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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3764-3771
Altered Development and Cytokine Responses of Myeloid Progenitors in
the Absence of Transcription Factor, Interferon Consensus Sequence
Binding Protein
By
Marina Scheller,
John Foerster,
Clare M. Heyworth,
Jeffrey F. Waring,
Jürgen Löhler,
Gary L. Gilmore,
Richard K. Shadduck,
T. Mike Dexter, and
Ivan Horak
From the Abteilung für Molekulare Genetik, Forschungsinstitut
für Molekulare Pharmakologie, und Universitätsklinikum
Benjamin Franklin, Freie Universität Berlin, Berlin, Germany;
Heinrich-Pette Institut der Universität Hamburg, Hamburg,
Germany; Paterson Institute for Cancer Research, Manchester, UK; and
The Western Pennsylvania Cancer Institute, Pittsburgh, PA.
 |
ABSTRACT |
Mice deficient for the transcription factor, interferon consensus
sequence binding protein (ICSBP), are immunodeficient and develop
disease symptoms similar to human chronic myeloid leukemia (CML). To elucidate the hematopoietic disorder of
ICSBP / mice, we investigated the growth,
differentiation, and leukemogenic potential of ICSBP/
myeloid progenitor cells in vitro, as well as by cell-transfers in
vivo. We report that adult bone marrow, as well as fetal liver of
ICSBP-deficient mice harbor increased numbers of progenitor cells,
which are hyperresponsive to both granulocyte macrophage colony-stimulating factor (GM-CSF) and G-CSF in vitro. In contrast, their response to M-CSF is strongly reduced and, surprisingly, ICSBP/ colonies formed in the presence of M-CSF are
mostly of granulocytic morphology. This disproportional differentiation
toward cells of the granulocytic lineage in vitro parallels the
expansion of granulocytes in ICSBP/ mice and
correlates with a 4-fold reduction of M-CSF receptor expressing cells
in bone marrow. Cell transfer studies showed an intrinsic leukemogenic
potential and long-term reconstitution capability of
ICSBP/ progenitors. Further experiments demonstrated
strongly reduced adhesion of colony-forming cells from
ICSBP/ bone marrow to fibronectin. In summary,
ICSBP/ myeloid progenitor cells share several
abnormal features with CML progenitors, suggesting that the distal
parts of signaling pathways of these two disorders are overlapping.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTERFERON CONSENSUS sequence binding
protein (ICSBP)1 is a member of the interferon regulatory
factor family (IRF) of transcription factors.2,3 IRFs were
first described as regulators of the expression of interferons (IFNs)
and IFN-regulated genes. However, recent studies also indicated
additional growth regulatory functions. IRF-1 has been shown to have
some tumor suppressor activity and IRF-2 an oncogenic
function.4 All 7 members of this family identified to date,
IRF-1,5 IRF-2,6 IRF-3,7
IRF-4/Pip,8,9 IRF-7,10 ISGF3 ,11
and ICSBP1 possess a conserved N-terminal DNA binding
region and a more variable C-terminal part, which mediates protein
interactions and transcriptional activity.
In vitro experimental data initially suggested that ICSBP is a negative
regulator of -IFN and  / -IFN-induced genes.12 To
gain more insight into its in vivo function, we have generated ICSBP-deficient mice (ICSBP/) by gene
targeting. We reported that these mice are immunodeficient and develop
a myeloproliferative syndrome that is in some respects similar to human
chronic myeloid leukemia (CML).13,14 It manifests with high
counts of granulocytic and B-lymphoid cells, as well as an increased
number of immature cells in peripheral blood and in hematopoietic
organs. The composition and the cellularity of hematopoietic organs in
ICSBP/ mice changes progressively with their age.
The primary genetic lesions of the hematopoietic disorder in CML and in
ICSBP/ mice are clearly different. The
molecular hallmark of CML is the t9;22 chromosomal translocation,
resulting in bcr-abl fusion and activation of c-abl tyrosine
kinase.15,16 Previously, we have not been able to detect
any altered abl mRNA expression and the genomic organization of c-abl
in ICSBP/ mice.13 Nevertheless,
although the role of ICSBP in human CML has not yet been studied in
detail, its lack of or strongly reduced expression was found in 79% of
CML and 66% of acute myeloid leukemia (AML)
patients.17
Myeloid progenitor cells from CML patients exhibit several cellular
features in vitro potentially relevant for the development and/or
progression of clinical disease. These include exaggerated growth
responses to cytokines18,19 and an altered adhesion to
components of the extracellular matrix.20-22 Therefore, we
investigated the response of ICSBP/
hematopoietic progenitor cells to interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and
M-CSF in vitro, as well as their adhesion to fibronectin and laminin.
We also studied their proliferative and differentiation potential in
vivo with a series of cell transfer experiments in lethally irradiated mice. Our results show several features shared by myeloid progenitor cells from CML patients and ICSBP-deficient mice and also demonstrate that ICSBP/ precursors harbor an intrinsic,
cell-autonomous leukemogenic potential. Thus, while the primary genetic
lesion underlying both syndromes differs, the cellular signaling
pathways driving a leukemic phenotype characterized by
hyperproliferation of myeloid cells may be partially overlapping.
| |
MATERIALS AND METHODS |
Mice.
ICSBP-deficient mice were generated as described.13 All
experiments reported in the present report were performed with
ICSBP/ mice bred on a mixed C57/BL6 × 129/Ola background.
Cell preparation.
Single-cell suspensions from bone marrow were prepared by flushing
femora with Iscove's modified Dulbecco's medium (IMDM) containing 2%
fetal calf serum (FCS). Single-cell suspensions from spleen and liver
were obtained by pressing the organs through a stainless steel sieve.
Fetal liver single-cell suspension were made by passing the homogenized
organ through a 22-G and subsequently a 27-G needle in the same medium.
Peripheral blood leukocytes were obtained by centrifugation after
erythrocyte lysis in 0.83% ammonium chloride solution and washing in
phosphate-buffered saline (PBS).
Quantification of c-fms expression in bone marrow.
Bone marrow cells were flushed out of femora with
fluorescence-activated cell sorting (FACS) buffer (PBS containing 2%
newborn calf serum, 2 mmol/L EDTA, 0.1% sodium azide), subjected to
red blood cell (RBC) lysis, and stained with a 1:100
dilution of monoclonal antibody (MoAb) 604B5 2E11,23
followed by staining with phycoerythrin (PE)-conjugated anti-rat
F(ab)2 (mouse-Ig cross-adsorbed; Jackson Immunoresearch,
West Grove, PA). After removal of unbound antibody, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-F 4/80 (Serotec, Oxford, UK). Flow cytometry was
performed on a FACS-calibur (Becton Dickinson, Heidelberg,
Germany); cells were gated for viability by propidium
iodide exclusion.
Enrichment of c-kit+-progenitor cells from bone marrow.
Bone marrow single-cell suspensions, prepared as detailed above, were
depleted with a cocktail of the following FITC-conjugated antibodies:
Gr-1, CD19, CD4, CD8, DX5, Thy-1.2, CD11b, F4/80, and Ter 119 (all from
Pharmingen, Hamburg, Germany) followed by magnet-activated cell sorting (MACS)-depletion on an
AS-depletion column (Miltenyi Biotec, Bergisch Gladbach,
Germany) according to the instructions of the
manufacturer. Absence of lineage markers and c-kit expression in
depleted fractions was documented by FACS analysis (see Results).
In vitro colony-forming assays.
The assays were performed essentially as described by Heyworth and
Spooncer.24 Single-cell suspensions of bone marrow (5 × 104/mL), spleen (105/mL), fetal or
adult liver (5 × 104/mL or
105/mL), and blood (105/mL), were plated in
triplicate in 1 mL IMDM (ICN, Eschwege, Germany) supplemented with 20% FCS (Biochrom, Berlin,
Germany), 1% bovine serum albumin (BSA) (Boehringer,
Mannheim, Germany), glutamine, 2-mercaptoethanol (5 × 105 mol/L), and 0.3% bacterial grade soft
agar (DIFCO, Augsburg, Germany) in 35-mm petri dishes
and incubated at 37°C in an humidified incubator gassed with 5%
CO2 and 5% O2. For assays of committed myeloid
progenitors, purified recombinant murine (rm) IL-3,
rmGM-CSF, rmG-CSF, rm-stem cell factor (SCF) (TEBU
GMBH, Frankfurt, Germany), rmM-CSF (R & D, Wiesbaden,
Germany) were used at the concentrations indicated in
the Results. Individual colonies (defined by >50 cells) were scored
at 7 days postplating. For the determination of colony morphology, agar
cultures were fixed, dried onto glass slides, and stained with Giemsa
solution. In all cases, triplicate determinations were performed on
each sample. Statistical significance compared with the
ICSBP+/+ populations was determined using Student's
t-test.
Colony-forming unit-spleen (CFU-S) assay and determination of
self-renewal capacity.
CFU-S assays were performed as described by Lord.25 Bone
marrow cells from ICSBP/ and
ICSBP+/+ mice (1 × 105 cells/mouse) were
injected intravenously (IV) into lethally irradiated (C57/BL6 × 129/Ola) F2 mice (for the irradiation protocol, see below).
After 12 days, the recipient mice were killed, the spleens were
removed, and the number of colonies per spleen (CFU-S12) was counted (s). The seeding efficiency (f) was
measured by a double transplantation technique.25 The total
number of spleen colony-forming cells (CFC-S) is given by
CFU-S12 divided by f.
Determination of self-renewal.
A known fraction (1/k) of each primary recipient spleen was
injected into lethally irradiated secondary recipient mice. The colonies per spleen in each secondary recipient (m) were
counted after 12 days as detailed above for the CFU-S assay.
Self-renewal capacity of the CFU-S colonies in the primary recipient
mice was defined as average CFU-S per primary colony as follows:
(k × m/s).
Long-term reconstitution assay.
Adult recipient mice (>8 weeks old) were irradiated with a dose of 12 Gy total body irradiation using a 18 MeV photon beam from a linear
electron accelerator with a dose rate 0.18 Gy/min. These mice were
reconstituted IV with 5 × 106 or 1 × 106 freshly isolated bone marrow or fetal liver cells in
0.2 mL PBS within 24 hours of irradiation. Reconstituted mice were
tail-bled every 2 weeks beginning 4 weeks after transplant and every 4 weeks begining at 18 weeks posttransplant. Differential white blood cells counts were performed on May-Grünwald-Giemsa-stained blood smears.
Cell adhesion assay.
c-kit+-enriched bone marrow fractions were prepared as
described above. Forty-eight-well plates (Falcon, Heidelberg,
Germany) were coated with 50 µg/mL of mouse
fibronectin (Chemicon, Hofheim, Germany), 50 µg/mL
laminin (Boehringer Mannheim), or with 1% BSA (Sigma, Deisenhofen,
Germany) in PBS overnight at 4°C. Nonspecific binding was blocked
with PBS supplemented with heat-denaturated 1% BSA for 2 hours at room
temperature. Cells were resuspended to 2 × 106
cells/well in serum-free IMDM and plates were incubated for 60 minutes
at 37°C in a 5% CO2 incubator in the presence or in
the absence of the cytokines indicated in Results. Nonadherent cells were removed by shaking the plate and washing twice with warm IMDM.
Adherent cells were harvested using trypsin/EDTA solution (GIBCO)
followed by 3 additional washes with warm IMDM. Nonadherent cells were
treated with trypsin/EDTA solution and IMDM medium as well. Both
adherent and nonadherent cell fractions were then washed in IMDM,
counted, and cultured in soft agar for CFC in the presence of rmIL-3
(10 ng/mL), as described above. Percentage of adherent CFC was
calculated as the number of CFC in the adherent fraction divided by the
number of CFC in both fractions × 100%.
FACS analysis of integrins on c-kit+ bone marrow cells.
Bone marrow cells of ICSBP+/+ and
ICSBP/ mice were prepared as detailed above
including RBC lysis. Staining was performed by incubation of
single-cell suspension with MoAbs against 1 (9EG7),
4 (R1-2), or 5 integrins (MFR5) at a
dilution of 1:100 simultaneously with biotinylated anti-c-kit and
R-PE-labeled anti-GR-1 (all from Pharmingen) for 30 minutes at
4°C followed by staining with anti-rat FITC-conjugated F(ab)
(cross-adsorbed to mouse; Jackson ImmunoResearch) and
streptavidin-allophycocyanin (APC) conjugate
(Pharmingen). Control samples were only stained with secondary
reagents. Flow cytometry was performed on a FACSCalibur (Beckton
Dickinson); viability gates were set by propidium iodide exclusion.
Functional determination of
41 and
51 integrins on
committed myeloid progenitor cells.
MoAbs against 1 (9EG7), 4 (R1-2), or
5 integrins (MFR5) (Pharmingen) were coated on 48-well
plates in PBS, pH 7.4, at a concentration of 2 µg/mL overnight at
4°C. IGg2a and IGg2b (Pharmingen) were used as isotype-matched
controls. Nonspecific binding was blocked with 0.2% BSA. Cells were
added to the coated plates at 106 cells in 200 mL per well
in IMDM and incubated for 1 hour at 37°C. Nonadherent and adherent
cells were processed exactly as detailed above and subjected to CFC
assay in the presence of rmIL-3 as described above.
| |
RESULTS |
Hypersensitivity of hematopoietic progenitors from
ICSBP/ mice to IL-3, GM-CSF, and G-CSF.
To dissect the impact of the ICSBP mutation on hematopoietic activity,
we analyzed colony-forming ability of progenitor cells in soft agar
cultures containing cytokines important in myeloid development.
Hematopoietic organs and peripheral blood of
ICSBP/ mice contain higher numbers of cells
that generate in vitro colonies in the presence of either IL-3, GM-CSF,
or G-CSF in comparison to ICSBP+/+ mice
(Fig 1A and B). In addition, our results
show the postnatal persistence of myeloid progenitors in peripheral
blood, as well as in adult liver and spleen of
ICSBP/ mice. A significant increase of CFC is
seen in 17-day-old ICSBP/ mice, progressing
with age. The increase of myeloid progenitor cells was observed in
fetal livers as early as 12.5 days postcoitum (dpc)
(Fig 1C), demonstrating a critical role of ICSBP in the early stages of
hematopoiesis.
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| Fig 1.
Increased number of myeloid progenitor cells generated in
ICSBP/ mice in response to IL-3 and GM-CSF in vitro.
Single-cell suspensions from hematopoietic organs and peripheral blood
from either ICSBP/ or from wild-type mice were plated
in soft agar in the presence of the rmIL-3 (10 ng/mL) and GM-CSF (10 ng/mL). Colonies of more than 50 cells were scored on day 7 postplating. Data shown represent the mean ± standard error of mean
(SEM) of colonies counted in 2 independent experiments performed in
triplicate with 4 mice per group in each experiment. (A) Bone marrow,
spleen, and liver cells at 17 days and 6 weeks of age. (B) Peripheral
blood leukocytes of 17-day-old and 6-week-old mice (G-CSF, 100 ng/mL).
(C) Fetal liver cells (12.5 dpc).
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Dose-response curves obtained in the presence of IL-3, GM-CSF, and
G-CSF, respectively (Fig 2A), indicate that
the increased number of ICSBP/ colonies
observed in Fig 1 is correlated with a significant hyperresponsiveness of ICSBP/ bone marrow progenitor cells to
GM-CSF and, to a lesser extent, to G-CSF. As much as 10-fold lower
concentrations of GM-CSF or G-CSF supported maximal growth of
ICSBP/ progenitor cells, as compared with
wild-type cells. We also noted that the
ICSBP/ colonies generated in response to
G-CSF and, to some extent, in response to GM-CSF, were much larger than
wild-type colonies, indicating increased proliferative potential of the
CFCs (data not shown). The hyperresponsiveness of
ICSBP/ cells to GM-CSF parallels the behavior
of progenitor cells in patients with juvenile CML.18
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| Fig 2.
Dose-response curves of colony formation in the presence
of cytokines in vitro. Colony formation was performed exactly as
described in the legend to Fig 1. Data shown represent mean ± SEM of
1 of 4 independent experiments with 3 mice per group in each
experiment. (A) Bone marrow cells from 6-week-old
ICSBP/ (- -) or ICSBP+/+ (- -)
mice were plated in the presence of rmIL-3, rmGM-CSF, or rmG-CSF at the
indicated concentrations. (B) As in (A), with the addition of 10 ng/mL
of rmSCF to the medium.
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Because SCF has been found to synergize with GM-CSF in promoting
proliferation of CML progenitors,19 we tested the effect of
SCF in combination with either IL-3, GM-CSF, or G-CSF. The addition of
a low concentration of murine SCF (at 10 ng/mL) did not significantly
increase the maximal number of CFCs generated (data not shown).
However, it did potentiate the hypersensitive pattern of colony growth
with all 3 cytokines tested, as is evident from the left shifted dose
response (Fig 2B, lower panels). SCF alone did not support colony
growth at this concentration (data not shown). We also examined whether
the enhanced colony formation of ICSBP/
progenitors in response to SCF could be due to an autocrine production of other growth factors. However, conditioned media harvested after 24 hours of incubation with ICSBP/ bone marrow
cells with SCF (100 ng/mL) did not support colony formation of
ICSBP+/+ bone marrow cells (data not shown). Therefore, the
growth potentiation elicited by SCF is not due to a costimulatory
effect of other extracellular factors released from the
ICSBP/ cells.
These data suggest that the increased sensitivity of
ICSBP/ progenitor cells to the growth factors
is mediated by intracellular signaling events and may contribute to the
dramatic expansion of myeloid progenitors observed in vivo.
Quantitatively reduced and qualitatively altered response to M-CSF.
In contrast to the elevated number of CFCs in response to IL-3, GM-CSF,
and G-CSF described above, the number of CFCs generated in the presence
of M-CSF was significantly reduced in ICSBP/
mice (Fig 3A). The reduced response to
M-CSF is detectable as early as 12.5 dpc (Fig 3B).
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| Fig 3.
Reduced response of hematopoietic progenitor cells from
ICSBP/ mice to M-CSF. (A) Dose-response curves of
bone marrow cells from 6-week-old ICSBP+/+ (--) and
ICSBP/ (--) mice cultured in the presence of
rmM-CSF. Data shown represent mean ± SEM of 1 of 6 independent
experiments performed in triplicate with 2 mice per group. (B) Fetal
liver cells isolated at day 12.5 postcoitum were
cultured in the presence of M-CSF at 10 ng/mL. (C) Reduced number of
M-CSFR-expressing cells in ICSBP/ bone marrow.
Freshly prepared cells from wild-type (left) or
ICSBP/ (right) bone marrow were stained with
antibodies recognizing the murine M-CSF receptor (c-fms) and the
macrophage marker F 4/80 and analyzed by flow cytometry as described in
Materials and Methods.
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The response of ICSBP/ cells to M-CSF is not
only quantitatively reduced, but also qualitatively altered
(Table 1). A severely altered
differentiation profile of ICSBP/ progenitors
is seen in the presence of M-CSF, where 69% of colonies are
granulocytic and 22% monocytic as compared with 5% granulocytic and
89% monocytic colonies in control animals. A less pronounced reduction
of monocytic colonies is also seen for GM-CSF, whereas the
differentiation profile of colonies made in response to IL-3 is
unchanged.
Reduced number of M-CSF receptor-expressing cells in
ICSBP/ mice.
The reduced response of ICSBP/ progenitors to
M-CSF prompted us to analyze the expression of the M-CSF receptor
(c-fms), which is known to be specifically expressed on myeloid cells
and is upregulated during monocytic differentiation.26 By
FACS analysis, we found that the percentage of M-CSF receptor-positive
cells was reduced 4-fold in ICSBP/ mice (Fig
3C). Thus, survival and/or proliferation of cells responsive to M-CSF
may also be reduced in vivo. This is probably not due to a direct
transcriptional regulation of c-fms by ICSBP, as expression of c-fms on
mature peritoneal macrophages was normal (data not shown).
High self-renewal capacity of multipotential progenitors.
The increased number of committed myeloid progenitors in
ICSBP/ mice posed the question as to whether
more primitive multilineage progenitors are also affected. Therefore,
we performed a CFU-S assay according to Lord.25
ICSBP/ and control bone marrow cells were
injected into lethally irradiated wild-type mice, and the number of
spleen colonies was determined. Table 2
shows that ICSBP/ and control bone marrow
cells generated equal amounts of spleen colonies both when expressed as
CFU-S12 per femur and when corrected for seeding efficiency
(CFC-S). We then tested self-renewal capacity of the primitive
multilineage progenitors by injecting fractions of CFU-S-containing
spleens into secondary lethally irradiated recipients. This experiment
showed a drastic difference in that ICSBP/
cells generated 3-fold higher number of secondary CFU-S as compared with controls (Table 2). This result shows that not only committed precursors, but also multipotential progenitor cells, are affected by
the lack of ICSBP.
Long-term reconstitution.
To investigate whether the microenvironment of bone marrow contributes
to the altered properties of progenitors in
ICSBP/ mice and to determine their
leukemogenic potential in vivo, we performed a series of cell transfer
experiments. Lethally irradiated ICSBP+/+ recipient mice
were reconstituted with bone marrow cells from ICSBP/ mice and their survival was followed
(Fig 4). The results show that
ICSBP/ marrow cells are capable of long-term
hematopoietic reconstitution, as 50% of mice injected with
106 cells lived longer than 6 months (Fig 4A). However,
mice transplanted with ICSBP/ bone marrow had
a decreased life span, depending on the number of transplanted cells.
This was associated with the development of a dramatic granulocytosis
in peripheral blood (Fig 4B). The leukemogenic potential was also
established for ICSBP/ fetal liver cells as
early as day 12.5 dpc (data not shown). These data show that the
microenvironment of adult bone marrow is not critical for the
hematopoietic aberration of ICSBP/ progenitor
cells.
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| Fig 4.
Long-term reconstitution of lethally irradiated mice. (A)
Percent survival of lethally irradiated wild-type mice transplanted
with 2 different amounts of bone marrow cells from
ICSBP/ or ICSBP+/+ mice as indicated.
(B) Induction of granulocytosis in wild-type mice transplanted with
ICSBP/ bone marrow cells. The graph shows the
percentage of circulating neutrophils in peripheral blood from
reconstituted mice. The results shown represent mean ± SEM of
wild-type mice transplanted with 5 × 106
ICSBP/ bone marrow cells (dashed line; n = 14) or
with 5 × 106 ICSBP+/+ bone marrow cells
(solid line; n = 9). Total leukocyte number in peripheral blood of
wild-type recipient mice at 8 weeks posttransplant was 20.9 × 106/mL (ICSBP+/+) and 21.4 × 106/mL (ICSBP/), respectively. (C)
Peripheral leukemia in ICSBP/ mice is rescued by
lethal irradiation and subsequent reconstitution with wild-type bone
marrow cells. The graph shows the percentage of circulating neutrophils
in peripheral blood from reconstituted mice. The results represent mean ± SEM of lethally irradiated ICSBP/ mice (dashed
line; n = 16) or wild-type mice (solid line; n = 9) transplanted
with 5 × 106 wild-type bone marrow.
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In a reciprocal experiment, lethally irradiated
ICSBP/ mice were reconstituted with bone
marrow cells from ISCBP+/+ donors. Figure 4C shows that
ICSBP/ mice are effectively cured by bone
marrow transplantation, as documented by a rapid decrease of
granulocytes in the peripheral blood of transplanted animals and their
long-term survival.
Taken together, these data argue against a stromal involvement in
leukemogenesis and suggest that this is an intrinsic defect of
ICSBP/ hematopoietic cells.
Reduced number of fibronectin-adhering hematopoietic progenitors in
ICSBP/ mice.
The presence of ICSBP/ progenitor cells in
peripheral blood and their persistence in adult spleen and liver
suggested that a defective homing and adhesion could contribute to
their altered distribution. Therefore, we measured the number of
progenitors in c-kit+-enriched bone marrow cells adhering
to fibronectin, a major extracellular matrix component with a known
role in the adhesion of hematopoetic progenitor cells.27 As
seen in Fig 5A, the percentage of myeloid CFCs adhering to fibronectin is greatly reduced in
ICSBP/ c-kit+-enriched bone
marrow cells. In contrast, the percentage of
ICSBP/ CFCs adhering to laminin was increased
2-fold (Fig 5B), thus documenting that the adhesion deficiency of
ICSBP-deficient precursor cells is specific for fibronectin. Both the
reduced adhesion to fibronectin and increased adhesion to laminin are
also characteristics of CML progenitors.21
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| Fig 5.
Hematopoietic progenitors from ICSBP/
mice display reduced adhesion to fibronectin, but not to laminin.
c-kit+-enriched cell fractions were prepared as
described in Materials and Methods. The percentage of
c-kit+ cells in lineage-depleted fractions was as
follows: 92%, 88.5% (ICSBP+/+), and 89%, 81%
(ICSBP/) in 2 independent sorting experiments,
respectively. (A) c-kit+-enriched bone marrow cells from
wild-type (black bars) or ICSBP/ mice (gray bars)
were plated on 48-well plates precoated with either fibronectin or BSA
in the presence or the absence of the cytokines indicated.
Subsequently, adherent, as well as nonadherent fractions, were cultured
in the presence of IL-3 (10 ng/mL). Percent adherence was calculated as
the number of CFC in the adherent fraction divided by the number of CFC
in both fractions × 100% (see Materials and Methods). Data shown
represent mean ± SEM of 2 independent experiments. (B) Bone marrow
cells from wild-type (black bars) or ICSBP/ mice
(gray bars) were incubated on 48-well plates precoated with laminin
(see Materials and Methods). Adherent cells were subjected to CFC assay
as described above. Data shown represent mean ± SEM of 2 independent
experiments performed in triplicate with 2 mice per group.
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Adhesion to fibronectin is mediated via specific receptors,
41-and
51-integrins. Therefore, we also analyzed
the presence of integrin receptors on progenitor cells. By FACS
analysis, we observed no difference in the surface expression of
integrins on c-kit-positive bone marrow cells
(Fig 6A). To assess integrin surface
expression only on CFCs, we performed a CFC assay in vitro with bone
marrow cells capable of binding to immobilized monoclonal integrin
antibodies (Fig 6B). Similar to the adhesion studies performed with
fibronectin, we observed a reduction in the number of CFCs in
ICSBP/ bone marrow binding to immobilized
integrin antibodies (Fig 6B).
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| Fig 6.
Expression of  4, 5,
and 1-integrins on ICSBP-deficient progenitor cells. (A)
Gating of bone marrow cells according to surface expression of c-kit,
as measured by FACS. (B) FACS-analysis of 4- (top
panel), 5- (middle panel), and 1-integrin
(lower panel) expression on c-kit+-gated (see A) bone
marrow cells from ICSBP+/+ (left) and
ICSBP/ (right) mice. Filled histograms represent
control traces from cells stained only with FITC-labeled secondary Ig.
(C) Adhesion of CFCs to immobilized antiintegrin antibodies. Bone
marrow cells from wild-type (black bars) or ICSBP/
mice (gray bars) were incubated in 48-well plates precoated with
antibodies to the indicated integrins. Adherent cells were subjected to
a CFC assay as described in Materials and Methods and in the legend to
Fig 5A. Data shown represent mean ± SEM of 2 independent experiments
performed in triplicate with 2 mice per group.
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Because the activation state of integrins can be modulated by
cytokines,28,29 we tested whether the deficient adhesion to
fibronectin in ICSBP/ progenitor cells could
be upregulated by IL-3, GM-CSF, and SCF. As shown in Fig 5A, treatment
of ICSBP/ with SCF for 1 hour upregulated
adhesion of ICSBP/ CFCs, but not of
ICSBP+/+ controls to fibronectin, while GM-CSF and IL-3 had
no effect on cells from either genotype, demonstrating the specificity
of the effect elicited by SCF. These data support the notion that the
low adhesion to fibronectin of ICSBP/ CFCs is
not due to transcriptional downregulation, but rather to decreased
surface presentation and/or function of integrin receptors.
| |
DISCUSSION |
Previous analysis of ICSBP-deficient mice showed altered
differentiation and proliferation of several hematopoietic cell
lineages, dominated by the expansion of granulocytic cells, and
suggested that ICSBP is an important regulator of
hematopoiesis.13 Congruently, we show here that
hematopoietic organs and peripheral blood of ICSBP/ contain significantly higher numbers
of cells generating in vitro colonies in the presence of either IL-3,
GM-CSF, or G-CSF in comparison to ICSBP+/+ mice. Moreover,
the CFC analyses showed that ICSBP deficiency affects hematopoiesis not
only in adult mice, but already in very early stages of embryonal
differentiation. The observed increase of CFCs results from the
enhanced sensitivity of ICSBP/ progenitors to
GM-CSF, and G-CSF in vitro, and and may contribute to the expansion of
myeloid progenitors observed in vivo. It is likely that the altered
responses to the growth factors are mediated by intracellular signaling
events, and that ICSBP might be a part of the signaling network for
several cytokines. Hypersensitivity to GM-CSF is a marker of
progenitors from juvenile-CML patients18 and has also been
noted in hematopoietic disturbances in mice deficient for NF-1 or
SH2-containing inositol-5-phosphatase
(SHIP).30,31 An increase of myeloid cells
in these 2 mouse models was ascribed to elevated Ras
signaling.30-32
A striking result is the quantitatively and qualitatively reduced
response of ICSBP/ cells to M-CSF. The low
numbers of monocytic cells generated in response to M-CSF in vitro
reflects most likely the observed strong reduction of the M-CSF
receptor-bearing cells in bone marrow. Importantly, the expression of
the M-CSF receptor is not affected in ICSBP-deficient peritoneal
macrophages (data not shown). Thus, the lack or reduced level of M-CSFR
expression in bone marrow cells is not likely due to a direct effect of
ICSBP on M-CSFR transcriptional regulation. Rather, it suggests that
this is due to a differentiation defect of an earlier progenitor cell
in this compartment.
On the basis of our results, one can hypothesize that the observed
reduction of macrophage lineage-committed progenitors, together with
the altered response to M-CSF, may lead to a disproportional differentiation towards cells of the granulocytic lineage. Thus, ICSBP
plays a role during an early differentiation step that influences the
lineage commitment. Recently, other transcription factors such as CEBP
,  , and, egr-1 have been identified, which influence the
differentiation of myeloid progenitors along the granulocytic or
macrophage lineage.33-35 Whether ICSBP also acts as
lineage-switch module is actively being studied at present.
The cell transfer-experiments presented show that bone marrow cells of
ICSBP/ mice are competent in long-term
reconstitution and capable of transferring the leukemic phenotype into
wild-type recipients independent of their environment. We conclude that
the primary defect, which leads to the myeloproliferative syndrome in
ICSBP/ mice, is due to an expansion and
deregulated differentiation of early multilineage progenitors.
Aberrations in adhesive interactions affecting cell homing and
migration can also affect cell proliferation and differentiation and
lead to pathological disorders.20,21 The interactions of normal and malignant hematopoietic progenitors with fibronectin has
been extensively studied. It has been shown that direct adhesion to
bone marrow stroma via fibronectin receptors
(41 and 51 integrins) inhibits proliferation of hematopoietic progenitor cells.36 The number of CFCs in ICSBP-deficient mice that
bind to fibronectin is severely reduced. Our experiments indicate that this could be due to either a reduced surface presentation or function
of 41 and
51 integrin-bearing progenitors. The
reduced adhesion could therefore be an additional factor contributing to hematopoietic alterations of ICSBP-deficient mice.
The hematopoietic disorder caused by the ICSBP gene defect in mice has
distinct similarities to CML.13 We have shown here that
several characteristics of CML progenitors, hypersensitivity to
GM-CSF18, synergistic proliferation in the presence of
SCF,19 and the altered adhesion to fibronectin and
laminin21 are also shared by the
ICSBP/ progenitors. Together, these
observations suggest that despite different primary lesions, the
signaling pathways leading to deregulated myeloid proliferation in CML
and in ICSBP/ mice may overlap in their
distal parts. Because the signaling cascades for both disorders have
not yet been elucidated, detailed knowledge of the ICSBP gene function
is desired.
| |
ACKNOWLEDGMENT |
We thank Drs Peter Rosenthal and Wolfgang Hinkelbein (Department of
Nuclear Medicine, Free University Berlin) for access to the irradiation facility.
| |
FOOTNOTES |
Submitted March 4, 1999; accepted July 27, 1999.
Supported by the Deutsche Forschungsgemeinschaft (SFB 506) and by the
Wilhelm-Sander-Stiftung.
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.
Address reprint requests to Ivan Horak, MD, Department of Molecular
Genetics, FMP, Krahmerstr. 6, 12207 Berlin, Germany; e-mail:
horak{at}fmp-berlin.de.
| |
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ABSTRACT
INTRODUCTION