Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 353-361
Specific Signals Generated by the Cytoplasmic Domain of the Granulocyte
Colony-Stimulating Factor (G-CSF) Receptor Are Not Required for
G-CSF-Dependent Granulocytic Differentiation
By
Jason Jacob,
Jeffery S. Haug,
Sofia Raptis, and
Daniel C. Link
From the Division of Bone Marrow Transplantation and Stem Cell
Biology, Department of Medicine and Pathology, Washington University
Medical School, St Louis, MO.
 |
ABSTRACT |
Granulocyte colony-stimulating factor (G-CSF) is the principal
growth factor regulating the production of neutrophils, yet its role in
lineage commitment and terminal differentiation of hematopoietic
progenitor cells is controversial. In this study, we describe a system
to study the role of G-CSF receptor (G-CSFR) signals in granulocytic
differentiation using retroviral transduction of G-CSFR-deficient,
primary hematopoietic progenitor cells. We show that ectopic expression
of wild-type G-CSFR in hematopoietic progenitor cells supports
G-CSF-dependent differentiation of these cells into mature
granulocytes, macrophages, megakaryocytes, and erythroid cells.
Furthermore, we show that two mutant G-CSFR proteins, a truncation
mutant that deletes the carboxy-terminal 96 amino acids and a chimeric
receptor containing the extracellular and transmembrane domains of the
G-CSFR fused to the cytoplasmic domain of the erythropoietin receptor,
are able to support the production of morphologically mature,
chloroacetate esterase-positive, Gr-1/Mac-1-positive neutrophils in
response to G-CSF. These results demonstrate that ectopic expression of
the G-CSFR in hematopoietic progenitor cells allows for multilineage
differentiation and suggest that unique signals generated by the
cytoplasmic domain of the G-CSFR are not required for G-CSF-dependent
granulocytic differentiation.
| |
INTRODUCTION |
GRANULOCYTE colony-stimulating factor
(G-CSF) is the principal growth factor regulating the production of
mature neutrophils. In fact, G-CSF is widely used to ameliorate
neutropenia in a variety of clinical settings.1 Multiple
actions of G-CSF have been described that may contribute to the
neutrophilic response. First, G-CSF stimulates the proliferation of
granulocytic precursors.2,3 Second, it reduces the average
transit time through the granulocytic compartment.3-5
Although the effect of G-CSF on neutrophil production is well
established, its role in the commitment of multipotent hematopoietic
progenitors to the myeloid lineage and their subsequent terminal
differentiation into mature neutrophils is controversial.
The biological effects of G-CSF are mediated through its interaction
with the G-CSF receptor (G-CSFR), a member of the cytokine receptor
superfamily.6 To define further the role of G-CSF in the
control of granulopoiesis, we recently generated G-CSFR-deficient mice.7 Similar to G-CSF (cytokine)-deficient
mice,8 G-CSFR-deficient mice have chronic neutropenia with
a uniform decrease in myeloid cells in the bone marrow.7,9
No accumulation of immature granulocytic cells in the bone marrow was
observed, suggesting that the residual granulocytic precursors present
in these mice were able to differentiate normally into mature
neutrophils. In agreement with this conclusion, G-CSFR-deficient
myeloid progenitors demonstrated normal granulocytic differentiation in
vitro in response to interleukin-3 (IL-3) or granulocyte-macrophage
colony-stimulating factor (GM-CSF).7 Surprisingly, near
normal numbers of myeloid-committed progenitors were observed in the
bone marrow of these animals. These data demonstrated that G-CSFR
signals are not required for lineage commitment or terminal
differentiation.
On the other hand, several studies have shown that the addition of
G-CSF to cultures of certain multipotential hematopoietic cell lines
results in their granulocytic differentiation.10-14 Furthermore, a carboxy-terminal region of the cytoplasmic domain of the
G-CSFR was identified that was required for granulocytic differentiation.11-13 These observations lead to the
hypothesis that G-CSFR signals play an active (instructive) role in
directing granulocytic differentiation. This conclusion is compatible
with data from the G-CSFR-deficient mice, because alternative
granulocytic differentiation signals (eg, from other hematopoietic
cytokines) may be able to compensate for the loss of G-CSFR signals in
vivo. In contrast, other studies have shown that granulocytic
differentiation can occur in a cytokine-independent fashion.
Suppression of apoptosis by ectopic expression of bcl-2 allowed for
granulocytic differentiation of hematopoietic cell lines in the absence
of added cytokines (although certain features of granulocytic
differentiation seemed to require G-CSF).15,16 These
conflicting data highlight the controversy as to the presence and
contribution of specific G-CSFR signals to granulocytic
differentiation.
In this study, we describe a system to study the role of G-CSFR signals
in the granulocytic differentiation of primary hematopoietic progenitor
cells that lack endogenous G-CSFR protein. In addition to wild-type
G-CSFR, two mutant G-CSFR were studied: a truncation mutant that
deletes the carboxy-terminal 96 amino acids (including the putative
maturation domain) and a chimeric receptor containing the extracellular
and transmembrane domains of the G-CSFR fused to the cytoplasmic domain
of the erythropoietin receptor. The effect of ectopic expression of
these receptors on G-CSF-dependent hematopoietic proliferation and
differentiation was assessed.
| |
MATERIALS AND METHODS |
Cytokines, cell lines, and mice.
Human flt-3 ligand and murine kit ligand were generously provided by
Immunex (Seattle, WA). Human thrombopoietin was a generous gift from Dr
John DiPersio (Washington University, St Louis, MO). The amphotropic
and ecotropic packaging cell lines GP+EnvAm12 and GP+E86,17
respectively, were maintained in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS),
15 µg/mL of hypoxanthine, 250 µg/mL of xanthine, and 25 µg/mL of
mycophenolic acid (HXM media) at 37°C in a 5% CO2,
humidified atmosphere. The generation of G-CSFR-deficient mice has
been described previously.7 All mice were housed in a
specific pathogen-free environment.
Construction of G-CSFR retroviral plasmids.
The wild-type and mutant G-CSFR cDNAs were subcloned into the
retroviral vector pMPncrdlneo.18 The d716 G-CSFR mutation was generated using a polymerase chain reaction (PCR)-based method to
introduce a C to T mutation at nucleotide 2403, as
described.19 The murine G-CSFR cDNA was used as the
template in PCR reactions with the following oligonucleotide primer
pairs: exon 16 forward primer (5
-CCCACAGTAGCCTGAGCTCC-3)
and exon 17 reverse mutagenesis primer
(5-AGAGGAATTCTAGGACTGGTTGGA-3); exon 17 forward
mutagenesis primer (5-CCAGTCCTAGAATTCCTCTCGCAC-3) and
exon 17 reverse primer (5-CCCCAAAGTTCTAGAAACCC-3). The
primary PCR products were purified by gel electrophoresis, annealed in
a 1:1 molar ratio, and amplified with the exon 16 forward and exon 17 reverse primers listed above. The resulting product was digested with
Sac I and Xba I and subcloned into a plasmid containing
the murine G-CSF cDNA that had been digested with Sac I and
Xba I. The GEpoR construct was generated as follows. The murine
erythropoietin cDNA was used as the template in a PCR reaction using a
forward primer (5-AAGAAAGACTTCCAAGATCTGGCCTGGCA-3) containing a Xmn I site and a reverse primer
(5-AATCTAGACTAGGAGCAGGCCACATAG-3) containing an
Xba I site. The resulting 690-bp amplicon was digested with
Xmn I and Xba I and subcloned into a plasmid containing
the murine G-CSFR cDNA that had been digested with Xmn I and
Xba I. Note that this subcloning strategy retains the first
four amino acids of the G-CSFR. Sequence analysis was performed to
confirm the fidelity of each of these constructs.
Retroviral infection of bone marrow cells.
The retroviral constructs were linearized with Sac-2, transfected into
GP+EnvAm12 cells using lipofectamine (GIBCO BRL, Gaithersburg, MD), per
the manufacturer's recommendations, and selected in HXM media
supplemented with 800 µg/mL geneticin (GIBCO BRL). The amphotropic retrovirus-rich supernatant from geneticin-resistant cells was used to
infect GP+E86 cells. Individual geneticin-resistant GP+E86 clones were
derived, and their supernatant was tested for viral production on
NIH3T3 cells. Clones producing 0.5 to 1.0 × 106
infectious particles/mL were obtained for each construct.
Hematopoietic cells were harvested from the femurs and tibiae of 5- to
8-week-old G-CSFR-deficient mice, and the light-density (progenitor-enriched) fraction was collected after centrifugation through a Histopaque 1077 density gradient (Sigma, St Louis, MO). Cells
were cocultured on irradiated (1,500 cGy) GP+E86 viral producer cells
in 
-minimum essential medium (-MEM) supplemented with 20% FCS,
murine IL-3 (10 ng/mL; R&D systems, San Diego, CA), human flt-3 ligand
(50 ng/mL), human thrombopoietin (50 ng/mL), murine kit ligand (50 ng/mL), and polybrene (6 µg/mL) for 4 days at 37°C in a 5%
CO2, humidified atmosphere.
Flow cytometry.
To assess surface G-CSFR expression, nonadherent cells from the
cultures described above were incubated at 4°C for 1 hour with
biotinylated G-CSF (generated as described7; 5 ng per
106 cells) in the presence or absence of a 100-fold molar
excess of nonlabeled G-CSF, followed by incubation with phycoerythrin (PE)-conjugated streptavidin (GIBCO BRL). Cells were coincubated with
the following cocktail of lineage-restricted fluorescien isothiocyanate
(FITC)-conjugated rat monoclonal antibodies: antimouse B220 (M1/70,
IgG2b), antimouse CD3 (M1/70, IgG2b), and
antimouse CD11b (M1/70, IgG2b). In other experiments,
PE-conjugated rat antimouse CD11b (M1/70, IgG2b; PharMingen, San Diego,
CA) and FITC-conjugated rat antimouse Gr-1 (RB6-8C5, IgG2b) were used. In all experiments, cells were incubated with actinomycin D, 7-amino (7AAD) to exclude nonviable (7AAD-positive) cells from analysis. All
antibodies were purchased from PharMingen. All cells were analyzed
using a FACScan flow cytometer and CellQuest version 1.2.2 software
(Becton Dickinson, Mansfield, MA).
Cell sorting.
Nonadherent cells from the cultures described above were incubated with
biotin-conjugated rat antimouse CD34 (RAM34, IgG2a) and the
same cocktail of FITC-conjugated lineage-restricted antibodies as
described above. After incubation with PE-conjugated streptavidin, CD34+ lineage
cells were sorted using a
Coulter Elite ESP cytometer (Coulter, Hialeah, FL).
Progenitor assays.
Seven hundred fifty to 2,000 CD34+
lineage cells were plated in 2.5 mL of
methylcellulose media (MethoCult 3230; Stem Cell Technologies,
Vancouver, British Columbia, Canada) supplemented with G-CSF at 1, 10, or 100 ng/mL (Amgen, Thousand Oaks, CA) or with 2.5 mL of
methylcellulose media supplemented with erythropoietin and pokeweed
mitogen-stimulated murine spleen cell-conditioned medium (MethoCult
3430; Stem Cell Technologies) and placed at 37°C in a humidified
chamber with 5% CO 2 for 10 days. Colonies containing at
least 50 cells were scored on day 10.
Cytological analysis.
Entire methylcellulose cultures were harvested on day 10 and leukocyte
differentials were performed on Wright-stained cytospin preparations.
Acetylcholine esterase20 and 2,7-diaminofluorene (DAF)21 stains were performed as described. For flow
cytometry, cells were washed extensively and incubated with Fc block
(PharMingen) per the manufacturer's recommendations before incubating
with the indicated antisera.
Statistical analysis.
Data represent the mean ± SD. Statistical significance was assessed
by the Student's t-test.
| |
RESULTS |
High efficiency transduction of G-CSFR-deficient hematopoietic cells
with G-CSFR-expressing retrovirus.
Most current models of granulocytic differentiation use immortalized
cell lines; however, these models are often limited by incomplete
differentiation and inappropriate gene expression. A system was
developed to study the effect of wild-type (or mutant) G-CSFR
expression on the granulocytic differentiation of primary hematopoietic
cells using retroviral transduction. Hematopoietic cells isolated from
G-CSFR-deficient mice were used in these studies because they lack
endogenous G-CSFR. In initial experiments, the ability of three
different G-CSFR proteins to support hematopoietic proliferation and
differentiation were examined (Fig 1). In
addition to wild-type murine G-CSFR (WT), two mutant receptors were
studied. The d716 mutation truncates the distal 96 amino acids of the
carboxy-terminal tail and reproduces a mutation of the G-CSFR found in
a patient with severe congenital neutropenia.22 Expression
of a similarly truncated G-CSFR in a myeloid cell line blocked
G-CSF-dependent granulocytic differentiation.22 The GEpoR
mutation produces a chimeric receptor with the extracellular
(ligand-binding), transmembrane, and the first four amino-acids of the
cytoplasmic domain derived from the G-CSFR and the remaining
cytoplasmic domain derived from the murine erythropoietin receptor. The
first four amino acids of the cytoplasmic domain of the G-CSFR were
retained solely to facilitate the subcloning of the GEpoR construct.
This chimeric receptor is predicted to transmit erythropoietin-specific
signals in a G-CSF-dependent fashion.
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| Fig 1.
Structure of mutant G-CSFR proteins. The extracellular
(ED), transmembrane (TM), and cytoplasmic domain (CD) of the murine G-CSFR are shown. The position of tyrosine residues (Y) in the cytoplasmic domain also are shown. The d716 construct contains a point
mutation at nucleotide 2403 of the murine cDNA that introduces a
premature stop codon resulting in the truncation of the G-CSFR at
amino-acid 716. The GEpoR mutation represents an in frame fusion of the
extracellular, transmembrane, and first four amino acids of the
cytoplasmic domain of the murine G-CSFR with the cytoplasmic domain of
the murine erythropoietin receptor.
|
|
Hematopoietic cells isolated from the bone marrow of G-CSFR-deficient
mice were cocultured with irradiated retroviral-producing cell lines
for 4 days in the presence of hematopoietic growth factors (see the
Materials and Methods). Transduction efficiency was assessed by
measuring the percentage of cells that specifically bound G-CSF
(Fig 2). In most experiments, approximately
50% of the immature (lineage) cells expressed
G-CSFR protein on their surface; no difference in transduction
efficiency was observed with the different retroviruses. After
transduction, CD34+ lineage progenitor
cells were isolated by flow cytometry (to eliminate mature cells) and
cultured in methylcellulose media in the presence of G-CSF (or control
cytokines) for 7 to 10 days. The number, size, and composition of the
resulting colonies were evaluated.
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| Fig 2.
Assessment of transduction efficiency. Nonadherent cells
were harvested after 4 days of coculture on irradiated
retroviral-producing fibroblasts, incubated with biotinylated G-CSF and
a cocktail of lineage-restricted antisera, and analyzed by flow
cytometry. The binding of biotinylated G-CSF by lineage+
cells (upper-right quadrant) was seen even in the presence of a
100-fold molar excess of nonlabeled G-CSF (data not shown) and therefore represents nonspecific binding. Transduction efficiency was
assessed by determining the percentage of lineage cells
that specifically bound G-CSF (upper left quadrant). No specific G-CSF
binding was detected in cells transduced with the empty retroviral
vector (neo). Nonviable cells were excluded from the analyses. Shown
are representative results of one of five experiments.
|
|
All three G-CSFR proteins support G-CSF-dependent hematopoietic
colony formation.
Hematopoietic cells transduced with WT, d716, GEpoR, or control (neo)
retrovirus produced similar numbers of colonies in response to the
cytokines present in pokeweed mitogen-stimulated spleen conditioned
media (Fig 3A). No colonies were detected
in any culture without added cytokine (Fig 3B). As expected, the
neo-transduced cells did not produce any colonies in response to G-CSF
even at the highest concentration (100 ng/mL). Cells transduced with
the WT, d716, or GEpoR constructs produced similar number of colonies in response to G-CSF at concentrations of 10 or 100 ng/mL.
Interestingly, at the lowest concentration of G-CSF (1 ng/mL),
significant differences were detected in the frequency of colony
formation. Compared with wild-type transduced cells, significantly
fewer colonies were detected in cultures of GEpoR-transduced cells; in
contrast, significantly greater numbers of colonies were detected in
cultures of d716-transduced cells. A nonsignificant trend to increased
colony size was observed with GEpoR-transduced cells compared with WT-
or d716-transduced cells; on day 10 of G-CSF stimulation (100 ng/mL),
the mean number of cells per colony ± SD was as follows: WT, 1,504 ± 322; d716, 2,068 ± 693; and GepoR, 3,098 ± 1,403.
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| Fig 3.
Production of hematopoietic colonies. CD34+
lineagelo cells transduced with the indicated retrovirus
were cultured in the presence of pokeweed mitogen-stimulated spleen
conditioned media (A) or the indicated amount of human G-CSF (B). The
total number of hematopoietic colonies (CFU-C) observed after 10 days
of culture are shown. *P value < .05 compared with cultures
of WT-transduced cells. Data represent the mean ± SD.
|
|
All three G-CSFR proteins support a similar degree of granulocytic
differentiation.
Hematopoietic colonies stimulated by G-CSF were harvested on day 10 of
culture and their cellular composition was analyzed. In cultures of
WT-, d716-, or GEpoR-transduced cells the predominant cell types
observed in the G-CSF-stimulated colonies were macrophages and cells
of the granulocytic lineage (Fig 4A
through C and Table 1). In fact, a similar number of
mature-appearing neutrophils were observed in each of these cultures.
The only consistent difference observed in these experiments was a
delay in granulocytic differentiation of GEpoR transduced cells; after
7 days of G-CSF stimulation, a greater fraction of cells were immature
granulocytic precursors (data not shown). To confirm the presence of
myeloid cells, the expression of Mac-1 and Gr-1 (proteins that are
expressed predominantly on myeloid cells) was examined by flow
cytometry (Fig 5). The majority of cells in
cultures of WT-, d716-, or GEpoR-transduced cells stained brightly for
Mac-1 and Gr-1, a pattern consistent with mature neutrophils and late
granulocytic precursors.23 Another characteristic of mature
neutrophils is the presence of chloroacetate esterase.24
Although the intensity of staining was reduced relative to normal
murine neutrophils, the majority of granulocytic cells in each of these
cultures clearly stained positive for chloroacetate esterase (Fig 4D
through F). Collectively, these data indicate that all three G-CSFR
proteins are able to support a similar degree of granulocytic
differentiation.
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| Fig 4.
Cytological analysis of hematopoietic colonies. Cells
were recovered from G-CSF-stimulated methylcellulose cultures of WT- (A and D), d716- (B and E), or GEpoR- (C and F) transduced cells. Cells
were analyzed by Wright stain (A through C) or chloroacetate esterase
stain (D through F). Shown are representative results of one of four
experiments. Original magnification × 1,110.
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| Fig 5.
Expression of Mac-1 and Gr-1. Cells were recovered from
the indicated G-CSF-stimulated methylcellulose culture on day 10 and stained with FITC-conjugated Mac-1 (CD11b) and PE-conjugated Gr-1 or
isotype controls. Distinct populations of Gr-1+
Mac-1+ (neutrophil lineage) cells and Gr-1lo
Mac-1+ (monocytic) cells were seen in each culture. Shown
are representative results of one of four experiments.
|
|
Ectopic expression of the G-CSFR in hematopoietic progenitor cells
allows for G-CSF-dependent multilineage differentiation.
In G-CSF-stimulated cultures of WT-, d716-, or GEpoR-transduced cells,
a small number of granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit (CFU-GEMM) and burst-forming unit-erythroid (BFU-E) colonies were observed (data not shown). In
agreement with this observation, erythroid cells and megakaryocytes
were consistently detected in these cultures (Table 1 and data not shown). The presence of megakaryocytes was confirmed by the detection of acetylcholine esterase-positive multinucleated cells (data not
shown). Likewise, the presence of erythroid cells was confirmed by the
demonstration of DAF-positive (hemoglobinized) cells (data not shown).
Either the autocrine production of growth factors by the hematopoietic
cells or the trace amounts of growth factors present in fetal calf
could contribute to the multilineage differentiation observed. We
therefore investigated whether neutralizing antibodies to
erythropoietin and thrombopoietin could block erythroid or megakaryocytic differentiation in G-CSF-stimulated cultures. After 10 days of culture, a similar number of erythroid and megakaryocytic cells
were observed (data not shown). Collectively, these data indicate that
the G-CSFR is able to generate signals in progenitor cells that support
the production of mature granulocytes, macrophages, megakaryocytes, and
erythroid cells.
| |
DISCUSSION |
Hematopoietic cytokines clearly play an important role in the
regulation of hematopoiesis, yet the mechanisms by which they exert
their control are unclear. In this study, we describe a system to study
the role of G-CSFR signals in granulocytic differentiation using
retroviral transduction of G-CSFR-deficient, primary hematopoietic progenitor cells. This approach has several advantages over the use of
immortalized myeloid cell lines to study granulocytic differentiation. First, the target cell population is comprised of primary progenitor cells (not cells that have been adapted to long-term culture). Second,
the target cells completely lack endogenous G-CSFR. Using this system,
we show that ectopic expression of wild-type G-CSFR in hematopoietic
progenitor cells supports G-CSF-dependent differentiation of these
cells into mature granulocytes and macrophages. Furthermore, we show
that two mutant G-CSFR proteins, d716 (a truncation mutant that deletes
the carboxy-terminal 96 amino acids) and GEpoR (a chimeric receptor
containing the extracellular and transmembrane domains of the G-CSFR
fused to the cytoplasmic domain of the erythropoietin receptor), also
are able to support the production of morphologically mature,
chloroacetate esterase-positive, Gr-1/Mac-1-positive neutrophils in
response to G-CSF. Surprisingly, along with granulocytes and macrophages, these G-CSFR proteins also were able to support the production of mature megakaryocytes and erythroid cells.
The role of G-CSFR signals in the terminal differentiation of myeloid
precursors to neutrophils is controversial. In the present study, we
show that the truncated G-CSFR mutant (d716) is able to support
G-CSF-dependent granulocytic differentiation of primary hematopoietic
cells. The d716 mutation was derived from a patient with severe
congenital neutropenia and acute myelogenous leukemia.22 This mutation deletes the putative maturation domain of the G-CSFR and
disrupts G-CSF-dependent activation of SHC and the JNK/SAPK pathway.22,25,26 Our results demonstrate that this region and these signal transduction pathways are not required for
G-CSF-supported granulocytic differentiation. In agreement with this
conclusion, we recently have shown that mice homozygous for a similar
targeted knock-in mutation of their G-CSFR have normal resting
granulopoiesis (manuscript submitted).
Recent studies showed that transduction of primary hematopoietic
progenitor cells with a retrovirus encoding either for c-fms or
for the nonhematopoietic prolactin receptor allowed for the generation
of erythroid colonies in response to macrophage colony-stimulating factor or prolactin, respectively.27,28 These results
suggested that signals uniquely generated by the erythropoietin
receptor were not required for erythrocytic differentiation. Likewise, in the present study, we show that expression of the GEpoR chimera in
hematopoietic progenitor cells allowed for G-CSF-dependent granulocytic differentiation. This result suggests that signals generated by the cytoplasmic domain of the erythropoietin are able to
substitute for those generated by the G-CSFR. In contrast, recent
studies have shown that ectopic expression of the full-length erythropoietin receptor,29 thrombopoietin
receptor,30 or c-fms27 in primary
murine hematopoietic cells does not support granulocytic differentiation. In addition to differences in the retroviral vectors
and culture conditions, the major difference between these studies and
the current study is the retention of the extracellular and
transmembrane domains of the G-CSFR in the GEpoR chimera. It therefore
is possible that these regions of the G-CSFR are sufficient to generate
the signals required for granulocytic differentiation in this system
(see discussion below). Studies are in progress to explore this
hypothesis.
Smith et al31 recently showed that multiple regions of the
-common chain of the GM-CSF receptor may contribute to
GM-CSF-dependent macrophage differentiation in a cell-specific manner.
Their data suggested that both the strength of the differentiative
signal and the pool of available signaling intermediates determined the differentiation response. It is therefore possible that the high level
of receptor expression achieved with retroviral promoters may allow for
an otherwise weak (and possibly nonphysiological) signal to initiate a
differentiation program. For example, multiple regions of the G-CSFR
may normally be required to induce granulocytic differentiation; the
loss of one region (ie, membrane-distal region) may be compensated for
by the overexpression of the remaining receptor regions. A confirmation
of our findings in mice carrying a similar targeted G-CSFR mutation in
which the level of GEpoR expression is physiological and
lineage-restricted is therefore warranted.
Two general models for the role of cytokines in hematopoietic
differentiation have been proposed.32 In the instructive
model, cytokines transmit specific signals to multipotential
hematopoietic cells directing lineage commitment and differentiation,
whereas, in the stochastic model, lineage-commitment and terminal
differentiation are intrinsically determined with cytokines providing
only growth and survival signals. Studies of mice carrying targeted
null mutations of the G-CSFR7 or erythropoietin
receptor33,34 have shown that the production of the
relevant lineage-committed progenitors is largely preserved. Likewise,
megakaryocyte progenitors are present in thrombopoietin
receptor-deficient mice, although at a much reduced level compared with
wild-type mice.35,36 Collectively, these data indicate that
the signals generated by these receptors are not required for lineage
commitment. In the present study, we show that ectopic expression of
the G-CSFR in hematopoietic progenitor cells supports multilineage
differentiation with the production of mature granulocytes,
macrophages, megakaryocytes, and erythroid cells. This result suggests
that signals generated by the G-CSFR are able to substitute for those
normally generated by the erythropoietin and thrombopoietin receptors
to support erythrocytic and megakaryocytic differentiation,
respectively. In agreement with this conclusion, ectopic expression of
the receptors for erythropoietin,37-39
thrombopoietin,30 or GM-CSF40,41 in primary
hematopoietic progenitor cells does not result in preferential lineage
commitment and allows for ligand-dependent multilineage differentiation
(with the possible exception of granulocytes, see above).
Collectively, these data provide new and strong evidence in support of
the stochastic model of hematopoietic differentiation in which
cytokines provide important growth and survival signals but do not
direct terminal differentiation. If specific signals directing terminal
differentiation (or lineage commitment) are not being generated, then
why do the cytoplasmic domains of members of the cytokine receptor
family share so little homology? Two recent studies provide a potential
explanation. Huffman et al42 recently showed that
GM-CSF-deficient mice have a defect in macrophage function that leads
to a clinical syndrome with features of pulmonary alveolar
proteinosis.42-44 Likewise, we recently have discovered that neutrophils isolated from G-CSFR-deficient mice have significant but selective functional defects45 (and manuscript in
preparation). These data suggest that the GM-CSF and G-CSF
receptors are providing nonredundant signals essential for mature
macrophage or neutrophil function, respectively. The need to
selectively regulate the function of specific populations of mature
hematopoietic cells may explain the diversity within the cytokine
receptor family.
| |
FOOTNOTES |
Submitted March 19, 1998;
accepted April 21, 1998.
Supported by a grant from Monsanto/Searle.
Address reprint requests to Daniel C. Link, MD, Washington University
Medical School, Division of Bone Marrow Transplantation and Stem Cell
Biology, Box 8007, 660 S Euclid Ave, St Louis, MO 63110-1093; e-mail:
link{at}IM.wustl.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Mark Sands for his assistance in the generation of
high-titer retrovirus. We also thank Jennifer Poursine-Laurent for her
expert technical assistance.
| |
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Abstract
Introduction
Abstract