From the Department of Medicine, Rheumatology Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Granulocyte-macrophage colony-stimulating factor
(GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is
comprised of an -chain (GM-CSFR
), which specifically binds
GM-CSF, and a
-chain (
c), shared with the
interleukin-3 and interleukin-5 receptors. GM-CSFR
exists in both a
transmembrane (tmGM-CSFR
) and a soluble form (sGM-CSFR
). We
designed an sGM-CSFR
-Fc fusion protein to study GM-CSF interactions
with the GM-CSFR
. The construct was prepared by fusing the coding
region of the sGM-CSFR
with the CH2-CH3 regions of murine IgG2a.
Purified sGM-CSFR
-Fc ran as a monomer of 60 kDa on reducing
SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200
kDa under nonreducing conditions. The sGM-CSFR
-Fc bound specifically
to GM-CSF as demonstrated by standard and competitive immunoassays, as
well as by radioligand assay with 125I-GM-CSF. The
sGM-CSFR
-Fc also inhibited GM-CSF-dependent cell growth
and therein is a functional antagonist. Kinetics of sGM-CSFR
-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFR
-Fc was bound to immobilized staphylococcal protein A on the
biosensor surface, and binding kinetics of GM-CSF in solution were
determined. This revealed a rapid koff of
2.43 × 10
2/s. A second set of experiments was
performed with GM-CSF immobilized to the sensor surface and the
sGM-CSFR
-Fc in solution. The dissociation rate constant
(koff) for the sGM-CSFR
-Fc trimer from
GM-CSF was 1.57 × 10
3/s, attributable to the higher
avidity of binding in this assay. These data indicate rapid
dissociation of GM-CSF from the sGM-CSFR
-Fc and suggest that
in vivo, sGM-CSFR
may need to be present in the local
environment of a responsive cell to exert its antagonist activity.
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INTRODUCTION |
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Granulocyte-macrophage colony-stimulating factor
(GM-CSF)1 plays an important
role in myeloid differentiation and is involved in many inflammatory
and immune processes. GM-CSF is a member of the four-helix bundle
family of cytokines which also includes growth hormone, interleukin-2
(IL-2), IL-4, and IL-5. GM-CSF activity is mediated by specific
cellular receptors (GM-CSFR), which belong to a supergene family
(1-7). The GM-CSFR consists of an -chain (GM-CSFR
), specific for
GM-CSF (4), and a
-chain (
c), which can also
associate with IL-3 and IL-5 receptor
-chains (5). The GM-CSFR
expressed in the absence of
c binds GM-CSF but with a
lower affinity than the heterodimeric receptor (8).
Naturally occurring soluble forms of cytokine receptors, including a
soluble form of GM-CSFR (sGM-CSFR
), have been described (9-11).
sGM-CSFR
has been cloned from human placenta (11) and human
choriocarcinoma cells (9) and has been detected in the supernatant of a
human choriocarcinoma cell line (10). Previous work in our laboratory
utilized polymerase chain reaction (PCR) to amplify and clone both the
transmembrane and the soluble forms of the GM-CSFR
from a
human GM-CSF-dependent myelomonocytic cell line
(AML193). Supernatants from sGM-CSFR
-transfected cells, but
not transmembrane GM-CSFR
(tmGM-CSFR
)-transfected cells, inhibited GM-CSF immunoprecipitation by neutralizing monoclonal antibody (mAb) 126.213 and inhibited GM-CSF-dependent
cellular proliferation. These experiments indicated that sGM-CSFR
binds GM-CSF and exhibits functional antagonist activity in
vitro (12).
The GM-CSF antagonist activity of sGM-CSFR could play a role in the
regulation of biological responses to GM-CSF. Biological effects
mediated by the sGM-CSFR
in vivo would be dependent on its binding characteristics. For example, if the sGM-CSFR
binds GM-CSF with high affinity, forming a stable complex, the sGM-CSFR
would be able to sequester GM-CSF away from tmGM-CSFR
-bearing cells.
In this way, sGM-CSFR
could act at a distance, perhaps by
transporting GM-CSF away from responsive cells. In contrast, if the
sGM-CSFR
binds with lower affinity, particularly with a rapid
off-rate, sGM-CSFR
would need to be present in the local cellular
environment where GM-CSF is present to exert its antagonist activity.
Thus, the binding kinetics of the sGM-CSFR
will have a major impact
on its in vivo functional role, and determining the kinetic
properties of the protein would help determine the mechanistic nature
of its biological role. Unfortunately, production and purification of
sufficient sGM-CSFR
for binding studies have been problematic
because production by cell lines is limited, and simple
purification methods are not available. Therefore, we prepared a
DNA-construct fusing the sGM-CSFR
to the Fc region of mouse IgG2a.
Here we describe the cloning, production, purification, binding, and
biological activity of this sGM-CSFR
-Fc fusion protein, with
preliminary analyses of binding kinetics.
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MATERIALS AND METHODS |
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Cloning of sGM-CSFR-Fc
sGM-CSFR cDNA clones were available as described
previously (12). sGM-CSFR
cDNA and the CH2-CH3 cDNA region
of mouse anti-reovirus type 3 mAb 9BG5 (IgG2a) (13) were amplified
using primers containing restriction endonuclease sites for restriction
enzymes BamHI and SalI for cloning the construct
into the pBabe-puro vector and including sequences that allowed their
use in the splicing by overlap
extension (SOE) step for the preparation of the construct. sGM-CSFR
cDNA from clone 9 was PCR amplified using these primers (restriction sites underlined): sGM-CSFR
(BamHI),
5'-TGACCGGATCCATGCTTCTCCTGGTGACAA; and sGM-CSFR
(SOE),
3'-CCACCCAAGAGGTTAGGTGCGTTTGTCTTTGATCTGTGGAA.
The CH2-CH3 region (Fc region) of mouse anti-reovirus mAb 9BG5 was amplified from cDNA prepared from the hybridoma according to our prior protocols (12) with the following primers: Fc (SOE), 5'-TTCCACAGATCAAAGACAAACGCACCTAACCTCTTGGGTGG; Fc (SalI), 3'- ATGCCGTCGACCTATTTACCCGGAGTCCGGGA.
These primers were used with cDNAs and Taq polymerase as
described previously (14, 15) to amplify the sGM-CSFR and Fc. The
program of amplification was 94 °C for 1.5 min followed by 25 cycles
at 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, with a
final cycle at 72 °C for 5 min. The PCR products were stored at
4 °C until used. A 2% analytical agarose-gel electrophoresis revealed a band of 1,100 bp and a band of 650 bp, respectively, for
sGM-CSFR
- and CH2-CH3-amplified products, which were therefore purified on a 1% preparative low melting agarose gel (NuSieve, FMC
BioProducts, Rockland, ME), extracted with phenol/chloroform (16), and
used in the SOEing step.
The SOEing step program consisted of two phases. In the first phase
(94 °C for 1 min followed by five cycles at 94 °C for 1 min,
60 °C for 1 min, 72 °C for 1 min) the two amplified cDNA products worked as primers for each other to obtain the construct. In
the second phase (94 °C for 1 min followed by 25 cycles at 94 °C
for 1 min, 60 °C for 1 min, 72 °C for 1 min) the primers sGM-CSFR 5' and Fc 3' were added and the construct amplified. A 2%
analytical agarose gel indicated a SOEing product of the desired size.
After treatment of SOEing mixture with proteinase K to destroy
Taq polymerase, the construct was extracted following the
phenol/chloroform protocol and precipitated with ethanol. The construct
and the pBabe vector (17, 18) were digested with restriction enzymes
BamHI and SalI. After alkaline phosphatase treatment of pBabe purified on a 1% preparative low melting
temperature agarose gel, ligation was performed following standard
procedures (16, 19).
The product was transformed into Escherichia coli DH5
cells that were plated into LB plates; and minipreparations were made from colonies. Plasmid DNA from minipreps were digested with
BamHI/SalI to identify the presence of the
desired insert. After amplification of cells with the right insert,
plasmid DNA was purified on a Qiagen column (Qiagen Inc., Chatsworth,
CA). The purified DNA was checked using two different pairs of
restriction enzymes (BamHI/SalI and
BamHI/XbaI), and the expected size of the inserts
was confirmed on a 2% analytical agarose gel (~350 bp and ~4,750
bp for the BamHI/SalI digestion, and
~1,200-1,300 bp, ~2,500 bp, and ~4,300 bp for the
BamHI/XbaI digestion). The sequence of the insert
was also confirmed by sequencing according to previously published protocols (20).
Transfection of PA317 Cells, Transduction into SP2/0 Cells
PA317 cells (a retroviral packaging line (17, 18)) were transfected with the purified DNA using LipofectAMINE reagent (Life Technologies, Inc.) and the described protocol (21). Briefly, 2 × 105 cells in 2 ml of DMEM (DMEM containing L-Gln, oxalate/pyruvate/insulin, penicillin/streptomycin, and 10% fetal calf serum) were seeded in each well of a six-well culture plate and grown until 80% confluence. 1.5 µg of DNA in 100 µl of serum-free DMEM was added to 12 µl of LipofectAMINE reagent diluted to 100 µl with serum-free DMEM. After gentle mixing and incubation at room temperature for 45 min to allow the DNA-liposome complex to form, the complex solution was diluted to 1 ml with serum-free DMEM and added to the rinsed adherent cells. Cells were kept in a 37 °C 5% CO2 incubator for 5 h and then rinsed, and serum-containing DMEM was added. 72 h after transfection, the cells were passed 1:10 into DMEM containing 5 µg/ml puromycin. Cells were grown until confluent, split twice, and grown for 1 week in medium without puromycin. The supernatant was then used to transduce SP2/0 cells.
3 × 106 SP2/0 cells, grown in RPMI medium, containing L-Gln, sodium pyruvate, penicillin/streptomycin, 25 mM Hepes, and 15% fetal calf serum, was resuspended in 2 ml of medium with 0.5 ml of PA317 supernatant and 10 µl of Polybrene (0.4 mg/ml). After 3 h of incubation, 8 ml of RPMI medium was added; after 2 days of incubation, cells were grown in medium containing puromycin. SP2/0 cells were then cloned and the presence of insert confirmed by PCR. An enzyme-linked immunosorbent assay (ELISA) (see below) was performed on PCR-positive clone supernatants to identify the clones producing the fusion protein.
Purification and Characterization of sGM-CSFR-Fc
Clones that presented the highest binding to GM-CSF in two ELISAs were expanded, and the fusion protein from the supernatants was purified on a staphylococcal protein A (SPA) column. To 140 ml of filtered supernatant glycine and NaCl were added to reach, respectively, 1.5 and 3 M final concentrations; the pH was adjusted to 8.0. After loading the sample, the column was washed with 10 mM boric acid, 3 M NaCl, pH 8.9, and eluted with 0.1 M sodium citrate, pH 3.5 (fractions collected into tubes containing 1 M Tris, pH 9.5). Elution fractions containing proteins were concentrated using Centricon-30 (30 kDa cutoff) filtration systems (Amicon, Beverly, MA).
The sGM-CSFR-Fc fusion protein was analyzed by SDS-polyacrylamide
gel electrophoresis. An 8% acrylamide gel, 1.5 mm thick, was used in
an electrophoresis system to analyze both oxidized and reduced forms of
the fusion protein. 45 µg of purified sGM-CSFR
-Fc fusion protein
purified from various cell clones was loaded into gels, run at 100 volts for ~1.5 h using 25 mM Tris, 250 mM
glycine, 0.1% SDS, pH 7.5, and then stained with Coomassie Blue.
A Sephacryl S-200HR gel filtration column (Pharmacia Biotech Inc.) was packed into a C-16/70 column (215 ml of resin) to analyze 500 µl of 0.676 mg/ml fusion protein preparation from clone 25, using 50 mM Tris, 150 mM NaCl, pH 7.5, buffer, 0.8 ml/min flow rate, 1 mm/min chart speed, and collecting 1.2-ml fractions.
Western Blot Analysis
Western blot analysis was performed using mAbs against GM-CSFR.
75 µg/well of both oxidized and reduced SPA-purified sGM-CSFR-Fc fusion protein were loaded into three wells of each of two 8% polyacrylamide gels. After the electrophoretic run as reported previously, the gels were cut in thirds, each strip containing two
wells, one with the sample and one with the high molecular mass
markers, and transferred to Immobilon-P membranes (Millipore, Bedford,
MA) for 1 h at room temperature, under stirring conditions, using
50 mM Tris, 150 mM NaCl, 20% methanol, pH 7.5, as the transfer buffer. The membranes were then blocked overnight at
4 °C with 5% non-fat dry milk in 50 mM Tris, 150 mM glycine, 0.05% Tween, pH 7.5 (5% NFDM in TTBS). The
next morning they were washed three times for 10 min with TTBS, and the
three pairs of oxidized and reduced membrane strips were incubated for
2 h at room temperature with one of the following monoclonal
antibodies diluted in TTBS: (a) 7 µg/ml mouse IgG2a
anti-human GM-CSFR (Pharmingen, San Diego) (this antibody binds GM-CSFR
on the same site recognized by GM-CSF); (b) 7 µg/ml mouse
IgM anti-human GM-CSFR (Pharmingen) (this antibody binds GM-CSFR on a
site different from that recognized by GM-CSF); (c) no first
antibody. The membranes were then washed three times for 2 min with 5%
NFDM in TTBS and twice for 2 min with TTBS. Samples from a
and b were then incubated for 2 h at room temperature, under shaking conditions, with a 1:40,000 dilution of biotin-goat anti-mouse IgG (Fab-specific) (Sigma) in TTBS. After washing the membranes three times for 2 min with 5% NFDM in TTBS and twice for 2 min with 1 × TTBS, they were incubated for 2 h at room
temperature, under shaking conditions with 0.5 µg/ml
avidin-horseradish peroxidase (HRP; Pierce) in 1 × TTBS; sample
c was incubated under same conditions with goat anti-mouse
IgG (H+L) HRP (Life Technologies, Inc.). The membranes were washed six
times with 1 × TTBS, and the TMB substrate solution was added
(Kirkegaard and Perry Laboratories, Gaithersburg, MD) until color
development. The color reaction was then stopped by distilled water
rinsing.
ELISAs
Clone supernatants were tested in three types of ELISAs. In the first type, ELISA plates (Dynatech Laboratories, Chantilly, VA) wells were coated with 50 µl of 10 µg/ml GM-CSF (Sargamostin Leukine, Immunex Corporation, Seattle) in 0.1 M NaHCO3, and after overnight incubation at 4 °C they were washed five times with 1 × PBST and blocked with 200 µl of 2% bovine serum albumin in 1 × PBST at 37 °C for 1 h. The plates were washed five times with 1 × PBST, and 50 µl of clone supernatants was added, incubated overnight at 4 °C, and washed seven times with cold 1 × PBST. 100 µl of 1/3,000 cold dilution in 1 × PBST of goat anti-mouse IgG (H+L) HRP was added per well and the plate kept overnight at 4 °C. After seven washes with cold 1 × PBST, 100 µl of 0.1 mg/ml substrate TMB dihydrochloride (Sigma) in 0.05 M phosphate citrate, 0.03% sodium perborate buffer, pH 5.0, was added per well. After color development at room temperature for 10 min, the enzymatic reaction was stopped with 20 µl of 2 N H2SO4 per well and the plate read at 450 nm.
In the second ELISA the wells were coated with 50 µl of 5 µg/ml SPA (Sigma) in 0.1 M NaHCO3 for 1 h at 37 °C, washed five times with 1 × phosphate-buffered saline with 0.1% Tween-20 (PBST), and blocked with 200 µl of 2% bovine serum albumin in 1 × PBST at 37 °C for 1 h. The wells were washed five times with 1 × PBST, 150 µl of clone supernatants was added, and the wells were incubated overnight at 4 °C and washed seven times with cold 1 × PBST. 50 µl of 10 ng/ml biotinylated GM-CSF (developed by standard methods (22)) was added per well, and the plate was incubated overnight at 4 °C, washed seven times with cold 1 × PBST, and 100 µl of 0.1 µg/ml avidin-HRP conjugate added per well. After incubation overnight at 4 °C and seven washes with cold 1 × PBST, 100 µl of substrate was added to each well, and after color development at room temperature for 10 min, the enzymatic reaction was stopped with 20 µl of 2 N H2SO4 per well and the plate read at 450 nm.
The third was a competitive ELISA, used to test highly concentrated
fusion protein preparations after purification on an SPA column. An
ELISA plate was coated with SPA and blocked as reported previously. 50 µl of 1 mg/ml sGM-CSFR-Fc was added per well and incubated
overnight at 4 °C. After three washes with 1 × PBST, 50 µl
of unmodified ("cold") GM-CSF was added at various dilutions and
incubated for 1 h at 37 °C. 50 µl of biotinylated-GM-CSF at 10, 1, and 0.1 ng/ml was added per well, and the plate was incubated for another hour at 37 °C. After washing, binding was detected by
avidin-HRP as reported above.
Radioligand Assay
Binding of purified sGM-CSFR to 125I-GM-CSF was
also tested as follows. The wells of a radioimmunoassay plate (Wallac,
Gaithersburg, MD) were coated with 50 µl of 5 µg/ml SPA overnight
at 4 °C, washed three times with PBST, and blocked with 200 µl of
2% bovine serum albumin in 1 × PBST at 37 °C for 2 h.
The plate was washed three times with PBST and then 50 µl of purified
fusion protein per well was added at 500, 250, 125, 62.5, 31, 15.5, and
0 µg/ml in 2% bovine serum albumin and PBST; as positive control
anti-GM-CSF mAb 126.213 was used at same dilutions. After a 3-h
incubation at 37 °C, the plate was washed three times with cold PBST
and incubated with 25 µl of 388 pM
125I-GM-CSF (1 × 105 to 3 × 105 cpm) per well, with or without a saturating amount (100 nM) of cold GM-CSF at room temperature for 1 h. The
plate was quenched on ice, washed three times with ice-cold PBST, and
the wells were cut out from the plate, transferred into tubes, and the
cpm counted.
Proliferation Assay
Inhibition of GM-CSF-dependent cells line MO7E (24 h) (from R. Zollner, Genetics Institute, Cambridge, MA) by sGM-CSFR
was tested as follows. 104 cells/well were incubated for 1 day with or without 250 pM GM-CSF in the presence of
different concentrations of sGM-CSFR
(250, 50, 10, 2, and 0 µg/ml). 20 µl of 5 mg/ml MTT (Sigma) in 1 × PBS was added per
well, and after 5 h 100 µl of 10% SDS in 0.1 N HCl was used to solubilize the precipitated crystals. After overnight incubation at 37 °C, A570 nm was detected. A
parallel assay was set as control, using IL-2-dependent
CTLL cells (5 × 103 cells/well) in the presence of
20% concanavalin A supernatant as the stimulus.
Biosensor Assay
An IAsys biosensor was used to characterize binding kinetics of
sGM-CSFR-Fc to GM-CSF. All experiments were done at 25 °C.
Immobilization and Regeneration Conditions
GM-CSF and SPA immobilization on a carboxymethyl-dextran cuvette (IAsys) was performed in the instrument using reagents supplied in the coupling kit as recommended by the manufacturer. Briefly, the cuvette was first washed with 200-µl washes of alternatively deionized water and 10 mM HCl to swell the cuvette matrix and finally equilibrated in PBST. The carboxymethyl-dextran layer on the sensor chip was then activated by a 10-min incubation with a mixture of 100 µl of 13 mg/ml N-hydroxysuccinimide and 100 µl of 76 mg/ml EDC to form succinimidyl esters that are reactive with amino groups. After washes with PBST, 200 µl of 10 µg/ml GM-CSF or SPA in 10 mM sodium acetate, pH 5, was added to the cuvette, and after a response plateau was reached signifying completion of the reaction, the cuvette was washed out with PBST. The remaining activated groups were blocked by injection of 200 µl of 1 M ethanolamine, pH 8.5, for 2 min. After reequilibration with PBST, the cuvette was ready for the binding experiments. The cuvette matrix was regenerated to remove bound ligand using 200 µl of either 0.5 M sodium carbonate, pH 11.0, or 10 mM HCl and 0.5 M NaCl and reequilibrated with PBST.
Binding Assays
Immobilized GM-CSF--
200 µl of sGM-CSFR-Fc at 75, 300, 600, and 900 nM in PBST was added to the cuvette. After a
binding plateau was achieved it was incubated with PBST and the cuvette
regenerated for the next experiment.
Immobilized SPA--
A 200-µl solution containing 20 µg of
sGM-CSFR-Fc in PBST was added to the cuvette and allowed to bind for
6 min (phase 1). PBST was added for 5 min (phase 2). GM-CSF at various
concentrations in PBS was then added and allowed to bind for 5 min
(association phase, phase 3) for 5 min. PBST was then added for an
additional 5 min (dissociation phase, phase 4). No regeneration was
performed between runs in these assays.
Data Analysis
Data were collected automatically and analyzed subsequently
using the Microsoft Excel program (Microsoft, Redmond, WA). Plots were
fitted using Cricket Graph (Cricket Software, Malvern, PA). The
sGM-CSFR-Fc surface prepared by capturing sGM-CSFR
-Fc on immobilized SPA was corrected for a downward drift as dissociation of
sGM-CSFR
-Fc from the SPA occurred along with GM-CSF binding to
sGM-CSFR
-Fc. Accordingly, control experiments were run in which
sGM-CSFR
-Fc was allowed to bind to SPA for 6 min (phase 1), the
receptor-associated surface was washed for 5 min (phase 2), and then
phosphate-buffered saline was added for an additional 5 min (to
simulate the association phase of GM-CSF, phase 3) followed by a final
5-min wash (simulating the dissociation phase of GM-CSF from
sGM-CSFR
-Fc, phase 4). In these experiments, the negative slope of
the dissociation of GM-CSFR
-Fc from SPA decreased slightly after
each of the washes (in phases 2, 3, and 4). Therefore, correction for
sGM-CSFR
-Fc dissociation from SPA was performed separately for each
phase. The slope of dissociation of sGM-CSFR
-Fc from SPA in the
final 200 s of phase 2 was calculated for each experiment and used
to normalize this portion of the sensorgrams. Similarly, the slope of
the final 200 s of phase 4 was calculated and used to correct the
dissociation phase of GM-CSF from sGM-CSFR
-Fc. A weighted average of
the slopes from phases 2 and 4 was used to predict a slope for phase 3, and this was used to correct the association phase of GM-CSF to the
sGM-CSFR
-Fc. Evaluation of these corrections on the mock experiments
gave reasonable agreement with the observed data (data not shown). This
triphasic correction was used for each experiment with immobilized
SPA.
Kinetic analysis was performed as described previously (23). In the case of a bimolecular interaction of two species A and B whose association and dissociation are regulated, respectively, by kon and koff for giving the final product AB,
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
In the case of the dissociation phase, any free A from dissociation of the product is assumed to be removed by the buffer so that there is no reassociation ([A] = 0). Therefore,
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(Eq. 4) |
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(Eq. 5) |
The equilibrium dissociation constant (KD) was calculated from the equation
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(Eq. 6) |
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RESULTS |
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Cloning of sGM-CSFR-Fc
We had previously cloned a soluble form of the GM-CSFR into a
retroviral vector (12). We used this clone, which encodes for
sGM-CSFR
, to prepare a fusion protein of the sGM-GM-CSFR
with the
Fc portion of murine IgG. The sGM-CSFR
-Fc construct (shown in Fig.
1) was obtained as follows. sGM-CSFR
cDNA from clone 9 and the CH2-CH3 cDNA region of mouse
anti-reovirus mAb 9BG5 (IgG2a) were PCR amplified using the primers
described under "Materials and Methods." An analytical 2% agarose
gel indicated a band of ~1,100 bp and a band of ~650 bp,
respectively, for sGM-CSFR
and CH2-CH3. After purification on a 1%
low melting temperature agarose gel the two products were fused in a
PCR-SOEing step, obtaining a construct of ~1,700 bp. The construct
was digested and ligated into the BamHI/SalI site
of the pBabe-puro vector. After transformation of E. coli
DH5
cells, a colony with the right sized insert was selected, grown,
and DNA purified on a Qiagen column. The construct was verified by
restriction enzyme mapping and sequencing as described under
"Materials and Methods."
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Transfection and Expression of the Fusion Protein
sGM-CSFR-Fc in the pBabe vector was transfected into a
retroviral packaging cell line, and the virus-containing medium was used to transduce SP2/0 cells. These SP2/0 cells were selected and
subcloned as described under "Materials and Methods." Clones were
tested for the presence of the construct by reverse transcriptase PCR,
with 9 of 96 clones expressing the sGM-CSFR
-Fc by this assay. Several subclones were isolated and grown to confluence; the selective media were removed and supernatants collected. These supernatants were
screened for production of sGM-CSFR
-Fc by two different ELISAs as
described under "Materials and Methods." An example of one ELISA is
shown in Fig. 2, comparing the binding of
clone supernatants with that of neutralizing anti-GM-CSF mAb 126.213 (24). In this example, the ELISA plate was coated with SPA to which the
sGM-CSFR
-Fc was bound. Biotinylated GM-CSF was then added, and
binding was detected by streptavidin-HRP conjugate. This assay shows
significant binding of several clones to GM-CSF. Similar results were
obtained in an assay in which the ELISA plate was coated with GM-CSF,
then the sGM-CSFR
-Fc was bound and binding detected by anti-mouse
Ig-HRP (data not shown). Clones that consistently displayed binding
were chosen for further analysis.
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Purification and Characterization of sGM-CSFR-Fc
sGM-CSFR-Fc was purified from ~140 ml of clone supernatant on
an SPA column, as described under "Materials and Methods." The
partially purified sGM-CSFR
-Fc was analyzed in an 8% acrylamide gel
(Fig. 3). In the gel run under oxidizing
conditions there is a high molecular mass band of ~160-200 kDa,
representing multimers of the fusion protein, whereas in the gel run
under reducing conditions there are two main bands, at ~60 kDa and
~43 kDa (sGM-CSFR
-Fc monomer and uncharacterized contaminant,
respectively). We scanned the gel using Image 1.41 (Wayne Rasband,
NIMH, Bethesda, MD) and performed densitometry, allowing us to estimate
that the sGM-CSFR
-Fc fusion protein represents ~50% of the total
protein purified by the SPA column. Gel filtration chromatography
(using a Sephacryl S-200HR column) was used to evaluate the molecular
mass of the sGM-CSFR
-Fc multimer. The chromatogram indicated that
the oxidized sGM-CSFR
-Fc runs at ~160 kDa (data not shown). When
compared with the reducing SDS-polyacrylamide gel electrophoresis of
the fusion protein, this suggests that the sGM-CSFR
-Fc forms
trimers. We have seen the ~60-200-kDa form from several
sGM-CSFR
-Fc preparations consistently and reduce to ~60 kDa upon
reduction consistently.
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Western blot analysis with anti-human-GM-CSFR mAbs to detect the
receptor portion of the fusion protein and anti-mouse-IgG to detect the
Fc portion of the fusion protein indicated a high molecular mass band
for the oxidized forms (sGM-CSFR-Fc trimer) and a strong band at 60 kDa for the reduced form (sGM-CSFR
-Fc monomer) (Fig.
4).
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Binding Analysis of sGM-CSFR-Fc
The SPA-purified sGM-CSFR-Fc was evaluated next for binding to
GM-CSF and for bioactivity. A competitive ELISA assay (Fig. 5) was carried out to confirm that the
sGM-CSFR
-Fc bound to native GM-CSF. An ELISA plate was coated with
SPA followed by the sGM-CSFR
-Fc. We then added unlabeled GM-CSF at
various dilutions, followed by biotinylated GM-CSF, with binding
detected by an avidin-HRP conjugate. We saw increasing binding with
increasing amounts of biotinylated GM-CSF (compare the
A450 nm without competitor for 7, 70, and 700 pM biotinylated GM-CSF), and this was inhibited competitively by increasing amounts of unlabeled GM-CSF. This indicates
that unmodified GM-CSF binds to our fusion protein.
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A radiolabeled binding assay (Fig. 6)
confirmed the binding of the sGM-CSFR-Fc fusion protein to
125I-GM-CSF. In this experiment, increasing the amount of
added fusion protein increased the binding 125I-GM-CSF, and
this binding was blocked fully by excess cold GM-CSF. This confirms the
binding of GM-CSF to the sGM-CSFR
-Fc fusion protein.
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Bioactivity of sGM-CSFR-Fc
We next evaluated the ability of the sGM-CSFR-Fc to inhibit the
biological activity of GM-CSF using a GM-CSF-dependent cell line. MO7E cells are a myelomonocytic cell line and are dependent on
GM-CSF for growth. We evaluated the growth of MO7E cells in the
presence of GM-CSF with or without added sGM-CSFR
-Fc using the MTT
assay (see "Materials and Methods"). The presence of increasing amounts of sGM-CSFR
-Fc resulted in increasing inhibition of
proliferation of the MO7E cells (Fig. 7).
We found that 250 µg/ml sGM-CSFR
-Fc produced 75% inhibition in
cellular proliferation. In contrast, the sGM-CSFR
-Fc had no effect
on the growth of the IL-2-dependent cell line CTLL (data
not shown). This indicates that the sGM-CSFR
-Fc is a specific
biological antagonist of GM-CSF.
|
Biosensor Analysis
GM-CSF Binding to sGM-CSFR-Fc--
The sGM-CSFR
-Fc was
evaluated for the kinetics of binding using a biosensor. Initially, the
binding of sGM-CSFR
-Fc to immobilized SPA was analyzed to assure
that the Fc portion of our receptor was functional. A typical
sensorgram obtained is shown in Fig. 8A. After the addition of
sGM-CSFR
-Fc, a bulk phase effect is seen initially (increase in the
response from bulk phase refractive index) followed by an association
phase. With washing, an initial rapid response decrease (likely from
bulk phase refractive index decrease) becomes relatively exponential.
Fig. 8B shows a typical dR/dt plot for
the association phase. The initial rapid response shift (likely bulk
phase effect) is shown in open circles, followed by a
relatively linear association shown in filled circles. The plot of dR/dt for the linear phase shows a
ks of 2.51 × 10
3/s for this concentration of sGM-CSFR
-Fc.
|
|
Immobilized GM-CSF--
The binding of various concentrations of
the sGM-CSFR-Fc trimer to immobilized GM-CSF was measured using the
conditions described under "Materials and Methods." Fig.
10A shows an overlay of
sensorgrams obtained with 75, 300, 600, and 900 nM
sGM-CSFR
-Fc. After an association phase showing the binding of
sGM-CSFR
-Fc to immobilized GM-CSF, a washing step with buffer alone
was used to effect the dissociation phase.
|
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DISCUSSION |
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In this paper we report the preparation and characterization of a
fusion protein obtained by expression of a construct made from
sGM-CSFR and the Fc portion of a mouse antibody. The fusion protein
is produced easily by transduced cells, recovered from their medium,
and purified on an SPA column via the Fc portion. This protein should
provide a useful means for the study of the interaction of GM-CSF or
GM-CSF mimics with the GM-CSFR
.
After amplification and SOEing of the sGM-CSFR cDNA and the
CH2-CH3 region of mouse anti-reovirus mAb 9BG5 (IgG2a), a construct of
the expected size was obtained and ligated into pBabe-puro vector (Fig.
1). This construct was used to produce recombinant retrovirus, which
was used to transduce SP2/0 cells. After subcloning the
sGM-CSFR
-Fc-transduced SP2/0 cells, supernatants were tested for
their binding to biotinylated GM-CSF. For all supernatants we obtained
low values in the ELISAs (Fig. 2 and data not shown), probably due in
part to the low concentration of fusion protein in the supernatants and
in part to the rapid off-rate of GM-CSF (see below). However, several
positive clones were obtained which allowed more extensive analysis of
purified sGM-CSFR
-Fc.
Denaturing polyacrylamide gel analysis of SPA-purified sGM-CSFR-Fc
under reducing and nonreducing conditions indicated a similar pattern
of molecular masses for sGM-CSFR
-Fc purified from two different
clones (Fig. 3). Moreover, both polyacrylamide gel electrophoresis
under nonreducing conditions and gel filtration chromatography of the
purified oxidized protein indicated that the fusion protein was present
as a multimer, probably a trimer of ~160-200 kDa. SDS-polyacrylamide
gel electrophoresis performed under reducing conditions indicated a
~60-kDa protein (Fig. 3), which represented ~50% of the total
protein in the preparation. Also seen was a ~45 kDa band (which was
absent in other preparations) and a smaller band of ~25 kDa which is
seen at the dye front of the gel in Fig. 3. A gel run in the same
conditions alongside fetal calf serum as a control shows bands of ~60
and ~25 kDa in the fetal calf serum (data not shown). Thus, it is
likely that there is antibody contamination from the fetal calf serum
used to grow our sGM-CSFR
-Fc-transduced SP2/0 cells. Bovine IgG
under oxidizing conditions likely co-migrates with the multimeric
sGM-CSFR
-Fc, with the bovine IgG heavy chain also co-migrating with
the sGM-CSFR
-Fc under reducing conditions. Alternatively, the other
bands seen on the reducing gel could represent proteins that covalently
attach to the sGM-CSFR
-Fc. We think this is unlikely as the quantity of contaminating proteins varies in our different preparations. In
addition, a two-dimensional gel run under reducing conditions showed a
single band with an estimated pI of 6.7 (data not shown).
The nature of the ~60 kDa band was investigated by Western blot
analysis, using antibodies against the GM-CSFR and murine IgG. The
experiment indicated that the ~60 kDa band is detected by two
different anti-human GM-CSFR monoclonal antibodies, recognizing the
receptor portion of the fusion protein, and by anti-mouse IgG,
recognizing the Fc portion of the sGM-CSFR
-Fc fusion protein (Fig.
4). The other contaminating bands were not recognized by either
reagent, making it unlikely that these represent breakdown products.
This biochemical and immunochemical analysis strongly suggests that the
sGM-CSFR-Fc exists as a trimer in the oxidized state. The reason for
this is unclear. The fusion protein has a total of 16 cysteines: 12 from the sGM-CSFR
and 4 from the IgG2a Fc sequence. These are
thought to participate in intramolecular disulfide bridges in native
GM-CSFR
and murine IgG, respectively. However, some of these
cysteines appear to form intermolecular disulfide bridges in the
sGM-CSFR
-Fc. We are uncertain of the disulfide bonding pattern of
the sGM-CSFR
-Fc which results in trimer formation. It is interesting
to speculate that such trimers of the GM-CSFR
may exist naturally,
possibly through noncovalent interactions. However, no data to support
or refute this possibility are currently available. The trimers seen
here show that trimer formation can occur without abrogating binding of
the sGM-CSFR
-Fc to GM-CSF. We are uncertain if this is an artifact
of the sGM-CSFR
-Fc construct or a reflection of a physiologic
capability for receptor aggregation to occur.
The binding capacity of the fusion protein for GM-CSF was tested in
several experiments. Competition of "cold" and biotinylated GM-CSF
on an ELISA plate coated with sGM-CSFR-Fc indicated that the fusion
protein specifically binds to GM-CSF (Fig. 5). This binding was also
confirmed by a radioligand binding assay, using 125I-GM-CSF
(Fig. 6), which again showed specific binding that was abrogated by
cold GM-CSF. This indicates that the fusion protein possesses active
binding site(s) for GM-CSF and that this binding is specific as it is
inhibited competitively by unlabeled GM-CSF. It was also of interest to
determine whether the sGM-CSFR
-Fc was active in a bioassay. Because
the sGM-CSFR
-Fc binds GM-CSF, it would be expected to block binding
of GM-CSF to cell surface receptors. This indeed appears to be the
case, as the sGM-CSFR
-Fc also prevented proliferation of the
GM-CSF-dependent cell line MO7E when present in the medium
together with the growth factor (Fig. 7). Thus, the sGM-CSFR
-Fc is
similar to sGM-CSFR
, as we described previously (12), in possessing
biological antagonist activity.
The parameters regulating the association and dissociation between
GM-CSF and sGM-CSFR-Fc were evaluated in preliminary studies of the
binding of GM-CSF to sGM-CSFR
-Fc in turn bound to immobilized SPA
using an IAsys optical biosensor (Figs. 8 and 9). This was only
possible because of the relatively linear dissociation of sGM-CSFR
-Fc from SPA, as shown in Fig. 8. The advantage of this experimental design is that GM-CSF exists as a monomer in solution, and
a 1:1 interaction with a single receptor site is likely. This eliminates the problem posed by the trimeric nature of the
sGM-CSFR
-Fc, which may contribute an avidity component to binding.
The disadvantage is that a complicated correction for the dissociation
of sGM-CSFR
-Fc from SPA was needed. In these experiments,
association phase analysis revealed nonlinear plots of
dR/dt versus R (data not shown). Although the
reason for this departure from linearity is obscure, given this
experimental configuration, it is unlikely to be the result of the
trimer formation by sGM-CSFR
-Fc. This prevented formal kon calculations. We were able to obtain
reproducible values for koff which averaged
2.43 × 10
2/s (Fig. 9, C-F). When used
to solve for t1/2 in Equation 4 for the case of
r = 1/2 R0, the estimated
half-time for receptor dissociation ranges from 27 to 30 s.
Similar analyses were carried out for immobilized GM-CSF with the
sGM-CSFR-Fc in solution. The association phase failed to follow the
predicted theoretical model (23) as demonstrated by the plots of both
dR/dt versus R and
ks versus concentration. In the dR/dt versus R plot (Fig. 10B)
we represented the first linear portion of the curves for 75, 300, and
600 nM concentrations, but we omitted from the plot the 900 nM concentration. This high sGM-CSFR
-Fc concentration
resulted in very rapid binding in the initial association phase (Fig.
10A). It was therefore impossible to obtain enough data
points to draw a reliable dR/dt versus R plot for
this concentration. Moreover, with such a rapid association rate, mass
transfer becomes the rate-limiting factor in the initial stage of the
binding. We therefore declined to calculate a formal kon from these data.
For analysis of the dissociation phase, we chose to analyze only the
plot for the highest concentration of sGM-CSFR-Fc, 900 nM, because this had the highest response/background ratio.
This analysis did not fit the expected exponential decays shown by the
curvature of the plot in Fig. 10C. Nonlinearity in
dissociation phase analysis has also been seen in the interaction of
insulin-like growth factor and insulin-like growth factor-binding
protein (26). In this case the effect was attributed to either long
term maturation of the analyte-ligand complex or functional
heterogeneity of the immobilized ligand. Other potential explanations
include multiple sites with different affinities, cooperativity in
binding, rebinding, and mass effects. Curve-fitting the data in Fig.
10C showed a fit to the sum of exponentials, and this is
perhaps the result of differential binding characteristics of sites
within the sGM-CSFR
-Fc trimer, depending on the number of active
receptor sites already involved in binding to immobilized GM-CSF. In
the absence of a definite physical explanation the data were analyzed
by taking the slope of the curve in the first 120 s. In support of
this method of analysis are its simplicity and the consistency of the calculated koff with the average value from
experiments with different sGM-CSFR
-Fc concentrations. The
koff value determined by this method is
1.57 × 10
3/s. This is ~1 order of magnitude
slower than for the reverse experimental configuration (see Fig. 9,
C-F). This indicates that the trimer formation of
sGM-CSFR
-Fc slows the off-rate, likely because of an avidity
effect.
Thus, our data indicate that the sGM-CSFR-Fc interaction with GM-CSF
is characterized by a rapid dissociation phase, implying that the major
energy of binding is contributed by a fast on-rate. These kinetics have
relevance to the biological activity of the sGM-CSFR
. The ability of
the sGM-CSFR
to function as a biological antagonist is likely caused
by competition for free GM-CSF with the transmembrane form of the
receptor (tmGM-CSFR
) present on cells. Given the relatively fast
off-rate of the sGM-CSFR
-Fc, the antagonist activity of sGM-CSFR
is then dependent on its continued presence, because once the
sGM-CSFR
diffuses away, the GM-CSF would dissociate and then be
available for binding to the tmGM-CSFR
. As noted above, the
t1/2 for dissociation is on the order of 27-30
s. Thus, for sGM-CSFR
to sequester GM-CSF and remove it from the
local environment of a responsive cell, diffusion away from the cell
would have to be more rapid than this dissociation rate. Slower rates
of diffusion would imply that the sGM-CSFR
needs to be present in
the vicinity of the cell to act as a competitive inhibitor.
In terms of understanding the high affinity sites formed by
tmGM-CSFR and
c, these data would suggest that the
contribution of
c is mostly to slow the off-rate, as the
on-rate is already relatively rapid. This is supported by experiments
reported by Gearing et al. (4), where binding of
125I-GM-CSF to low affinity sites on HL-60 cells was lost
after a short (10-min) incubation in the absence of
125I-GM-CSF, but the high affinity sites remained. We would
postulate, based on our data and theirs, that the primary role of the
GM-CSFR
in the complex is to capture GM-CSF with a rapid association
phase and that the
c functions primarily to slow the
dissociation phase. It is interesting to speculate on the role of the
-subunit (
c) in slowing the dissociation of GM-CSF
from the heterodimeric receptor. Studies of GM-CSF mutants support a
key role for Glu-21 in binding to the high affinity but not the low
affinity receptor (8, 27-31). These authors would argue for direct
binding of the GM-CSF A-helix, centered on residue Glu-21, to the
c. Such direct binding would account for the slower
off-rate seen with the heterodimeric receptor. However, direct evidence
is lacking for such an interaction, and conformational changes or
aggregation of the GM-CSFR
imparted by
c could also
account for the slower off-rate seen. Clarification of this matter
awaits direct binding data with GM-CSFR
,
c, and GM-CSF.
When compared with data from other cytokine receptor interactions, the
kinetics of binding for GM-CSF to the sGM-CSFR-Fc are similar to
those reported for other cytokines, such as IL-5 (25). The
characteristics of rapid on-rates and somewhat slower, but still rapid,
off-rates may be a general characteristic of cytokine-receptor
interactions for four-helix bundle cytokines.
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ACKNOWLEDGEMENTS |
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We thank Joann Kwah, Christine Myers, and Joan VonFeldt for technical and intellectual assistance and J. C. for inspiration.
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FOOTNOTES |
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* This study was supported by grants from the American Cancer Society and the Arthritis Foundation (to W. V. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Grants R01 AI/GM 36502 and AI/GM 40462.
§ To whom correspondence should be addressed: 913 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-662-2789; Fax: 215-349-5572; E-mail: rheum{at}sunspark.med.upenn.edu.
1 The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR, GM-CSF receptor; sGM-CSF, soluble GM-CSF; tmGM-CSF, transmembrane GM-CSF; IL, interleukin; PCR, polymerase chain reaction; mAb, monoclonal antibody; SOE, splicing by overlap extension; bp, base pairs; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; SPA, staphylococcal protein A; HRP, horseradish peroxidase; TMB, 3,3',5',5'-tetramethylbenzidine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
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REFERENCES |
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