten thousand monocytes were cultured for 6 days with 10% FCS IMDM with
10 ng/mL of M-CSF. In panel A, the effect of immobilized and
nonimmobilized ligand was compared by adding the soluble
Deltaext-myc (0, 0.3, 1, 3, and 10 µg/mL) to plastic
wells that were previously coated ( ) or not coated ( ) with 10 µg/mL of 9E10 F(ab')2 anti-myc antibody fragments. In panel B,
controls consisting of purified control medium from noninfected NSO
cells and control antibody fragments are shown. Monocytes
(5 × 103) were cultured with M-CSF in the presence
of 1 µg/mL of Deltaext-myc or control medium in wells
coated with 9E10 F(ab')2 or anti-CD20 F(ab')2 antibody
fragments. (*) represents statistical differences between cultures
containing Deltaext-myc in the presence of 9E10
F(ab')2 antibody fragments and either cultures
containing control medium in the presence of 9E10
F(ab')2 antibody fragments or cultures containing
Deltaext-myc in the presence of control, anti-CD20
F(ab')2 antibody fragments.
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Time course of reduction of monocyte numbers
We next determined the time course of the reduction of cell numbers
due to immobilized Deltaext-myc. Monocytes were incubated
in wells with 9E10 antibody F(ab')2 fragments
attached to the surface containing either Deltaext-myc or
control medium. M-CSF was then added to the wells and, after 1, 2, 4, and 6 days, the cells were collected and counted. The number of
monocytes in the presence of Deltaext-myc decreased
dramatically until day 3, where 36% of the cell numbers in the control
well was seen, and afterward declined at a more gradual rate similar to
that seen in control cultures (Figure 5).
This rapid reduction of the number of cells during the initial 2 days
of culture was observed using monocytes obtained from each of 3 different individuals (Table 1).
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| Fig 5.
Effect of Deltaext-myc on monocyte survival.
The number of monocytes present in cultures containing 10% FCS IMDM
and 10 ng/mL M-CSF in the presence of 1 µg/mL of
Deltaext-myc ( ) or control (). Tissue culture wells
were coated with anti-myc antibody, 9E10 F(ab')2 to
attach the myc containing Deltaext-myc to the plastic
surface. Ten thousand monocytes were added to each well and after 1, 2, 4, and 6 days, cells were collected and counted. (*) represents
statistical difference compared with the respective control.
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Apoptosis of monocytes in the presence of
Deltaext-myc and macrophage colony-stimulating factor
To determine whether the lower numbers of cells present in cultures
of monocytes with immobilized Deltaext-myc and M-CSF
resulted from an increase in programmed cell death, apoptosis was
evaluated using annexin V and, as an indication of cell viability,
propidium iodide. As shown in Figure 6A,
cultures containing the immobilized Deltaext-myc showed an
approximately 3-fold increase in the proportion of cells undergoing
apoptosis that stained positively for annexin V but did not stain
with propidium iodide, compared with cells from control cultures at 12 hours. By 48 hours, there was an increase in the portion of cells
staining positively with propidium iodide as well as annexin V,
suggesting the cells proceeded to a necrotic state (Figure 6). This
result was seen with cells from 2 additional individuals (data
not shown).
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[in this window]
[in a new window]
| Fig 6.
Induction of apoptosis in monocytes by
Deltaext-myc in the presence of M-CSF.
Ten thousand monocytes were cultured with 10% FCS IMDM containing
M-CSF (10 ng/mL) in the presence of Deltaext-myc (1 µg/mL) or as a control, control material from nontransfected NSO
cells. All wells were coated with anti-myc 9E10
F(ab')2 fragments. Cells were harvested at 12, 24, and 48 hours, washed with binding buffer, and incubated in binding
buffer containing annexin V-FITC and PI. The x-axis represents log
fluorescence intensity with PI staining; the y-axis represents log
fluorescence intensity with annexin V staining.
|
|
We also investigated the morphology and nonspecific esterase staining
of cells surviving in the presence or absence of immobilized Delta.
After 6 days of culture with M-CSF and immobilized
Deltaext-myc, cytocentrifuge slides of the cells were
prepared and stained with hematoxylin and eosin, and with nonspecific
esterase. Cells obtained from cultures with or without ligand had the
appearance of macrophages and stained positively with nonspecific
esterase, indicating that in each case these cells had differentiated
into macrophages (data not shown).
Apoptosis occurs in the presence of macrophage colony-stimulating
factor but not granulocyte-macrophage colony-stimulating factor
To determine whether the induction of apoptosis by
Deltaext-myc was specific for M-CSF, we evaluated the
effect of another cytokine known to support the proliferation and
differentiation of monocytes, ie, GM-CSF.36-38 Although
substantial proportion of cells in cultures with M-CSF and immobilized
Deltaext-myc underwent apoptosis (annexin V+ propidium
iodide) after 12 hours, we saw no significant difference between
cultures containing GM-CSF, or GM-CSF plus M-CSF (Figure
7). This result was seen with cells from 2 additional individuals (data not shown). These data indicate that
apoptosis due to Deltaext-myc is induced in a
cytokine-specific manner. Furthermore, because apoptosis was not seen
in the presence of GM-CSF and M-CSF, GM-CSF appears to abrogate the
expected apoptosis induced by Deltaext-myc in the presence
of M-CSF.
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
Induction of apoptosis in monocytes by
Deltaext-myc in the presence of M-CSF compared to GM-CSF.
Monocytes were cultured with 10% FCS IMDM containing M-CSF (10 ng/mL),
GM-CSF (100 ng/mL), or M-CSF plus GM-CSF in the presence of
Deltaext-myc (1 µg/mL) or purified control medium. All
wells were coated with anti-myc 9E10 F(ab')2
fragments. Cells were harvested at 12 hours, washed with binding
buffer, and incubated in binding buffer containing annexin V-FITC and
PI. The mean and standard error of annexin V positive cells in the PI
negative window for the 3 wells are shown. (*) represents statistical
difference compared with respective control.
|
|
| |
Discussion |
To determine cell lineages in which Notch-mediated interactions may
be relevant in cell-fate decisions, we evaluated the
maturation-dependent expression of 2 members of the Notch receptor
family, Notch-1 and Notch-2, using immunocytochemistry and quantitative
immunofluoresence. We detected low levels of Notch-1 and Notch-2
protein on both CD34+ and CD34+ Lin cells and found that these
levels in CD34+ cells increased as they matured into monocytes, but
decreased as they differentiated into granulocytes. Surprisingly, the
greatest amounts of Notch receptor expression were in the more mature
cells, with monocytes expressing the highest amounts of Notch-1 within the myeloid lineages. It was thought possible that the Notch activity in monocytes may affect their survival and/or subsequent decisions regarding further differentiation into macrophages. Because the survival of monocytes and their differentiation into macrophages are
promoted by the cytokine, M-CSF, we first determined the effect of a
truncated Notch ligand, Delta-1, on monocytes in the presence of this
cytokine.34,35 Previous studies in our
laboratory have indicated that the presentation of this ligand,
Deltaext-myc in immobilized form, but not in solution,
induced inhibition of muscle differentiation by C2 myoblast cells,
indicative of Notch activation (Varnum-Finney et al, submitted). In
those studies, a second construct, Deltaext-IgG, consisting
of the extracellular domain of Delta-1 and the Fc portion of human
IgG1, and a control construct, ControlIgG,
consisting of the Jagged-1 signal peptide fused to the Fc portion of
human IgG1, were also studied. Immobilized
Deltaext-IgG, as well as Deltaext-myc, but not
immobilized ControlIgG, inhibited C2 myoblast
differentiation, further demonstrating the specificity of the
effect (Varnum-Finney et al, submitted). It was not
possible, however, to use these latter constructs for studies of
monocytes, because the Fc domain of the constructs affected monocyte
survival. Deltaext-myc was therefore presented in an
immobilized form tethered to the surface of the culture well by
plastic-bound anti-myc F(ab')2 antibody fragments,
which recognized myc-tags included in the engineered ligand.
In this study, we observed significant apoptosis in monocytes after
incubation with immobilized Deltaext-myc in the presence of
M-CSF. Although immobilization of the truncated ligand was required for
the induction of cell death in monocytes, other studies have found that
nonimmobilized soluble forms of a Notch ligand can inhibit
oligodendrocyte differentiation or neurite retraction in vitro as
indicators of Notch activation.39,40 Moreover, conflicting
data has also been observed in vivo, where soluble, truncated forms
have caused inhibitory as well as activating effects on Notch
signaling.41,42 Further studies will be required to
determine the responsible mechanisms and reasons for differences between different cell systems and possibly between the different ligand forms used in these experiments.
Monocytes are known to undergo spontaneous apoptosis in the absence of
specific survival signals, but on differentiation into macrophages,
become more resistant to cell death.43-45 Because M-CSF is
known to promote the survival of monocytes and their differentiation
into macrophages,34,35 it is tempting to
speculate that Notch activation inhibited M-CSF-induced signaling,
thereby enhancing apoptosis. A portion of cells did, however, survive in cultures containing Deltaext-myc. These cells, which
were nonspecific esterase positive macrophages, may have been derived
from more mature cells that are more resistant to apoptosis, or from
cells less sensitive to effects of Deltaext-myc.
We further found that apoptosis was induced in the presence of M-CSF
but not GM-CSF. Thus, the effect of Notch signaling is a cytokine
specific one. Although the mechanism for this specificity is not known,
studies in 32D cells have shown that the constitutively active forms of
the intracellular domains of Notch-1 and Notch-2 inhibited the
differentiation of 32D cells in response to specific cytokines.28 These studies found that the
activated, intracellular domain of Notch-1 was inhibitory to
G-CSF-induced differentiation, and the Notch-2 intracellular domain
was inhibitory to GM-CSF-induced differentiation. These studies
further suggested that a specific Notch domain, termed the Notch
cytokine response region, contributed to this cytokine
specificity.28 Alternatively, Notch signaling inhibits the transcriptional expression of genes that are normally induced by M-CSF and are part of the differentiation and survival program required for M-CSF-induced terminal macrophage
differentiation. In this case, the expression of those genes induced by
GM-CSF-induced signaling may not be inhibited by the Notch pathway.
Consistent with this notion is the observation that M-CSF and GM-CSF
generate macrophages that are phenotypically and functionally
distinct.37,38 We are currently exploring whether, in the
presence of GM-CSF, Notch signaling allows the survival of cells with
the appearance of macrophages, but which are not terminally committed
and retain the capacity to either further differentiate along the
macrophage lineage, or along an alternative cellular pathway, ie,
dendritic cells.46-48
A role for Notch signaling in preventing apoptosis has already been
determined in T-cell development. Notch-1-mediated signaling has been
found obligatory for T-cell development,49 and further evidence indicates an antiapoptotic effects of Notch-1 signaling in
immature CD4+ CD8+ T cells and T-cell lines. In one study, overexpression of constitutively active intracellular domain of Notch-1
inhibited glucocorticoid-induced apoptosis of a T-cell leukemic cell
line.50 Further studies have shown that Notch-mediated effects on cell survival in T cells are mediated by
Nur-77.51 The finding in this study of an inductive rather
than inhibitory effect on apoptosis in monocytes, taken together with
results of studies of T cells, clearly indicates a more general role
for Notch signaling in regulating hematopoietic cell survival.
The widespread expression of at least 2 Notch receptors in developing hematopoietic cells and their lineage-restricted expression by the
progeny of multipotent precursors is consistent with a role for Notch
signaling in a variety of cell-fate decision during hematopoietic
development, including decisions by more mature blood elements.
Relatively little is known, however, about potential differences in
signaling induced by the different Notch receptors. We found that
Notch-2 staining levels were slightly higher relative to Notch-1 in
CD34+ and CD34+ Lin cells, whereas staining levels with Notch-1
were relatively greater than with Notch-2 on monocytes and T cells, and
only Notch-1 was detected in erythroid precursors. These differences in
the relative expression of Notch-1 and Notch-2 by cell subsets suggest
the possibility of distinct roles for the different Notch receptors.
Recent evidence that Notch-1 and Notch-2 may affect distinct
cytokine-activated signaling pathways further supports the notion of
distinct activities for the different Notch
paralogs.28 It is also not known whether the
ligand used in this study preferentially activates 1 or more of the
different Notch receptors.
Although this study has focused on Notch-1 and Notch-2, at least 4 members of the Notch family9-13 and 5 Notch
ligands14-21 have been identified in vertebrates. Thus, a
more complex story of Notch and Notch ligand interactions is likely to
unfold. Nonetheless, it is clear that future evaluations of the
ontogeny of expression of these molecules in hematopoietic development
may provide important insights into their role in development as well
as insights in guiding experiments to determine their physiologic role.
These studies also indicated a role for Notch signaling in regulating more mature myeloid elements, suggesting further investigations of the
role of Notch-mediated cellular interactions on monocyte differentiation.
| |
Acknowledgments |
We thank Monica Yu, Carolyn Brashem-Stein, Steven Staats, Doug Wilson,
and Rita E. Ryncarz for their excellent technical assistance; Dr Robert
G. Andrews and Dr Louise E. Purton for critically reviewing the
manuscript; and Lynn Planet for preparation of the manuscript.
| |
Footnotes |
Submitted November 22, 1999; accepted January 7, 2000.
Supported by NIH grants HL54881, CA-18029, and CA-15704. K.O. is a
Fellow of the Leukemia Society of America. I.D.B is supported as a
Clinical Research Professor by the American Cancer Society.
Reprints: Irvin D. Bernstein, Fred Hutchinson Cancer Research
Center, 1100 Fairview Ave N, C1-169, Seattle WA 98109.
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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T. Yamada, H. Yamazaki, T. Yamane, M. Yoshino, H. Okuyama, M. Tsuneto, T. Kurino, S.-I. Hayashi, and S. Sakano
Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells
Blood,
March 15, 2003;
101(6):
2227 - 2234.
[Abstract]
[Full Text]
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V. Orgogozo, F. Schweisguth, and Y. Bellaiche
Binary cell death decision regulated by unequal partitioning of Numb at mitosis
Development,
March 12, 2003;
129(20):
4677 - 4684.
[Abstract]
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B. Varnum-Finney, C. Brashem-Stein, and I. D. Bernstein
Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors with lymphoid and myeloid reconstituting ability
Blood,
March 1, 2003;
101(5):
1784 - 1789.
[Abstract]
[Full Text]
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L. Espinosa, J. Ingles-Esteve, A. Robert-Moreno, and A. Bigas
Ikappa Balpha and p65 Regulate the Cytoplasmic Shuttling of Nuclear Corepressors: Cross-talk between Notch and NFkappa B Pathways
Mol. Biol. Cell,
February 1, 2003;
14(2):
491 - 502.
[Abstract]
[Full Text]
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A. H. Campos, W. Wang, M. J. Pollman, and G. H. Gibbons
Determinants of Notch-3 Receptor Expression and Signaling in Vascular Smooth Muscle Cells: Implications in Cell-Cycle Regulation
Circ. Res.,
November 29, 2002;
91(11):
999 - 1006.
[Abstract]
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S. Weijzen, M. P. Velders, A. G. Elmishad, P. E. Bacon, J. R. Panella, B. J. Nickoloff, L. Miele, and W. M. Kast
The Notch Ligand Jagged-1 Is Able to Induce Maturation of Monocyte-Derived Human Dendritic Cells
J. Immunol.,
October 15, 2002;
169(8):
4273 - 4278.
[Abstract]
[Full Text]
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R. Hubmann, J. D. Schwarzmeier, M. Shehata, M. Hilgarth, M. Duechler, M. Dettke, and R. Berger
Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia
Blood,
May 15, 2002;
99(10):
3742 - 3747.
[Abstract]
[Full Text]
[PDF]
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L. Espinosa, S. Santos, J. Ingles-Esteve, P. Munoz-Canoves, and A. Bigas
p65-NF{kappa}B synergizes with Notch to activate transcription by triggering cytoplasmic translocation of the nuclear receptor corepressor N-CoR
J. Cell Sci.,
March 15, 2002;
115(6):
1295 - 1303.
[Abstract]
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X.-Q. Yan, U. Sarmiento, Y. Sun, G. Huang, J. Guo, T. Juan, G. Van, M.-Y. Qi, S. Scully, G. Senaldi, et al.
A novel Notch ligand, Dll4, induces T-cell leukemia/lymphoma when overexpressed in mice by retroviral-mediated gene transfer
Blood,
December 15, 2001;
98(13):
3793 - 3799.
[Abstract]
[Full Text]
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L. Walker, A. Carlson, H. T. Tan-Pertel, G. Weinmaster, and J. Gasson
The Notch Receptor and Its Ligands Are Selectively Expressed During Hematopoietic Development in the Mouse
Stem Cells,
November 1, 2001;
19(6):
543 - 552.
[Abstract]
[Full Text]
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A. C. Jaleco, H. Neves, E. Hooijberg, P. Gameiro, N. Clode, M. Haury, D. Henrique, and L. Parreira
Differential Effects of Notch Ligands Delta-1 and Jagged-1 in Human Lymphoid Differentiation
J. Exp. Med.,
October 1, 2001;
194(7):
991 - 1002.
[Abstract]
[Full Text]
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K. Ohishi, B. Varnum-Finney, R. E. Serda, C. Anasetti, and I. D. Bernstein
The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells
Blood,
September 1, 2001;
98(5):
1402 - 1407.
[Abstract]
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J. Wang, L. Shelly, L. Miele, R. Boykins, M. A. Norcross, and E. Guan
Human Notch-1 Inhibits NF-{{kappa}}B Activity in the Nucleus Through a Direct Interaction Involving a Novel Domain
J. Immunol.,
July 1, 2001;
167(1):
289 - 295.
[Abstract]
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B. K. Hadland, N. R. Manley, D.-m. Su, G. D. Longmore, C. L. Moore, M. S. Wolfe, E. H. Schroeter, and R. Kopan
gamma -Secretase inhibitors repress thymocyte development
PNAS,
June 19, 2001;
98(13):
7487 - 7491.
[Abstract]
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F. N. Karanu, B. Murdoch, T. Miyabayashi, M. Ohno, M. Koremoto, L. Gallacher, D. Wu, A. Itoh, S. Sakano, and M. Bhatia
Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells
Blood,
April 1, 2001;
97(7):
1960 - 1967.
[Abstract]
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N. Ohno, A. Izawa, M. Hattori, R. Kageyama, and T. Sudo
dlk Inhibits Stem Cell Factor-Induced Colony Formation of Murine Hematopoietic Progenitors: Hes-1-Independent Effect
Stem Cells,
January 1, 2001;
19(1):
71 - 79.
[Abstract]
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J. Ingles-Esteve, L. Espinosa, L. A. Milner, C. Caelles, and A. Bigas
Phosphorylation of Ser2078 Modulates the Notch2 Function in 32D Cell Differentiation
J. Biol. Chem.,
November 21, 2001;
276(48):
44873 - 44880.
[Abstract]
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Top
Abstract
Introduction