Blood, 15 August 2000, Vol. 96, No. 4, pp. 1588-1590
BRIEF REPORT
Reassessment of interactions between hematopoietic
receptors using common beta-chain and interleukin-3-specific receptor
beta-chain-null cells: no evidence of functional interactions with
receptors for erythropoietin, granulocyte colony-stimulating factor, or
stem cell factor
Clare L. Scott,
Lorraine Robb,
Bette Papaevangeliou,
Rachel Mansfield,
Nicos A. Nicola, and
C. Glenn Begley
From The Walter and Eliza Hall Institute of Medical
Research, The Cooperative Research Centre for Cellular Growth Factors,
and the Rotary Bone Marrow Research Laboratories Factors, PO Royal
Melbourne Hospital, Victoria, Australia.
 |
Abstract |
Mice lacking both the gene encoding the shared receptor for
granulocyte macrophage-colony-stimulating factor (GM-CSF),
interleukin-3 (IL-3), and IL-5 common 
-chain
(Bc) and the gene for the IL-3 specific
receptor (BIL3) were generated. This was achieved by targeting the Bc locus in embryonic stem cells
that were heterozygous for a null mutation of BIL3. Cells
from mice generated with the doubly targeted embryonic stem cells were
unresponsive to all 3 cytokines. Considerable previous data suggested a
role for common beta-chain (c) in modulating
signaling of cytokines including erythropoietin (EPO), G-CSF, and stem
cell factor (SCF). However, bone marrow cells from mice lacking
c and IL3 showed
normal responsiveness to these cytokines. Thus, there was no evidence
for a biologically significant interaction between signaling via
c or IL3 and
signaling by EPO, G-CSF, or SCF. Previously documented biochemical
phenomena, including receptor transmodulation, receptor
transphosphorylation, and even direct physical interaction, involving
the c/IL-3 receptor systems do not reflect genuine
interactions of physiological significance in primary hematopoietic
cells. This study provided results that challenge conclusions
previously established using a variety of biochemical assays.
(Blood. 2000;96:1588-1590)
© 2000 by The American Society of Hematology.
| |
Introduction |
Interleukin (IL)-3 has numerous effects on
hematopoietic cells including actions on precursors and mature
cells.1-4 The receptor for IL-3 consists of a unique
specific 
-chain, IL-3R, which binds IL-3 with low
affinity,5,6 and the common -chain (c) which is also used by granulocyte macrophage-colony-stimulating factor
(GM-CSF) and IL-5. Following the binding of IL-3 to IL-3R, c converts the interaction to one of high
affinity.7 In mouse cells, but not human cells, an
additional IL-3-specific -chain (IL3) is used in
preference to c for signaling by IL-38 and,
unlike c, uses low affinity to directly bind
IL-3.
Mice lacking c (c
null mice), have an eosinopenia and, like mice deficient in GM-CSF,
develop lung disease reminiscent of human pulmonary alveolar
proteinosis.9-12 Cells from these mice lack high-affinity
binding for GM-CSF and IL-5. Mice that lack IL-3
(IL-3 null mice)8,10 show decreased biological responsiveness of cells to IL-3 (via intact c
signaling).8 Ablation of IL-3 explained the
conflicting results observed for hierarchical receptor interactions in
mouse cells versus human cells. Although GM-CSF was not able to
"transmodulate" IL-3 receptors and alter IL-3 binding in wild-type
murine cells (because of the availability to IL-3 of
IL-3), it was able to trans-down-modulate IL-3 binding
in IL-3 null cells, as a result of competition between GM-CSF and IL-3 for binding to c chains.8
Accumulated evidence suggests a role for c in modulating
signaling of other hematopoietic cytokines including G-CSF,
erythropoietin (EPO), and stem cell factor (SCF). Both GM-CSF and IL-3
transmodulated binding of G-CSF and M-CSF in normal
cells.13 IL-3 also showed this effect in both
c null and in IL3
null cells.8 However, transmodulation by GM-CSF required a
functional c receptor. Based on these results we
proposed that c and IL-3 interacted
with the G-CSF receptor (G-CSFR) and M-CSFR and/or that the GM-CSF or
IL-3 activation of cellular signaling pathways modified G-CSFR and
M-CSFR, perhaps resulting in their internalization. However, this
phenomenon remains unexplained.8
Interaction between c and the EPO receptor
(EPOR) has been demonstrated. EPO stimulated tyrosine phosphorylation
of c in the UT-7 erythroleukemia cell
line,14 although neither GM-CSF14 nor
IL-315 stimulated tyrosine phosphorylation of EPOR. It was therefore suggested that EPO might activate the GM-CSF signaling pathway by phosphorylating c.14 Functional
and physical interactions between c and EPOR were
demonstrated using the murine IL-3-dependent cell line (Ba/F3), which
expresses IL-3R, c, and IL-3. The Ba/F3 cells transfected with murine EPOR acquired responsiveness to
EPO, and increased expression of murine c resulting in
heightened responsiveness to EPO. Conversely, inhibition of murine
c function in Ba/F3/EPOR cells inhibited both
IL-3-dependent and EPO-dependent cell growth. Moreover, an
EPO-independent physical interaction between c and EPOR
was demonstrated by coimmunoprecipitation.16 We sought to
address interactions involving the
c/IL-3R system using cells from
c/IL-3
null mice.
| |
Study design |
Generation of Bc/BIL3 null mice
The Bc and IL-3
loci are closely linked on mouse chromosome 15.17 To
generate mice with a mutation in both loci, the embryonic stem (ES)
cell line 3.15, which contains a targeted mutation of one allele of the
BIL3 locus, was electroporated with a linearized targeting
construct for the Bc locus. This construct was
as previously described,9 except that a cassette
containing the hygromycin-resistance gene18 was inserted
in exon 7, and a thymidine kinase cassette19 was ligated
to the 5'-end of the construct. Selection and screening of hygromycin
and FIAU-resistant ES cell clones were performed as previously
described. To detect homologous recombinants,
BamHI-digested DNA was hybridized with probe A (Figure
1) and a 3'-probe. Correctly targeted
clones were further analyzed by Southern blot analysis for the presence
of the BIL3 mutation.8 We used 3 ES cell lines
to create chimeric mice, which were screened for cosegregation of the
BIL3 and Bc mutations. Control mice
were C57BL/6 or 129/Sv, and the experiments were conducted on mice 6 to
14 weeks of age.
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| Figure 1.
Targeting the Bc and
IL-3 loci.
(A) Partial map of the Bc locus, targeting
construct, and predicted alteration of the Bc
locus after homologous recombination. Coding exons are numbered and
shown as black boxes. Noncoding exons are shaded gray. The position of
probe A is indicated. This probe was used to identify homologous
recombinants and to genotype mice by Southern blotting. Restriction
enzyme sites are shown, where B indicates BamHI; E,
EcoRI; S, SacI; V,
EcoRV. (B) Southern blot analysis of tail DNA from offspring
of a chimera generated by injection of ES cells containing targeted
mutations of the Bc and BIL3 loci.
DNA was digested with BamHI. Blots were probed with probe A
(top panel) or a probe that detects the targeted mutation of the
BIL3 locus (bottom panel).8 In the top panel,
the targeted Bc allele is a 6.7-kb (kilobase)
band, and the wild type allele is a 5-kb band. The probe
cross-hybridizes with the BIL3 locus, which is seen as a
10-kb band. In the bottom panel, the 9-kb band represents hybridization
of the probe to the Bc and BIL3 wild
type alleles. The targeted BIL3 allele is seen as a 2.5-kb
band. In the ES cell line used to generate these mice, the targeted
mutations in the Bc and BIL3 loci are
always observed in the same offspring, indicating that the homologous
recombination events in the 2 loci lie on the same chromosome.
|
|
Progenitor cell assays
Bone marrow (BM) progenitor cells were assayed in clonal culture
as previously described.9 Semisolid 1-mL agar cultures containing 5 × 104 BM cells or 105 spleen
cells in 0.3% agar in Dulbecco's modified Eagle's medium (DMEM) with
20% newborn calf serum were plated in triplicate and stimulated by
multiple combinations of purified recombinant growth factors. To
determine cytokine responsiveness, BM cells from
c/IL3 null
mice and control mice were cultured in agar using serial dilutions of
G-CSF (initial G-CSF concentration, 500 U/mL) or SCF (initial SCF
concentration, 100 ng/mL) for 7 days of incubation at 37°C in a fully
humidified atmosphere of 10% carbon dioxide (CO2) in air.
The colonies were enumerated using a dissection microscope. For
colony-forming unit-E (CFU-E) assays, bone marrow cells were
cultured using serial dilutions of EPO (initial EPO concentration, 4 U/mL) in methylcellulose cultures incubated for 2 days in 5%
CO2 in air. The colonies were enumerated using an inverted
microscope. Cultures were scored by an investigator blinded to the
genotype of the cells.
| |
Results and discussion |
Baseline hematopoiesis in
c/IL-3 null mice was
no different than that seen in c null mice
(C.L.S., L.R., and C. G. B., unpublished observations). This was in
keeping with previous reports of mice lacking either
c,9,10
IL-3,8,10 or the combination of
c and IL-3.20,21
Thus, in mouse cells and in spite of the presence of an additional
c specific for IL-3, which might imply an important
function, IL-3 did not have an essential role in steady-state hematopoiesis.
Hematopoietic progenitor cells from
c/IL-3 null mice were
examined. The lack of responsiveness to IL-3 and GM-CSF was confirmed. There was no proliferation when BM cells from
c/IL3 null mice were
stimulated by either cytokine. In contrast, colony formation in
response to stimulation with other hematopoietic cytokines, including
SCF and the combination of SCF, G-CSF, and IL-6, was normal. Analysis
of erythroid colonies in methylcellulose cultures revealed that the
number of erythroid progenitor cells (both BFU-E and CFU-E)
was also normal (C.L.S., L.R., and C.G.B., unpublished observations).
We have previously demonstrated that IL-3 was able to transmodulate
both G-CSFR and M-CSFR in the absence of either
c null or IL3 null
cells.13 However, transmodulation of G-CSFR and M-CSFR by
GM-CSF required the presence of
c.8 These results predict that a
biochemical analysis of transmodulation of G-CSFR and M-CSFR by IL-3 in
the absence of both chains would show a lack of transmodulation
capacity by IL-3. Indeed, in keeping with the lack of high-affinity
IL-3 binding that results from generating
c/IL-3 null
cells, we did not see transmodulation of G-CSF receptors by IL-3 on BM
cells from
c/IL-3 null
mice, even at doses of 100-nmol/L IL-3. This was in contrast to the
transmodulation of G-CSF receptors by IL-3 that was observed on
normal BM cells in the same experiments (N.A.N., unpublished observations).
The corollary of the G-CSFR transmodulation results predicted that the
response to G-CSF may, in part, be mediated by c or IL-3, although neither receptor was directly engaged by
G-CSF. This issue was addressed using
c/IL3 null cells. We
would have predicted that the absence of Bc
rendered cells less sensitive to stimulation with G-CSF. However, as
shown in Figure 2,
c/IL3 null cells
showed normal responsiveness to G-CSF. This indicated that biological
responsiveness to G-CSF was not dependent on c or IL3, implying that the biochemical
phenomenon of transmodulation was of no genuine biological
significance.
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| Figure 2.
Cytokine responsiveness of the
c null and
c/IL3 null mice.
Responsiveness to (A) 500 U/mL G-CSF (initial concentration), (B) 4 U/mL EPO, and (C) 100 ng/mL SCF with serial 2-fold dilutions. Results
are the colony number (the mean plus or minus SD) at each cytokine
dilution expressed as a percentage of maximal colony number, using 1-2 mice per genotype. Similar results were seen in 3 independent
experiments. A minimum of 3-4 mice were examined per genotype. Wild
type is indicated by open circles and
c/IL-3 null by
closed circles.
|
|
Several different observations have suggested a role for
c in signaling by EPO. In addition to the
phosphorylation and physical interaction data described above, IL-3 and
GM-CSF are known to cooperate with EPO in erythropoiesis in
vitro,22,23 and common signal transduction pathways
involving STAT5 are used by their receptors.24 In
addition, c has been implicated in signaling by SCF via
its receptor, c-kit, and SCF is able to induce serine/threonine phosphorylation of c.25
To determine the physiological significance of these observations,
responsiveness of
c/IL-3 null BM
cells to EPO and SCF was examined. However, there was no observed
difference in responsiveness to EPO (Figure 2). Nor was there a
difference in responsiveness to SCF for BM cells cultured from
c/IL-3 null mice
compared to wild-type mice (Figure 2).
We therefore conclude that previously documented biochemical phenomena,
including receptor transmodulation and receptor transphosphorylation, are not physiologically relevant in the context of
hematopoietic cell growth responses to individual cytokines.
Moreover, even the demonstration of direct physical interaction
involving the c/IL-3 receptor
systems in cell lines did not extrapolate to an interaction of
physiological significance in primary hematopoietic cells. This result
is important for interpreting the significance of biochemical
interactions between receptor molecules, particularly in studies in
which cell lines are employed.
| |
Acknowledgment |
The authors thank Louise Barnett for her work in generating the ES
cell line.
| |
Footnotes |
Submitted December 13, 1999; accepted April 10, 2000.
Supported in part by the Anti-Cancer Council of Victoria and
the Cooperative Research Centre for Cellular Growth Factors, Victoria,
Australia; the Bone Marrow Donor Institute; the Sylvia and Charles
Viertel Charitable Foundation; grant HL62275 from the National
Institutes of Health, Bethesda, MD; and the National Health and Medical
Research Council of Australia.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: C. L. Scott, The Walter and Eliza Hall
Institute of Medical Research, PO Royal Melbourne Hospital, Victoria
3050, Australia; email:
scottc{at}wehi.edu.au.
| |
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Abstract
Introduction
