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Blood, 15 November 2001, Vol. 98, No. 10, pp. 3165-3168
BRIEF REPORT
Perverted responses of the human granulocyte-macrophage
colony-stimulating factor receptor in mouse cell lines due to
cross-species  -subunit association
Barbara McClure,
Frank Stomski,
Angel Lopez, and
Joanna Woodcock
From the Cytokine Receptor Laboratory, Hanson Centre
for Cancer Research, Institute of Medical and Veterinary Science,
Adelaide, South Australia.
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Abstract |
Transfected murine cell lines are commonly used to study the
function of many human cytokine or receptor mutants. This study reports
the inappropriate activation of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor by the human GM-CSF antagonist, E21R, when the human receptor is introduced into the murine
cell line BaF-B03. E21R-induced proliferation of the BaF-B03 cells is
dependent on transfection with both hGM-CSF receptor  and
 c subunits. Studies on the underlying mechanism
revealed constitutive association between human and mouse
c and GM-CSF receptor-, tyrosine phosphorylation of
mouse and human c, and association of phosphorylated
mouse c into an activated human GM-CSF receptor complex
in response to E21R and GM-CSF. This interspecies receptor cross-talk
of receptor signaling subunits may produce misleading results and
emphasizes the need to use cell lines devoid of the cognate endogenous
receptors for functional analysis of ligand and receptor mutants.
(Blood. 2001;98:3165-3168)
© 2001 by The American Society of Hematology.
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Introduction |
Human granulocyte-macrophage
colony-stimulating factor (hGM-CSF) is a pleiotropic cytokine that
stimulates the proliferation, differentiation, and survival of myeloid
precursors and induces the effector functions of mature myeloid
cells.1,2 These multiple functions are mediated by binding
to high-affinity receptors that comprise a GM-CSF-specific chain
(hGMR-) and an affinity-converting, signal-transducing subunit
(hc), which is shared with the interleukin-3 (IL-3) and
IL-5 receptors.3,4 In the murine system there are 2 subunits, mc, which is analogous to hc,
transducing signals induced by mGM-CSF, mIL-3 or mIL-5, and
mIL-3, which is specific for mIL-3.5,6
Mouse myeloid cell lines, for instance FDCP-1 and BaF-B03, express both
mouse subunits.
Given the importance of hc in transducing signals
that regulate immune responses, hematologic recovery, and, in some
cases, leukemia, a significant amount of work has been devoted to
structure-function analysis of these cytokine-receptor systems. Studies
have led to the engineering of cytokine analogs with unique properties such as the hGM-CSF mutant E21R that behaves as a specific hGM-CSF antagonist.7 Characterization of the receptors has sought
to identify functional regions involved in receptor activation and has
identified regions in the cytoplasmic domain of hc that
couple to specific signaling molecules such as JAKs, STATs, and the
ras/MAP kinase pathway.8-10 However results from these
studies have in some cases been ambiguous or even conflicting.
Central to the analysis of cytokine and receptor mutants is the
choice of experimental system. Predominantly, mouse myeloid cell lines,
which are readily transfected with human receptor subunits, have been
used. A major problem is the expression of endogenous receptors for
these cytokines in these cell lines. For example, transfection of
hGMR- alone in murine FDCP-1 cells was initially reported to be
sufficient to mediate a proliferative signal despite only
displaying low-affinity hGM-CSF binding.11 However, it was
later shown that functional reconstitution of hGMR required both
hGMR- and hc subunits,12 and that the
initial observation with hGMR- alone was confounded by the
recruitment of endogenous mc.13 Likewise,
interaction of an extracellular point mutant of hc with
an endogenous mGMR- has been shown to lead to factor-independent
proliferation14 and chimeras between a constitutively
active erythropoietin receptor with the cytoplasmic domain of
GMR- promoted proliferation but only in presence of mc.15 Here we show an abnormal response of
the human GM-CSF antagonist E21R in transfected mouse cell lines and
the molecular basis for human-mouse GM-CSF receptor cross-talk. These
results emphasize the need for caution when interpreting data
from experiments using transfectants and the desirability of using
homologous systems.
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Study design |
Cell lines and proliferation assays
The human erythroleukemic cell line TF1.8, the
megakaryocytic leukemia human cell line, UT7, and the murine pro-B-cell
line, BaF-B03 (transfected with human GMR- and hc),
were grown as previously described.16,17 Following
cytokine starvation overnight, the proliferation of cell lines in
response to hGM-CSF or E21R was determined as previously
described.18 E21R, a single point mutant of hGM-CSF
(Gln21Arg) was donated by Bresagen (Adelaide, Australia).
Antibodies
Murine monoclonal antibodies 1C1 and 8E4, for detection of
hc, and 4H1 for immunoprecipitating hGMR- were
produced as previously described.16 1C1 was biotinylated
using a cellular labeling and immunoprecipitation kit (Boehringer
Mannheim, Rose Park, SA, Australia) and
streptavidin-horseradish peroxidase (HRP) was purchased from Amersham
Life Science (Little Chalfont, United Kingdom). Anti-mc rabbit polyclonal K-17 was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated antibodies specific for mouse or rabbit immunoglobulins were purchased from Pierce (Rockford, IL) and Dako (Botany, NSW, Australia),
respectively. The HRP-conjugated antiphosphotyrosine monoclonal
antibodies, PY20 and 4G10, were obtained from Transduction Laboratories
(Lexington, KY) and Upstate Biotechnology (Lake Placid, NY), respectively.
Immunoprecipitation, sodium dodecylsulfate-polyacrylamide gel
electrophoresis, and immunoblotting
Analysis of ligand-induced receptor complexes and their
phosphotyrosine status was determined using starved cells that were stimulated with factors for 5 minutes at room temperature at indicated concentrations. Cells were lysed and immunoprecipitated proteins were
run on reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting as previously described.19
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Results and discussion |
Human GM-CSF supports the proliferation of human cells by
activating endogenous hGMR.7 The antagonistic hGM-CSF
mutant, E21R, however, cannot mediate a proliferative response in human cells and is unable to bind hGMR with high affinity.7 We
observed that the biologic activity of E21R is strikingly different
when analyzed in hGMR-transfected murine cell lines. First, the
biologic activity of E21R was determined in comparison to wild-type
hGM-CSF on the human cell line, TF1.8, and the murine cell line BaF-B03 transfected with both hGMR- and hc (hGMR BaF-B03).
Human GM-CSF induces proliferation of TF1.8 cells with an effective
dose (ED50) of 0.03 ng/mL, whereas E21R is unable to elicit
a proliferative response and antagonizes the activity of wild-type
GM-CSF (Figure 1A).7
Surprisingly, however, E21R induced a proliferative response in the
hGMR BaF-B03 cell line at concentrations of E21R above 10 ng/mL
(Figure 1B).
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| Figure 1.
The human GM-CSF antagonist E21R behaves as an agonist
on mouse cell lines.
Factor-deprived human TF1.8 (A) and mouse hGMR BaF-B03 cells (B) were
stimulated with hGM-CSF ( ) or E21R ( ). Functional antagonism of
0.03 ng/mL GM-CSF with increasing concentrations of E21R on human TF1.8
cells is also shown ( ). Cells were cultured with cytokine for 48 hours and the resulting proliferation was measured by the incorporation
of 3H-thymidine. The results are expressed in
disintegrations per minute (dpm) and each point represents the
mean of triplicate determination with error bars representing 1 SD.
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It has been previously described that hGMR- can interact with either
hc or mc to transmit a growth signal in
response to hGM-CSF in BaF-B03 cells.13 However, the
interaction of hGMR- with mc, unlike
hc, does not form a high-affinity receptor. The
proliferation induced by E21R in hGMR BaF-B03 cells is also the result
of a low-affinity interaction with a dissociation constant of
approximately 4 nM (data not shown) as determined by Scatchard analysis
of saturation binding,18 identical to the affinity of E21R
measured on human neutrophils.7
The abnormal proliferative response of hGMR BaF-B03 cells to E21R
differs from the previously described cross-talk phenomenon between
hGMR- and mc because it is dependent on the
coexpression of both hGMR- and hc. This is
highlighted by the inability of BaF-B03 cells expressing hGMR- alone
to respond to E21R, even at concentrations of 100 µg/mL (data not
shown). Because the abnormal agonism was only observed in cell lines
where mc is endogenously expressed, it suggested that
the unexpected activity demonstrated by E21R may be the result of a
novel cross-species interaction, involving both hc and
mc.
Together with the inability of E21R to mediate a proliferative response
in human cells, it cannot stimulate tyrosine phosphorylation of
hc. The tyrosine phosphorylation status of
hc was investigated to determine if E21R can activate
hc in this murine system. Tyrosine phosphorylation of
hc was readily detected in the murine cell line hGMR
BaF-B03 after stimulation with E21R (Figure
2B), but not the human cell line
expressing endogenous hc, UT7 (Figure 2A), and TF1.8
(data not shown). In comparison, tyrosine phosphorylation of
hc was detected following stimulation with hGM-CSF in
both human UT7 cells and murine hGMR BaF-B03 cells (Figure 2A,B). The dose response of E21R-induced tyrosine phosphorylation of
hc is consistent with the proliferation data where
responsiveness occurs at a concentration of E21R above 10 ng/mL, with
no response at 1 ng/mL (Figure 1B).
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| Figure 2.
Transfected hc spontaneously associates
with endogenous mc and the c complex is
phosphorylated inappropriately in response to E21R.
Factor-deprived human UT7 cells (A) or mouse hGMR BaF-B03 cells (B-D)
were stimulated as indicated for 5 minutes at 25°C. Cells were lysed
and hc was immunoprecipitated with 8E4
anti-hc antibody (A-C). The presence of tyrosine
phosphorylated hc was detected using an
antiphosphotyrosine antibody, PY20 (A) or 4G10 (B,C). Filters were then
stripped and reprobed for the presence of hc with 1C1
anti-hc antibody (A,B). The presence of
mc following hc immunoprecipitation was
determined on a duplicate filter using anti-mc antibody,
K-17 (C). Tyrosine phosphorylated proteins were immunoprecipitated with
the 4G10 antiphosphotyrosine antibody (D) and the presence of
hc detected using 1C1 antibody. The filter was then
stripped and reprobed for the presence of mc with
K-17.
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Because mc functions when recruited to a
hGM-CSF-hGMR- complex, we investigated if this molecule plays a
role in facilitating the tyrosine phosphorylation of hc
in response to hGM-CSF or E21R in hGMR BaF-B03 cells. Stimulation of
hGMR BaF-B03 cells with either GM-CSF or E21R resulted in tyrosine
phosphorylation of hc (Figure 2C). Significantly,
mc was associated with hc and this
interaction appears to occur regardless of stimulation (Figure 2C). It
is interesting to note that dimerization of a least 2 hc
subunits is required for hGMR activation.19,20 The
interaction between mouse and human c may provide the
molecular basis to support the agonistic activity of E21R and mediate
this perverted receptor response observed in this murine cell line.
Phosphotyrosine immunoprecipitations were performed to address if
preassociation between the human and murine c subunits allows mc to be associated with an activated hGMR
complex. Human c was immunoprecipitated by an
antiphosphotyrosine antibody following stimulation of hGMR BaF-B03
cells with hGM-CSF but not when left unstimulated or with an
isotype-matched control antibody (Figure 2D). Mouse c
appears to be weakly phosphorylated in the absence of stimulation, but
interestingly was strongly coimmunoprecipitated with
antiphosphotyrosine antibody after stimulation with hGM-CSF (Figure
2D), suggesting an increase in phosphorylation of mc on
stimulation with the human ligand.
The hGMR subunits GMR- and hc have previously been
shown to exist as a preformed complex on human cells,20,21
and we have now shown that mc and hc are
also associated prior to ligand stimulation on hGMR BaF-B03 cells. To
determine whether mc is also a component of a preformed
complex on hGMR BaF-B03 cells, hGMR- was immunopreciptated from
nonstimulated and GM-CSF-stimulated cells. Immunoblotting revealed
that both hc and mc were associated with
hGMR- regardless of stimulation (data not shown) suggesting that
mc chain may be a component of a preformed hGMR receptor on hGMR BaF-B03 cells. Therefore activation of the receptor by GM-CSF
and E21R may be mediated by a preformed
hGMR--hc-mc complex in these cells.
The ability of human and mouse c subunits to interact
and influence responses of a hGM-CSF variant introduces a new level of
complexity in the analysis of ligand-receptor interaction and subsequent signaling capabilities. It also raises the question of the
influence this interaction has had on previous hGMR studies that have
been performed in murine cell lines. Clearly mc can become associated within an active hGMR complex, and it is important to
consider its contribution to the receptor's biologic response. This is
of concern especially when investigating the activation of downstream
signaling molecules.22 In light of the interaction between
hc and mc it will be difficult to
discriminate which signaling molecules have emanated from the
hc alone.23 In addition, the associated
mc subunit may facilitate signaling by an otherwise inactive hc mutant. This may explain the surprising
result seen where hc deficient in intracellular
tyrosines is still capable of responding to hGM-CSF.10 In
addition, the observation that a cytoplasmically truncated
hc can still respond to hGM-CSF when expressed in
BaF-B03 cells may be potentiated via signaling through mc.24 The contribution this interspecies
subunit interaction plays when distinguishing regions in hGMR
responsible for differentiation or proliferation in murine myeloid cell
lines remains unclear. It has been demonstrated previously that
hc can form homodimers that are activated in response to
hGM-CSF.20,21 The association between mc
and hc must result in a complex that has different properties to hc homodimers because E21R is unable to
activate the latter.
A number of receptors belonging to the cytokine receptor family show
cross-species specificity between human and murine components. Because
receptor dimerization is a common theme in receptor activation, it
cannot be ruled out that interaction of interspecies signaling components may obscure results when studying a wide range of human receptors in murine cells and similar precautions may need to be taken.
In addition to our findings that the use of transfected murine cell
lines may not be the best approach when studying hGM-CSF ligand
variants, other cytokines show similar discrepancies. An analogous
situation has been reported for a human IL-4 mutant that can mediate
either agonistic or antagonistic responses when studied in either a
murine or human cell line, respectively.25 Similarly,
mutational analysis of thrombopoietin shows conflicting data in
the identification of residues functionally important in interaction
with its receptor when screened on Mpl-transfected BaF-B03 cells
compared to enzyme-linked immunosorbent assay or Biacore
analysis.26,27
The demonstration of an inherent association between mouse and human
c shown here highlights the need for a careful selection of appropriate systems. In the case of the hGMR the availability of
c knockout mice permits the use of cells from these
animals for receptor reconstitution experiments as a better background to analyze structural and functional outcomes with the hGMR.
| |
Footnotes |
Submitted May 2, 2001; accepted July 16, 2001.
Supported by grants from the National Health and Medical Research
Council of Australia. J.W. is a Research Fellow of the Anti-Cancer Foundation of South 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: Joanna Woodcock, Cytokine Receptor Laboratory,
Hanson Centre for Cancer Research, Institute of Medical and Veterinary
Science, Adelaide, SA 5000, Australia; e-mail:
joanna.woodcock{at}imvs.sa.gov.au.
| |
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Abstract
Introduction