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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1504-1514
Activation of Granulocyte-Macrophage Colony-Stimulating Factor and
Interleukin-3 Receptor Subunits in a Multipotential Hematopoietic
Progenitor Cell Line Leads to Differential Effects on Development
By
Caroline A. Evans,
Andrew Pierce,
Sandra A. Winter,
Elaine Spooncer,
Clare M. Heyworth, and
Anthony D. Whetton
From the Department of Biomolecular Sciences, Leukaemia Research Fund
Cellular Development Unit; and CRC Section of Haemopoietic Cell and
Gene Therapeutics, Paterson Institute for Cancer Research, Christie
Hospital NHS Trust, Manchester, United Kingdom.
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ABSTRACT |
Activation of specific cytokine receptors promotes survival and
proliferation of hematopoietic progenitor cells but their role in the
control of differentiation is unclear. To address this issue, the
effects of human interleukin-3 (hIL-3) and human granulocyte-macrophage
colony-stimulating factor (hGM-CSF) on hematopoietic development were
investigated in hematopoietic progenitor cells. Murine multipotent
factor-dependent cell-Paterson (FDCP)-mix cells, which can
self-renew or differentiate, were transfected with the genes encoding
the unique  and/or shared  c human hIL-3 receptor
(hIL-3 R) or hGM-CSF receptor (hGM R) subunits by retroviral gene
transfer. Selective activation of hIL-3 R,c or hGM
R,c transfects by hIL-3 and hGM-CSF promoted
self-renewal and myeloid differentiation, respectively, over a range of
cytokine (0.1 to 100 ng/mL) concentrations. These qualitatively
distinct developmental outcomes were associated with different patterns
of protein tyrosine phosphorylation and, thus, differential signaling
pathway activation. The cell lines generated provide a model to
investigate molecular events underlying self-renewal and
differentiation and indicate that the subunits act in combination
with the hc to govern developmental decisions. The role
of the subunit in conferring specificity was studied by using a
chimeric receptor composed of the extracellular hIL-3 R and
intracellular hGM R subunit domains. This receptor promoted
differentiation in response to hIL-3. Thus, the subunit cytosolic
domain is an essential component in determining cell fate via specific
signaling events.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RECEPTORS OF THE CYTOKINE receptor
superfamily are present on primitive hematopoietic progenitor
cells1,2 and activation by their cognate cytokines
regulates hematopoietic cell survival and proliferation. The receptors
for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) are members of a subfamily of cytokine receptors and
are composed of  and subunits. They contain common structural
motifs found in many other cytokine receptors.3 The subunits that bind IL-3 or GM-CSF are related but unique to each of the
receptors. They bind their cognate ligand with low affinity and
interact with a common subunit (c) to form a
functional high affinity receptor complex.4 The
c subunit lacks the ability to bind either of these
cytokines independently, is structurally related to the IL-2 receptor
chain or gp130 subunit of the IL-6 receptor family, and is believed
to elicit most, if not all, of the signaling events emanating from
these receptors.5
IL-3 and GM-CSF both promote the survival and proliferation of
multipotent cells; IL-3 stimulates the development of multilineage colonies from normal bone marrow, and GM-CSF promotes the production of
granulocytes and macrophages.6-11 Thus, receptors for IL-3 and GM-CSF promote both overlapping and distinct biologic effects in
hematopoietic cells. The specificity of the cytokine-mediated response
may be conferred either by expression of the GM-CSF and/or IL-3
receptor subunits on different cells or by differential effects
elicited in response to receptor subunit interaction with the subunits. The precise role of the IL-3 and GM-CSF receptors in the
regulation of hematopoietic progenitor cell differentiation is unclear.
We have investigated the function of the IL-3 and GM-CSF cytokine
receptors in hematopoietic cell development by using the murine
multipotential hematopoietic cell line, FDCP-mix (clone A4). These
cells are nonleukemic, karyotypically normal, and their survival,
proliferation, and development are subject to regulation by cytokines.
Relatively high concentrations of murine IL-3 promote self-renewal12 and low concentrations of IL-3, in
combination with other cytokines such as GM-CSF or erythropoietin,
promote differentiation into granulocytes and macrophages or into
mature erythroid cells.13,14 Removal of IL-3 results in
cell death via apoptosis.15
IL-3 and GM-CSF are species specific; therefore, human IL-3 (hIL-3) and
human GM-CSF (hGM-CSF) selectively activate hIL-3 and GM-CSF receptors
only. The relative contributions of the hIL-3 receptor (hIL-3 R), hGM-CSF R (hGM R), and hc subunits
in hematopoietic progenitor cell development could, therefore, be investigated by transfection into the murine FDCP-mix cell line and
activation by addition of the cognate human cytokine. This experimental
approach allowed the function(s) of the human receptor subunits to be
studied in the context of a multipotential hematopoietic progenitor
cell line. Transfection of mutated receptor subunits also enabled
structure-function analysis of the human receptors to be performed.
Here, we specifically address the effects on IL-3 and GM-CSF on
development, unlike many previous studies on IL-3 and GM-CSF receptor
function, which have been performed in leukemic or differentiation
blocked cell lines16,17 and show a role for the subunits in determining cell fate.
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MATERIALS AND METHODS |
FDCP-mix (clone A4) cells were routinely cultured in Iscove's modified
Dulbecco's medium (IMDM) supplemented with 5% (vol/vol) medium
conditioned by the X63-Ag-653 cell line (used as a source of murine
IL-3)18 and 20% (vol/vol) horse serum. For differentiation assays, cells were cultured in IMDM, 20% fetal calf serum (FCS), and
the appropriate cytokine(s).13 In soft-gel assays, cells were cultured in IMDM, 20% (vol/vol) horse serum, 1% (wt/vol) bovine
serum albumin (BSA), the appropriate cytokine, and 0.33% (vol/vol)
agar.12 Colonies were incubated at 37°C in 5%
CO2, 5% O2, and N2 for 7 days
before analysis.
Cytokines.
Recombinant hIL-3 and hGM-CSF were gifts from Sandoz Pharma (Basel,
Switzerland) and Glaxo (Greenford, UK), respectively. Recombinant mIL-3
(4 × 107 U/mg) and hG-CSF (108 U/mg) were
obtained from R&D Systems (Abingdon, UK) and Chugai (Geneva,
Switzerland), respectively. Murine (m) GM-CSF
(1.25 × 107 U/mg) was a gift from Biogen (Geneva, Switzerland).
Transfection of FDCP-mix cells with subunits of the hIL-3 and
hGM-CSF receptors.
Retroviral transfections were performed by using the pM5 vector
containing the receptor subunit gene and an antibiotic resistance gene
as a selectable marker.19 In the case of the subunits, this was neomycin phosphotransferase (neo) and for the
hc, hygromycin phosphatase (hgr). All receptor
subunits were cloned into the BamHI site of the pM5 retroviral
vector. The chimeric receptor subunit was produced by introducing
an NheI restriction site (position 1093 for the hIL-3 R and
1142 for the hGM R) into the transmembrane domain by site-directed
mutagenesis. The mutagenesis was performed on double-stranded DNA with
the Chameleon ds mutagenesis kit (Stratagene, La Jolla, CA) in
accordance with the manufacturer's instructions. The chimeric receptor
subunit was produced by ligating the extracellular domain of the hIL-3
R to the intracellular domain of the hGM R at the NheI
site. All mutations and constructs were confirmed by DNA sequencing.
Retroviral vectors containing the antibiotic resistance gene(s) were
used as controls. The receptor constructs were lipofected into the
GP+envAM12 murine fibroblast packaging cell line20 by using
Lipofectamine (GIBCO, Paisley, UK). Retroviral
transfection of FDCP-mix cells was performed by coculture with the
packaging cell line for 48 hours. FDCP-mix cells were harvested,
washed, and selected for antibiotic resistance by culturing in medium
supplemented with 1 mg/mL G418 and/or 0.15 mg/mL hygromycin B, as
appropriate. Polyclonal cell populations were labeled for flow
cytometric analysis with antibodies directed against the extracellular
domain of the receptor subunits (see below). After flow cytometric
analysis, clonal populations were generated from single cells sorted
into 96-well culture plates by using the Automatic Cell Deposition Unit
facility of the fluorescence-activated cell sorter (FACS) Vantage flow
cytometer (Becton Dickinson, Cowley, UK). For each set of
experiments, data are shown from a clone representative of the multiple
transfectants tested.
Analysis of human IL-3 and GM-CSF receptor expression.
Ectopic receptor expression was confirmed by flow cytometry by using a
2-step antibody labeling procedure. Cells expressing the hGM R were
identified by using an anti-hGM R monoclonal antibody (Santa Cruz)
and detected after incubation with fluorescein isothiocyanate
(FITC)-conjugated anti-mouse IgG (Becton Dickinson). For hIL-3 R and
hc, biotinylated anti-hIL-3 R and
anti-hc antibodies (Cambridge BioScience, Cambridge,
UK) were used followed by incubation with FITC-avidin and
streptavidin phycoerythrin (PE) (Becton Dickinson), respectively. Flow
cytometric analysis was performed with a FACS Vantage flow cytometer
(Becton Dickinson).
Scatchard analysis.
FDCP-mix cells were washed once in IMDM (4°C), centrifuged, and
resuspended in phosphate-buffered saline (PBS), pH 3, for 1 minute to
remove cytokine bound to receptors. The cells were then pelleted and
resuspended in binding buffer (Dulbecco's modified Eagle's medium
containing 0.02% sodium azide, 25 mmol/L HEPES, 0.1% [wt/vol] BSA,
pH 7.4). Binding of [125I] hGM-CSF (specific activity,
1,820 Ci/mmol) or [125I] hIL-3 (specific activity, 543 Ci/mmol) was assayed.21 Nonspecific binding was calculated
by using a 100-fold excess concentration of unlabeled hGM-CSF or hIL-3,
as appropriate.
Measurement of proliferation.
DNA synthesis was used as a measure of proliferation and was performed
by determining incorporation of [3H] thymidine as
previously described.22 Briefly, cells were washed and
incubated with cytokines (5 × 104 cells/sample unless
otherwise stated) for 16 hours, pulsed for 4 hours with
[3H] thymidine, harvested by using a DYNATECH cell
harvester (DYNATECH, Billingshurst, UK), and the
incorporated radioactivity measured by scintillation counting.
Morphologic analysis.
A morphologic analysis of cells in liquid culture was performed as
described.12 Slides were prepared with a Shandon cytospin centrifuge (Shandon, Runcorn, UK) and stained with
May-Grünwald-Giemsa stain. At least 100 cells were scored for
each slide.
Granulocyte-macrophage differentiation assay of FDCP-mix cells.
FDCP-mix cells in the logarithmic growth phase were washed to remove
growth factors and resuspended in IMDM supplemented with 20% (vol/vol)
FCS, 0.01 ng/mL IL-3, and 10% (vol/vol) mouse lung conditioned
medium.23 After 7 days in culture, cells were counted and
cytospin preparations made.
Analysis of differentiation markers.
Cell-surface expression of Mac-1 (CD11b) was analyzed by flow
cytometry. Cells were labeled by using anti-Mac-1 antibody
(Pharmingen) followed by PE-conjugated anti-rat antibody (Becton
Dickinson). Nonspecific labeling was assessed with primary antibodies
of the corresponding isotype. Flow cytometric analysis was performed by
using a FACS Vantage flow cytometer (Becton Dickinson).
Analysis of protein tyrosine phosphorylation.
Tyrosine phosphorylation of intracellular proteins was analyzed by
Western blotting with a monoclonal mouse antiphosphotyrosine antibody
(TCS, Botolph Claydon, UK) as previously
described.22 Briefly, cell lysates were prepared in buffer
containing 50 mmol/L Tris Acetate (pH 7.5), 1% (vol/vol) NP-40, 1 mmol/L EDTA, 1 mmol/L EGTA, 120 mmol/L NaCl, 1 mmol/L Na3
VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL pepstatin A, benzamidine, antipain, aprotinin, TLCK, and TPCK.
Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to nitrocellulose (Hybond C;
Amersham), and immunoblotted by using a mouse monoclonal antiphosphotyrosine antibody. The protein-antibody complexes were visualized by ECL (Pierce, UK).
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RESULTS |
Retroviral mediated gene transfer of hIL-3 R and hGM R subunits into
FDCP-mix cells.
The use of 2 selectable markers and retroviral mediated-gene transfer
allowed the generation of FDCP-mix cell lines expressing the hIL-3 or
hGM-CSF receptor subunit genes, both alone and in combination with
the hc subunit. After antibiotic selection, the
polyclonal cell populations were analyzed for human receptor gene
expression by flow cytometry with antibodies directed against the
extracellular domain of the hIL-3 R and hGM R and/or
hc subunits. Dual transfectants expressing both the
hIL-3 R or hGM R together with hc were assessed by
using 2-color analysis with FITC- and PE-conjugated secondary
antibodies to label the transfected human and c
subunits, respectively. Clonal cell populations were generated from
cells identified and sorted on the basis of ectopic expression of hIL-3
R or hGM R subunits. Flow cytometric analysis was performed to verify
ectopic expression of the human receptor subunit in the clonal
populations obtained. No specific fluorescence labeling of the parental
FDCP-mix cells was detected with anti-hIL-3 R, anti-hGM R, or
anti-hc antibodies (Figs 1A-C). However, in the appropriate clones,
expression of hIL-3 R, hGM R, or hc was evident as
shown by a log increase in fluorescence (Figs 1D-F) relative to the
parental cells. Cells transfected with the control retroviral vector
containing the antibiotic resistance gene(s) showed a similar pattern
of labeling to the parental FDCP-mix cells (data not shown).
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| Fig 1.
FACS profiles of FDCP-mix cells transfected with hIL-3
and hGM-CSF receptor subunits. Cells were labeled with anti-hIL-3 and
hGM-CSF receptor subunit antibodies in a 2-step procedure. The black
histograms denote nonspecific fluorescence obtained with the secondary
fluorescent reagent only and are overlaid with the histograms obtained
after labeling with both the primary antireceptor subunit antibody and
secondary reagent. Isotype control antibodies gave similar flow
profiles to those obtained by using the secondary reagent only (data
not shown). Results are shown for expression of the (A and D)
hIL-3 R, (B and E) hGM R and (C and F) hc receptor
subunits by the (A-C) parental FDCP-mix cells and cells transfected
with (D-F) hIL-3 R, hGM R, and hc subunits and are
representative of least 6 such experiments using different clones
expressing hIL-3 R, hGM R, and hc subunits,
respectively. Similar profiles were obtained for cells coexpressing
hc subunits and either hIL-3 R or hGM R subunits.
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To avoid biasing the differentiation or developmental potential of the
cells before experimentation, cells were always cultured in mIL-3
rather than hIL-3 or hGM-CSF throughout the selection procedure. The
investigation of the human receptor subunits' effects in the control
of differentiation required that the developmental potential of the
FDCP-mix cells was maintained. The cell lines generated were checked
and found capable of differentiation in response to cytokines that
promote differentiation of the parental FDCP-mix cells.13
Characterization of the FDCP-mix hGM-CSF and hIL-3 receptor
transfects.
The hIL-3 and hGM-CSF responsiveness of the FDCP-mix hIL-3 R and hGM R
subunit transfects were initially assessed by determining the effects
of the human cytokines on cell viability. FDCP-mix cells expressing hGM
R or hIL-3 R (hGM R cells and hIL-3 R cells) survived in
response to the cognate human cytokine (Fig 2A). The addition of hIL-3 promoted
survival of hIL-3 R cells similar to that obtained in the presence
of 10 ng/mL mIL-3, whereas hGM-CSF provided a relatively weak survival
stimulus in hGM R cells. The relatively small response to hGM-CSF
was not because of a discrete subpopulation of the FDCP-mix cells
expressing the hGM R subunit; as flow cytometric analysis of this
clonal population before experimentation indicated, there was a single
population of cells expressing the hGM R subunit (Fig 1E). Notably,
the response of the hIL-3 R and hGM R populations was only
observed at relatively high concentrations (100 ng/mL) of the human
cytokines (Fig 2B). This is consistent with previous reports indicating that there is a weak interaction between ectopic human subunits and
endogenous murine subunits, resulting in formation of low affinity
receptors.24-26
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| Fig 2.
Effects of hIL-3 and hGM-CSF on survival of FDCP-mix cell
transfects. (A) Cell viability was assessed either in the absence of
cytokines ( ) or in the presence of 100 ng/mL hGM-CSF ( ) or 100 ng/mL hIL-3 ( ). Results are expressed as percentage of control (10 ng/mL mIL-3) and are the mean values from at least 2 experiments ± SEM. (B) Dose-response of FDCP-mix cells expressing (i) hIL-3 R
( ) or hIL-3 R/c ( ) to hIL-3 (ii) hGM R ( )
or hGM R/c ( ) to hGM-CSF. Cell viability was
assessed in the presence of hIL-3 or hGM-CSF (0 to 100 ng/mL) by trypan
blue exclusion after 48 hours culture. Results are expressed as
percentage of control (10 ng/mL mIL-3) and are the mean values from at
least 3 experiments ± SEM. Similar results were obtained with at
least 4 clones of each transfect.
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FDCP-mix cells expressing the hc subunit
(hc cells) were nonresponsive to hIL-3 or human GM-CSF.
However, cells coexpressing the hc subunit together with
the hIL-3 R subunit (hIL-3 R,c cells) or hGM R
subunit (hGM R,c cells) responded to the cognate cytokine at concentrations for which cells singly transfected with
hIL-3 R and hGM R cells were unresponsive (Fig 2B). The addition
of hIL-3 and hGM-CSF promoted cellular proliferation of the hIL-3
R,c and hGM R,c transfects,
respectively, as assessed by [3H] thymidine incorporation
assays (Fig 3). The dissociation constant (kd) values for the receptors in FDCP-mix cells
transfected with the hc subunit together with either the
hIL-3 R or hGM R were 269 pmol/L and 324 pmol/L, respectively, as
determined by the Scatchard analysis (data not shown). These results
are consistent with the presence of high affinity receptor
sites.27-30 The hIL-3 R and hGM-CSF R were expressed at
more than 1,000 receptors per cell with levels ranging between 1,200 to
17,000 receptors per cell. The same biologic response was stimulated by
the cognate human cytokine in all cell lines over a range of cytokine
concentrations (0.1 to 100 ng/mL) (Fig 6B). Thus, the transfected
receptors were expressed at high levels relative to endogenous cytokine
receptors on parental FDCP-mix cells and hematopoietic progenitor
cells, which are present at 20 to approximately 100 receptors per
cell.2,21,31
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| Fig 3.
Effects of hIL-3 and hGM-CSF on proliferation of FDCP-mix
cells coexpressing hIL-3 R,c or hGM
R,c. Proliferation was assessed by determining
[3H] thymidine incorporation after culturing for 16 hours
at 37°C. FDCP-mix cells expressing (A) hIL-3 R /c
and (B) hGM R /c were stimulated by hIL-3 (0.01 to
100 ng/mL) and hGM-CSF (0.1 to 100 ng/mL), respectively. Results are
expressed as percentage of control (10 ng/mL mIL-3) and are the mean
values from at least 3 experiments ± SEM. Similar results were
obtained with at least 4 clones of each transfect.
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hGM-CSF and hIL-3 stimulate differential signaling events in
transfected FDCP-mix cells.
Cytokine receptors mediate their effects by promoting intracellular
signaling pathways. Tyrosine phosphorylation of proteins is an initial
signal transduction event in response to IL-3 and GM-CSF.32
Tyrosine phosphorylation was assessed and compared by
antiphosphotyrosine Western blotting of cell lysates prepared from the
hGM R,c and the hIL-3 R,c cells
after stimulation with their cognate cytokines. Cells were stimulated
with hIL-3 or hGM-CSF at a concentration for which the singly
transfected hIL-3 R and hGM R were nonresponsive (10 ng/mL). The
effects of hIL-3 and hGM-CSF on the dual-transfected hGM
R,c and hIL-3 R,c cell populations
could, therefore, be attributed to interaction of the hIL-3 R and
hGM R subunits with hc and not endogenous murine subunits (Figs 2 and 3). There were differences in the patterns of
tyrosine phosphorylation observed in response to hIL-3 and hGM-CSF. The
major differences were that a protein of molecular weight
(Mr), approximately 80 kD, was tyrosine phosphorylated in
response to hGM-CSF but not hIL-3 and an Mr 46-kD protein was more heavily phosphorylated in response to hIL-3 than hGM-CSF (Fig
4). Next, it was determined whether or not
the differences in hIL-3 and hGM-CSF signaling were associated with
differential biologic responses.
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| Fig 4.
Protein tyrosine phosphorylation in response to hIL-3 and
hGM-CSF in hIL-3 R,c and hGM R,c
cells, respectively. Cells were washed and incubated growth-factor free
for 4 hours before stimulation with 10 ng/mL hIL-3 or hGM-CSF as
appropriate for 10, 30, and 60 minutes (lanes 2, 3, 4, and 6, 7, 8, respectively). Cell lysates were prepared and resolved by SDS-PAGE
using a 7.5% gel before Western blotting using an antiphosphotyrosine
antibody. Lanes 1 through 4 and 5 through 8 are cell lysates prepared
from hIL-3 R,c and hGM R,c cells,
respectively. Lane 1 and 5 are control samples (cytokine diluent only).
Arrows indicate molecular weights of the molecular-weight markers.
Similar results were obtained with at least 2 clones of each
transfect.
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The effects of hIL-3 and hGM-CSF on the clonogenic potential of
FDCP-mix cells coexpressing human subunits with
hc subunits.
To compare the effects of hIL-3 and hGM-CSF on development, their
potential to stimulate proliferation and long-term expansion of the
FDCP-mix receptor transfectants was assessed. The addition of hIL-3
promoted expansion of hIL-3 R,c cells at a rate
similar to that obtained in response to mIL-3, which is the cytokine in which FDCP-mix cells are routinely cultured (Table
1). In contrast, although hGM-CSF was
capable of stimulating [3H] thymidine incorporation over
a short period (Fig 3), it promoted only limited population expansion
compared with hIL-3 stimulation of hIL-3 R,c cells
(Table 1). Thus, although both hGM-CSF and hIL-3 promote proliferation
of cells expressing hGM R,c or hIL-3 R,c, respectively, only hIL-3 was able to stimulate a
persistent expansion or maintenance of the population.
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Table 1.
Population Expansion of hGM R,c Cells
and hIL-3 R,c Cells Continuously Cultured in the
Presence of 10 ng/mL hGM-CSF and hIL-3, Respectively
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The maintenance of clonogenic potential of hIL-3 R,c
and hGM R,c cells was assessed after 7 days liquid
culture in the presence of the appropriate human cytokine, either alone
or in combination with murine cytokines, which promote myeloid
differentiation of FDCP-mix cells. This was assayed by plating cells
into soft agar containing mIL-3 and determining the number of colonies
formed (Fig 5). When hIL-3
R,c cells were cultured in hIL-3 for 7 days before
plating in mIL-3, it was apparent that hIL-3 was more potent than high
concentrations of mIL-3 in maintaining clonogenic
potential.12,13 Furthermore, culture of hIL-3
R,c cells in granulocyte-macrophage differentiation
conditions (which promote a loss in clonogenic potential) in the
presence of hIL-3 had the effect of maintaining the clonogenic
potential of hIL-3 R,c cells (Fig 5A). Thus, hIL-3,
via interaction with hIL-3 Rc complex, promoted
prolonged stimulation of proliferation and suppression of clonogenic
extinction in the presence of cytokines, which promote differentiation.
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| Fig 5.
Clonogenic potential ability of (A) hIL-3
R,c cells and (B) hGM R,c cells.
(A) hIL-3 R,c cells and (B) hGM R,c
cells were cultured in the presence of the cognate cytokine (0.1 to 10 ng/mL) alone or in combination with cytokines that promote
granulocyte/macrophage development (G Diff) for 7 days before washing
free of growth factors and plating (at a cell density of 2,000 cells/plate) in triplicate into soft agar containing 5% (vol/vol)
mIL-3. Cells cultured in recombinant (r) mIL-3 (10 ng/mL)
for 7 days before plating were used as the positive control. Data are
from a single representative experiment of 3 and the values shown are
the mean of triplicates ± SD. Similar results were obtained with at
least 3 clones of each transfect.
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For hGM R,c cells, hGM-CSF was unable to sustain the
number of clonogenic cells compared with parallel cultures maintained in mIL-3 throughout the 7-day period. Culture of hGM
R,c cells with hGM-CSF together with
granulocyte-macrophage differentiation conditions was also unable to
support maintenance of colony-forming potential (Fig 5B). The hGM
R,c differed from hIL-3 R,c in being unable to support long-term proliferation and maintenance of
colony-forming cells.
Effects of human IL-3 and human GM-CSF on the development of FDCP-mix
cells in the presence of ectopically expressed human receptor subunits.
The effects of hGM-CSF and hIL-3 on the morphology of the receptor
transfectants were assessed after 7 days in culture with the
appropriate human cytokine. In the case of hIL-3 R,c
cells cultured in the presence of hIL-3 (10 ng/mL), the vast majority of the cells had a blast cell morphology (Fig 6A
[i]). Furthermore, culture
of these cells with hIL-3 in combination with granulocyte-macrophage differentiation conditions resulted in maintenance of their blast cell
morphology (Fig 6A [ii]). Thus, hIL-3 inhibited the acquisition of a
mature cell phenotype, which is normally obtained in
granulocyte-macrophage differentiation conditions and maintained the
clonogenic potential of the hIL-3 R,c cells even in
the presence of developmental stimuli (Fig 5). The morphology of hIL-3
R,c cells cultured in granulocyte-macrophage
differentiation conditions is shown in Fig 6A [iii] and confirms that
the cells were capable of differentiation.
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| Fig 6.
(A) Morphology of hIL-3 R,c cells and
hGM R,c cells in culture. Cells expressing hIL-3
R,c were cultured in (i) hIL-3 (10 ng/mL) alone or
(ii) hIL-3 (10 ng/mL) in combination with murine cytokines that promote
granulocyte-macrophage differentiation. Cells expressing hGM
R,c were cultured in (iv) hGM-CSF (10 ng/mL) alone or
(v) hGM-CSF (10 ng/mL) in combination with granulocyte-macrophage
differentiation conditions. Panels (iii) hIL-3 R,c
cells and (vi) hGM-CSF R,c cells show the morphology
of cells cultured in granulocyte-macrophage differentiation conditions
for comparison. Cytospin samples of cells were prepared after 7 days in
culture and the morphology examined after May-Grunwald-Giemsa staining.
Bar, 10 µm. Results are from an experiment representative of 3. Similar results were obtained with at least 3 clones of each transfect.
(B) Dose-response of hIL-3 and hGM-CSF effects on morphology of hIL-3
R,c cells and hGM R,c cells
respectively in culture. Cells expressing (i) hIL-3
R,c or (ii) hGM R,c were cultured
in hIL-3 or hGM-CSF (0.1 to 100 ng/mL), respectively. Cytospin samples
of cells were prepared after 7 days in culture and the morphology
examined after May-Grünwald-Giemsa staining. Results are
expressed as cell morphology (percentage of total cells scored). Cells
were scored as blast, early granulocyte (EG), late granulocyte (LG), or
macrophage (m/phage). Results are from a single experiment
representative of 3. Similar results were obtained with at least 3 clones of each transfect.
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The loss of clonogenic potential seen in the hGM R,c
cells, despite their initial marked proliferative response to hGM-CSF, suggested that the cells may have undergone maturation in response to
this human cytokine. Analysis of cell morphology after 7 days in
culture with hGM-CSF showed that the cells acquired a mature myeloid
cell morphology (Fig 6A [iv]), resembling that observed for cells
cultured in granulocyte-macrophage differentiation conditions shown in
Fig 6A (vi). Furthermore, cells cultured in hGM-CSF plus granulocyte-macrophage differentiation conditions led to the formation of mature cells (Fig 6A [v]). The effects of hIL-3 or hGM-CSF on the
morphology of the hIL-3 R,c and hGM
R,c cells, respectively, were similar at all
concentrations of human cytokine tested (0.1 to 100 ng/mL). This
indicated that the differential effects of hIL-3 and hGM-CSF were not
simply due to the dose of the cytokine used (Fig 6B).
To further characterize the developmental status of these cells, the
expression of markers for granulocyte-macrophage development was
assessed with flow cytometry. There was no significant change in
expression of the myeloid lineage marker, MAC-1, in the hIL-3 R,c cells cultured in hIL-3 (P  .05,
n = 2). However, there was a 2.3-fold increase in the level of
expression of MAC-1 in hGM R,c cells cultured in
hGM-CSF. This confirmed the differential effects of hGM-CSF and hIL-3,
respectively, on hGM R,c and hIL-3 R,c cells and suggested that the specificity of the
response may be conferred by the subunit of these heterodimeric receptors.
A chimeric receptor subunit consisting of the
extracellular domain from the IL-3 R subunit and the
intracellular domain of the GM R subunit promoted
myeloid development in FDCP-mix cells.
To investigate whether or not the signal specificity of the hGM-CSF
receptor is generated by the unique subunit, a chimeric hIL-3/GM
R was generated. This chimeric subunit was composed of the
extracellular domain of the hIL-3 R subunit ligated to the cytosolic
domain of the hGM R subunit. The fusion was made in the
transmembrane region to avoid possible interference with known
functional sequences proximal to the membrane. Thus, the chimeric
hIL-3/GM R contained the binding site for hIL-3, which is present in
the external domain of the hIL-3 R33,34 and the
cytosolic domain of the hGM R subunit. Expression of the chimeric
hIL-3/GM R subunit was detected by flow cytometry by using
anti-hIL-3 R antibody. The results are shown in Fig
7A and the flow
cytometric profile resembled that obtained for hIL-3R expressing
cells (Fig 1D) with a log increase in fluorescence relative to the
nonspecific binding obtained with the secondary antibody only. hIL-3,
but not hGM-CSF, promoted proliferation of these cells comparable in
magnitude with that observed in hGM R cells in response to hGM-CSF
(Fig 7B). Furthermore, exposure to hIL-3 led to a change in
the phenotype of the cells from blast cells to a more mature cell
phenotype (Fig 7C [i]), a response that was more similar to that
observed for hGM R cells (Fig 7C [iii]) than hIL-3 R cells (Fig
7C [ii]) cultured in hGM-CSF or hIL-3, respectively. Thus, replacing
the hIL-3 R subunit cytosolic domain with the hGM R subunit
cytosolic domain led to differential responses to hIL-3 in this cell
line compared with hIL-3 R cells, which maintain a blast cell
phenotype and undergo marked proliferation and population expansion in
the presence of hIL-3. The developmental response resembled that
obtained for hGM R cells, which undergo myeloid differentiation in
response to hGM-CSF, indicating that the specificity is conferred by
the cytosolic domain of the subunit.
View larger version (55K):
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| Fig 7.
(A) Flow cytometric profile of FDCP-mix hIL-3/GM R
cells. Expression of the chimeric receptor was determined by flow
cytometry by using an anti-hIL-3 R antibody as outlined in Fig 1.
(B) Effects of hIL-3 on proliferation of FDCP-mix cells expressing (i)
chimeric hIL-3/hGM R, (ii) hIL-3 R, and (iii) hGM R subunits.
Proliferation was assessed by determining [3H] thymidine
incorporation stimulated by hIL-3 and hGM-CSF (1 to 100 ng/mL) after 16 hours in culture. Cells were plated at 4 × 105 per sample
for the chimeric hIL-3/GM R cells and the hGM R cells. Results
are expressed as percentage of control (10 ng/mL mIL-3) and are the
mean values from at least 3 experiments ± SEM. Similar results were
obtained for at least 3 clones of each transfect. (C) Effects of hIL-3
on morphology of FDCP-mix cells expressing (i) chimeric hIL-3/hGM R
(ii) hIL-3 R, and (iii) hGM R subunits. Morphology was assessed
with May-Grünwald/Giemsa cytospin preparations of cells cultured
in either (i), (ii) 100 ng/mL hIL-3, or (iii) 100 ng/mL hGM-CSF for 7 days as appropriate; (i) hIL-3/GM R cells, (ii) hIL-3 R, and
(iii) hGM R cells. Bar, 10 µm. Similar results were obtained for
at least 3 clones of each transfect.
|
|
| |
DISCUSSION |
The requirement of specific cytoplasmic domains of the
hc for cell viability, proliferation, and
differentiation signaling has been well-defined in
vitro.16,17,26,35-37 The role of the c
subunit in vivo has also been investigated in transgenic mice null for
c, IL-3 or expressing a constitutively
activated hc38-42 (see below). However, to
date, little information on the role of the subunits of the GM-CSF
and IL-3 receptors in differentiation has been available. The
differential responses of murine multipotent FDCP-mix cells to mIL-3
(self-renewal) and mGM-CSF (weak proliferation and myeloid
differentiation) intrigued the investigators.13 Furthermore, constitutive mGM-CSF expression in FDCP-mix cells after
retroviral transfection with the mGM-CSF gene, stimulated differentiation that could be blocked by mIL-3.43 Such
differential effects must reside in receptor-mediated signaling events.
Because murine hematopoietic cells express two subunits, of which
only one can interact with mGM R, we decided a clear
structure/function analysis of the receptors could only be performed by
ectopically expressing human receptors and mutants in FDCP-mix cells.
Expression of hIL-3 and hGM-CSF receptor and c
subunits in FDCP-mix cells allowed both analysis and direct comparison
of their effects on hematopoietic cell development. The IL-3 and GM-CSF
cytokines are species specific; thus, addition of hIL-3 and hGM-CSF
selectively activated the ectopically expressed hIL-3 R and hGM R,
facilitating structure-function analysis of wild type and mutant
receptor subunits.
FDCP-mix cells transfected with the hIL-3 R or hGM R subunits
alone only responded to relatively high concentrations of their cognate
cytokines (Fig 2B). These results indicate the formation of low-affinity receptors because of interaction with the endogenous murine c and IL-3 as reported
previously.24-26 Coexpression of hc subunits
together with hIL-3 R or hGM R led to formation of high-affinity
receptors (Fig 2B), although cells transfected with hc
subunits alone were unable to respond to hIL-3 or hGM-CSF (Fig 2A).
These data are consistent with previous reports indicating that the
c subunit alone is unable to bind IL-3 or GM-CSF but acts to convert the initial low-affinity ligand binding by the subunit to a high-affinity receptor complex.4
The hIL-3 R,c and hGM R,c both
promoted survival and proliferation of FDCP-mix cells but their
developmental effects were distinct. The dual-transfected hIL-3
R,c cells survived and proliferated in response to
hIL-3 and maintained their clonogenic potential. Coaddition of hIL-3
with a combination of cytokines that promote granulocyte-macrophage
development of the hIL-3 R,c cells
"countermanded" the differentiation response (Figs 5 and 6). The
hIL-3 R,c, thus, promoted self-renewal and
maintenance of primitive morphology. In contrast, hGM-CSF promoted
myeloid development of cells cotransfected with hGM R and
hc subunits (Fig 6). Thus, the human receptors for hIL-3
and hGM-CSF promote differential effects in FDCP-mix cells,
self-renewal, and myeloid differentiation. The developmental responses
observed were similar throughout a wide dose-range of human cytokine
demonstrating that the effects are not concentration dependent. By
culturing hIL-3 R,c and hGM R,c
cells in concentrations of hIL-3 or hGM-CSF, at which the single hIL-3
R or hGM R are nonresponsive (Figs 2 and 3), the human IL-3 R
and hGM R subunits interact with hc and not
endogenous murine subunits. The differences in the biologic effects
observed with the hIL-3 R,c and hGM
R,c can, thus, be attributed to results from
interaction of the hIL-3 R or hGM R subunits with the
hc subunits.
Cells transfected with either the hIL-3 R or hGM R alone also
gave similar results when cultured with their respective cognate cytokines, with hIL-3 promoting self-renewal and hGM-CSF promoting myeloid differentiation (Fig 7). Interestingly, the hGM
R1 and 2 subunit isoforms have previously
been shown to promote myeloid differentiation in response to hGM-CSF in
FDCP-1 cells.25 However, the effects of activation of these
hGM R isoforms on clonogenic potential were not determined.
We have shown also that hIL-3 and hGM-CSF stimulate differential
cellular signaling events; they initiate different tyrosine phosphorylation responses (Fig 4). To our knowledge, this is the first
demonstration of differential developmental and cellular signaling by
these cytokines in primitive hematopoietic progenitor cells. Specific
activation of hIL-3 and hGM-CSF receptors by the addition of their
cognate cytokines to FDCP-mix cells provides a model system for the
analysis of the molecular pathways associated with hematopoietic
development. The magnitude of the responses elicited by activation of
the hGM R,c is much greater than that of the mGM-CSF
receptor in FDCP-mix cells. This is probably because of overexpression
of the hGM R compared with approximately 20 mGM
receptors/cell13 and facilitates analysis of the signaling events associated with GM-CSF-mediated differentiation. Although the
signal transduction pathways for survival and proliferation in response
to hGM-CSF are relatively well defined, their roles in differentiation
remain to be determined. For example, tyrosine phosphorylation of the
SHP-2, JAK2, Shc, Erk, and STAT5 signaling proteins correlates with
hGM-CSF-mediated proliferation but is not required for
differentiation.37,44 Evidence for the involvement of
specific signaling pathways in differentiation comes from several groups, including the demonstration that activation of the PKC isoform determines lineage commitment in bipotential
granulocyte-macrophage colony forming cells.45,46 The model
we have developed will now permit differentiation signaling in
multipotent cells to be investigated.
Is there a role for the unique subunit in hIL-3 and hGM-CSF
receptor function? Results obtained with a chimeric hGM R, composed of
the extracellular domain of hGM R and the cytoplasmic domain of the
hc subunit, have indicated that the cytoplasmic domain of GM Rc can functionally replace that of the hGM R
subunit to promote proliferation.47 However, studies on
deletion mutants of subunits of the IL-3 subfamily of receptors
have showed that their cytoplasmic domains play a key role in receptor
mediated signal transduction.34,48-50 For example, deletion
of the cytoplasmic domain of IL-3 R subunit resulted in a
high-affinity hIL-3 R that was unable to stimulate proliferation or
promote tyrosine phosphorylation of the c subunit and
STAT5 signaling proteins.34 Consistent with such data, the
GM R also plays a direct role in activation of the JAK/STAT
pathway.51 Recent studies on truncation mutants of the hGM
R subunits expressed in IL-3-dependent murine FDCP-1 cells indicate
that specific regions of the subunit are required for survival
proliferation and maturation.44 In this current study, the
role of the subunit in self-renewal/differentiation was
investigated by transfection of a chimeric receptor composed of the
extracellular domain of the hIL-3 R and the intracellular domain of
the hGM R into FDCP-mix cells. Cells expressing the chimeric
hIL-3/GM R construct responded to hIL-3 and not hGM-CSF. The cells
resembled the hIL-3 R cells, in that the response was observed only
at relatively high concentrations of hIL-3, which is consistent with
the formation of a low-affinity receptor. The biologic response more
closely resembled that of hGM R than hIL-3 R in that the chimeric
hIL-3/GM R provided a weak survival and proliferation stimulus and
promoted myeloid differentiation (Fig 7). These results suggest that
the cytosolic domain of the subunit may confer the specificity of
the biologic response.
How do these results compare to data derived from experiments on the
function of GM-CSF and IL-3 receptors in transgenic mice? A series of
knock-out mice have been generated that are defective for
c, IL-3 or both c and IL-3
ligand.38-41 Hematopoiesis is apparently normal in these
mice, although c and c -IL-3 null animals
have a pulmonary alveolar proteinosis-like disease,38,41,52 resembling that observed in GM-CSF ligand-deficient mice.53 The c null bone marrow does, however, display decreased
ability to initiate white blood cell recovery on transplantation into irradiated recipient mice.39 Interestingly, expression of
the hc in transgenic mice resulted in a
myeloproliferative disorder resembling polycythemia vera.42
Thus, there is a clear role for subunits from in vivo experiments.
The subunits are required for signal response coupling with the subunit.4,32 However, there is no direct evidence on the
role of the subunits in self-renewal or differentiation; indeed the
complexity of hematopoietic regulatory mechanisms in vivo would argue
against addressing this issue using whole animal experiments.
Therefore, we decided to investigate the receptor subunit functions in
the context of the multipotent FDCP-mix cell line. Although it has been
shown that the GM R subunit can alone promote glucose transport in
oocytes,54 GM-CSF is unable to mediate glucose transport in
neutrophils from mice deficient in the c
subunit.38 Our data suggest that the hIL-3 and hGM-CSF
receptor subunits play a role in conferring signal specificity and
eliciting different biologic responses within the context of the
c receptor subunit complex.
It is well known that IL-3 stimulates normal marrow cells to form some
blast colonies (corresponding with the data on the effects of
transfection of the hIL-3 receptor into FDCP-mix cells) but it also
promotes granulocyte-macrophage colony formation.10 This,
in part, reflects the age structure of hematopoiesis where a spectrum
of IL-3-responsive cells from the multipotent to late myeloid
progenitors are present. Thus, normal bone marrow gives a heterogeneous
response to IL-3 with some cells undergoing self-renewal. In FDCP-mix
cells just such a spectrum of cells at different stages of
differentiation can be generated by culture on stromal cells from
long-term marrow cultures. Some FDCP-mix cells retain their multipotent
phenotype and others differentiate to form mature granulocytes and
macrophages. Thus, the similarities between primary hematopoietic cells
and FDCP-mix are noteworthy, yet IL-3 is plainly not exclusively a
self-renewal factor for hematopoietic progenitor cells. The FDCP-mix
cells were first derived from murine long-term bone marrow cultures.
IL-3 was required to prevent the death of suspension cells removed from
these cultures. FDCP-mix cells self-renew indefinitely when cultured in
high concentrations of IL-3, presumably this is a feature of the cells
that allowed their isolation and cloning.
Nonetheless, FDCP-mix can be obtained in large quantities for
biochemical studies, important clues to the molecular mechanisms stimulated by cytokines in primitive hematopoietic cells can be derived, and the information can be used to study enriched progenitor cell populations, which can be isolated from bone marrow. Further work
is now needed to extend these observations that the cytoplasmic domain
of the subunit plays a role in conferring signal specificity for
self-renewal or differentiation. This current study indicates that
signaling pathways activated by interaction of hGM R or hIL-3 R
and the c receptor after cytokine activation are
different and regulate self-renewal/differentiation decisions.
| |
ACKNOWLEDGMENT |
Thanks to Professor T.M. Dexter (Paterson Institute for Cancer
Research) for the use of laboratory facilities and for helpful discussions with regard to this work.
| |
FOOTNOTES |
Submitted January 5, 1999; accepted May 3, 1999.
Supported by the Biotechnology and Biological Science Research Council
(UK), Cancer Research Campaign, and the Leukaemia Research Fund.
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 Anthony D. Whetton, PhD,
Leukaemia Research Fund Cellular Development Unit, Department of
Biomolecular Sciences, UMIST, Manchester M60 1QD, United Kingdom.
| |
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