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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 461-469
HEMATOPOIESIS
Identification of the soluble granulocyte-macrophage colony
stimulating factor receptor protein in vivo
Farzana Sayani,
Felix A. Montero-Julian,
Valerie Ranchin,
Jay M. Prevost,
Sophie Flavetta,
Weibin Zhu,
Richard C. Woodman,
Herve Brailly, and
Christopher B. Brown
From the Alberta Bone Marrow/Stem Cell Transplant Program and
Division of Hematology, Departments of Medicine and Oncology,
University of Calgary, Calgary, Alberta, Canada, and Immunotech, a
Beckman-Coulter Company, Marseille, France.
 |
Abstract |
On the basis of the finding of alternatively spliced mRNAs, the
 -subunit of the receptor for GM-CSF is thought to exist in both a
membrane spanning (tmGMR) and a soluble form (solGMR). However,
only limited data has been available to support that the solGMR
protein product exists in vivo. We hypothesized that hematopoietic
cells bearing tmGMR would have the potential to also produce
solGMR. To test this hypothesis we examined media conditioned by
candidate cells using functional, biochemical, and immunologic means.
Three human leukemic cell lines that express tmGMR (HL60, U937,
THP1) were shown to secrete GM-CSF binding activity and a
solGMR-specific band by Western blot, whereas a tmGMR-negative
cell line (K562) did not. By the same analyses, leukapheresis products
collected for autologous and allogeneic stem cell transplants and media
conditioned by freshly isolated human neutrophils also contained
solGMR. The solGMR protein in vivo displayed the same
dissociation constant (Kd = 2-5 nmol) as that of recombinant
solGMR. A human solGMR ELISA was developed that confirmed the
presence of solGMR in supernatant conditioned by the
tmGMR-positive leukemic cell lines, hematopoietic progenitor cells,
and neutrophils. Furthermore, the ELISA demonstrated a steady state
level of solGMR in normal human plasma (36 ± 17 pmol) and
provided data suggesting that plasma solGMR levels can be elevated
in acute myeloid leukemias.
(Blood. 2000;95:461-469)
© 2000 by The American Society of Hematology.
| |
Introduction |
A common theme amongst the cytokine receptors is the
existence of soluble isoforms1,2 mainly but not exclusively
comprising the low affinity, ligand-specific " -subunits" of
these multimeric receptor complexes. The potential for influence over
the biologic activity of each cytokine by these soluble receptors and
the precise cytokine specificity they display suggests that soluble
cytokine receptors could play a significant role in the modulation of
the response of cells to cytokine-mediated signaling.
The -subunit of the GM-CSF receptor is 1 such cytokine receptor that
is thought to exist both in a membrane-anchored (tmGMR) and in a
soluble form (solGMR).3,4 Current evidence for the
existence of solGMR rests most solidly on the finding of a truncated
mRNA species in all cells so far examined that also produce the full
length tmGMR mRNA and express tmGMR on their surface. The
solGMR mRNA arises by an alternative splicing mechanism that removes
the exon encoding the transmembrane domain.5 The splicing
event is such that the amino terminus 317 residues of solGMR remain
exactly the same as the extracellular domain of tmGMR; however, the
deletion and subsequent frameshift predicts the replacement of the
transmembrane and cytoplasmic domains of tmGMR with a unique 16 amino acid "tail" on solGMR. Recombinant solGMR binds to
GM-CSF in solution and can antagonize the biological activity of GM-CSF
in vitro.6,7
Despite the substantial information available regarding in vitro
properties of recombinant solGMR, its biologic relevance has been
questioned because the molecule has been difficult to demonstrate in
vivo. Sasaki et al8 demonstrated a soluble GM-CSF binding
moiety in supernatant conditioned by a choriocarcinoma cell line but
the exact nature of the binding molecule was not clarified and the
overall results of their experiments led them to suggest that solGMR
was not produced by hematopoietic cells. However, in this article, we
provide direct evidence for the production of solGMR by
hematopoietic cell lines and physiologic hematopoietic cells and show
that this molecule demonstrates the characteristics of the recombinant
solGMR. We also demonstrate that solGMR is a normal plasma
constituent whose levels can be altered in some cases of acute leukemia.
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Materials and methods |
Sample procurement
Blood samples were obtained with the informed consent of the donors.
Utilization of peripheral blood stem cell materials was reviewed and
approved by the Ethics Board of the Foothills Medical Center of the
University of Calgary.
Cell lines and culture conditions
All cell lines except × 63.Ag8.653 were maintained in
RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum
(FCS), 1% antibiotic-antimycotic solution, 50 mmol/L glutamine, 1 mmol/L sodium pyruvate, and 1% nonessential amino acids. The human
leukemic cell lines U937, THP-1, K562 additionally received 50 µmol
 -mercaptoethanol, the GM-CSF dependent human leukemic cell line TF-1
was maintained with rhGM-CSF (R&D Systems), 1 ng/mL. Dihydrofolate
reductase-/-Chinese Hamster Ovary (CHO) cells were
supplemented with 10 µg/mL of adenosine, deoxyadenosine, and
thymidine. Supernatants from appropriate cells lines were harvested
during the exponential phase of cell growth. × 63.Ag8.653 was
maintained in DMEM 10% FCS supplemented with 1%
antibiotic-antimycotic solution, 50 mmol/L glutamine, 1 mmol/L sodium
pyruvate, and 1% nonessential amino acids.
GM-CSF receptor cloning and expression
A cDNA corresponding to the extracellular domain of GMR
(eGMR, amino acids 1-317) was amplified by RT-PCR from mRNA
prepared from the GM-CSF dependent human leukemic cell line TF-1 and
was subcloned directly into the mammalian expression vector pKCR. eGMR was also shuttled into a pBluescript vector containing a cDNA
encoding human IL-2 (kindly provided by Dr M. Boneville, Nantes) such
that eGMR was upstream of IL-2, in-frame and separated by a sequence
encoding the dipeptide ala-gly. The eGMR/IL-2 sequence was
subsequently cloned into pKCR. The pKCR inserts were confirmed by sequencing.
Ten micrograms of the pKCR-eGMR and pKCR-eGMR/IL-2 constructs
were transfected separately into CHO cells by electroporation at 300 V
and 900 µF (Genezapper 450/2500 IBI, Kodak). Stable transfectants were induced by growth of the CHO cells in increasing concentrations of
methotrexate (10-50 nmol) and transformants were cloned by limiting
dilution. Production of the eGMR/IL-2 soluble fusion protein by
candidate clones was established initially on the basis of the
measurement of immunoreactivity in a human IL-2 ELISA (Immunotech Inc,
Marseille, France). Production of eGMR was initially established by
Western blot of media conditioned by candidate clones using anti-GMR
antibody SCO4 (see below).
eGMR/IL-2 was purified in 2 steps. Conditioned media was first
loaded onto a carboxymethyltrysacryl gel column and eluted with 50 mmol/L sodium acetate pH 4.5, 0.5 mol/L NaCl. IL-2 immunoreactive fractions were pooled and loaded onto an immunoaffinity column on which
anti-IL-2 antibody IL-2.66 (Immunotech Inc, Marseille, France) was
grafted onto CNBr-activated sepharose. The column was eluted with 50 mmol/L sodium acetate pH 4.5, 0.5 mol/L NaCl at a flow rate of 30 ml/h
and fractions containing the hybrid protein were detected by IL-2
ELISA. Purity was determined to be > 90% by SDS-PAGE and Coomassie
Blue staining and Western blot analysis with anti-IL-2 antibody
IL-2.66. The fusion protein was quantitated by amino acid analysis.
Production of anti-GMR antibodies
BALB/C mice (Iffa Credo, Les Oncines, France) were immunized
intraperitoneally twice, at 3 weekly intervals, with 5 µg of recombinant eGMR/IL-2 in complete Freund's adjuvant emulsified in
0.1 mL of sterile PBS. Spleen cells were fused with mouse myeloma cell
line × 63.Ag8.653 using polyethylene glycol 1500, and
hybridomas were established by conventional HAT selection. Anti-GMR
antibodies were selected by the detection of immunoreactivity against
microtiter wells coated with eGMR/IL-2 and the absence of
immunoreactivity to microtiter wells coated with recombinant IL-2.
Antibodies were purified with protein A sepharose (Pharmacia, Uppsala,
Sweden) and subclasses of antibodies were determined with a mouse
monoclonal antibody isotyping kit (Amersham, Les Ullis, France). Two
noncross reactive IgG1 anti-GMR antibodies, SCO4 and SCO6, were
identified for further use.
Enzyme immunometric assay
An enzyme-linked immunoassay (ELISA) was developed using a solid
phase coated with anti-GMR monoclonal antibodies SCO4 and biotinylated-SCO6. 96-well plates were coated with SCO4 at 5 µg/mL in
PBS after which the wells were blocked with PBS/BSA 3%. SCO6 antibodies were biotinylated with biotin- -amino-caproic
acid-N-hydroxysuccinimide ester (Boehringer Mannheim, Germany)
following manufacturer's instructions. The ELISA procedure was as
follows: 50 µL/well of standard or sample were incubated for 2 hours
at room temperature on an orbital shaker. The wells were rinsed 3 times
with an automatic washer (SLT, Salzburg, Austria) with 300 µL of a 9 g/L NaCl solution containing 0.05% Tween 80 after which 50 µL/well
of biotinylated anti-GMR antibody SCO6 and 100 µL of
streptavidin-peroxidase were added. The plates were incubated for 30 minutes at room temperature on an orbital shaker, washed 3 times and
100 µL/well of TMB peroxidase substrate was added. The color reaction
was allowed to develop in the dark for 20 minutes with agitation. The
reaction was then stopped by addition of 50 µL/well of 2N
H2SO4 and the absorbance was measured at 450 nm
with a microplate reader (Molecular Device, UK). The absorbance of the
substrate was subtracted from all values. All determinations were
performed in duplicate. For quantitation of solGMR in plasma
samples, polyclonal mouse immunoglobulins (Scantibodies, CA) were added
to the biotinylated antibody to a final concentration of 50 µg/mL.
Peripheral blood stem cell products
Plasma conditioned by human hematopoietic progenitor cells was
obtained from peripheral blood stem cell (PBSC) products collected as
previously described9,10 from G-CSF-primed stem cell donors undergoing leukapheresis for autologous or allogeneic stem cell transplant. The PBSC product was collected by a 3- to 8-hour
leukapheresis procedure, during which the cells accumulated in a small
volume of autologous plasma. The stem cell product was centrifuged at low speed and the cells cryopreserved for later transplantation. The
remaining supernatant, consisting of 50 to 200 mL of conditioned plasma, was frozen at  80°C for future use.
Isolation of neutrophils
Neutrophils from healthy donors were purified by dextran
sedimentation, followed by hypotonic lysis and Histopaque
centrifugation as previously described.11 Except for the
dextran sedimentation step, which was performed at room temperature,
the cells were kept at 4°C throughout the isolation precedure. Cell
preparations contained > 95% neutrophils with > 99% viability
using Trypan Blue dye exclusion. After isolation neutrophils were
resuspended at a final concentration of 1 × 107
cells/mL in PBS.
Purification of soluble GMR
Ligand affinity chromatography.
Supernatants (ranging from 200-1000 mL) conditioned by HL60, U937,
THP1, and K562 cell lines, PBSC-Con A eluates or supernatants conditioned by freshly isolated human neutrophils were applied to a
GM-CSF ligand affinity column constructed and used as we have
previously described.6 Eluted fractions containing
solGMR were pooled, dialyzed against 1% PBS at 4°C for 8 hours,
and lyophilized. The samples were then made up to either 500 µL or 1 mL with distilled water and stored at 4°C or 20°C.
Before passage over the GM-CSF ligand affinity column, the frozen
plasma from PBSC products was thawed at 4°C, spun at 16 000 rpm to
remove the cryoprecipitate, and gravity filtered using Whatman filter
paper to remove any particulate matter. The supernatant was then
applied to a Con A sepharose column (Pharmacia Biotech, Uppsala,
Sweden) at 4°C at a rate of 40 mL/h. The column was washed with 5 column volumes of binding buffer 20 mmol/L Tris-HCl, 0.5 mol/L NaCl, pH
7.4 and bound glycoproteins eluted with 40 mL of 0.3 mol/L methyl
D-glucopyranoside. The eluate was diluted 3-fold and applied to the
GM-CSF ligand affinity column.
Immunoaffinity chromatography.
Purification of eGMR from media conditioned by eGMR-transfected
CHO cells and of solGMR from 1 L of human serum was performed using
immunoaffinity chromatography. The anti-GMR monoclonal antibody SCO4
was grafted onto CNBr-activated Sepharose (Pharmacia Biotech, Uppsala,
Sweden), following the manufacturer's instructions, at 5 mg of
antibody per milliliter of gel. Samples were loaded at a flow rate of
20 mL/h. The column was washed with 20 mmol/L borate pH 8, 0.15 mol/L
NaCl containing 0.05 g/L of Tween 80, and eluted with citrate pH 3 at a
flow rate of 30 mL/h. The fractions were neutralized immediately with 1 mol/L Tris pH 10.
Gel filtration chromatography
Gel filtration chromatography was performed with a calibrated
Superdex 75 10/30 column and FPLC system (both from Pharmacia Biotech,
Uppsala, Sweden). The sample was eluted with PBS at 0.5 mL/min in 1 mL fractions.
125I-GM-CSF soluble receptor binding assay
Soluble receptor binding assays were performed and analyzed as
previously described.6
SDS polyacrylamide gel electrophoresis and Western blotting
Samples were size fractionated under reducing conditions on 10.5%
SDS polyacrylamide gels and electrophoretically transferred onto
Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA) and prepared for Western blotting as previously
described.12 The blots were incubated in a 1:5000 dilution
of the anti-GMR monoclonal antibody 8G6 at room temperature for 2 hours. After washing 3 times with TBS-Tween (20 mmol/L Tris, pH 7.6, 137 mmol/L NaCl, 0.1% Tween), the blots were incubated with a
horseradish peroxidase-labeled rabbit antimouse polyclonal antibody
(Amersham Life Sciences, Oakville Ontario, Canada) for 45 minute at
room temperature. After washes with TBS-Tween, blots were visualized with enhanced chemiluminescence detection reagents (ECL, Amersham Life
Sciences, Oakville Ontario, Canada) and exposed to x-ray film.
| |
Results |
Human leukemic cell lines express sol
We hypothesized that hematopoietic cells, which expressed tmGMR,
would also express solGMR. To test our hypothesis we collected supernatant conditioned by 3 human leukemic cell lines known to express
tm (HL60, U937, THP1) and 1 cell line that does not express tm
(K562).13-16 In serial experiments 200 to 800 mL of
conditioned media from each cell line was first subjected to ligand
affinity chromatography to enhance the possibility of identifying
solGMR. Fractions corresponding to the elution pattern of
recombinant solGMR were thereafter pooled and volume reduced by
dialysis and lyophylization. The samples were then analyzed for the
presence of solGMR. As shown in panel A of Figure
1 the supernatants of each of the
tmGMR-positive cell lines were shown to contain a GM-CSF specific
soluble binding moiety by solution phase receptor binding assays,
whereas the tmGMR-negative K562 cell line did not. As well, Western
blot analysis of these samples with the use of an anti-GMR
monoclonal antibody (Figure 1, panel B) showed that the supernatant of
each of the cell lines displaying soluble GM-CSF binding activity
contained a molecule that was recognized by the anti-GMR antibody
and that comigrated with recombinant solGMR on SDS-PAGE. The K562
cell line on the other hand showed no immunologic evidence for the
production of solGMR.
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| Fig 1.
The soluble GM-CSF receptor is produced by human leukemic
cell lines.
Equal volumes of supernatant conditioned by the cell lines as shown
were applied to a GM-CSF ligand affinity column and eluted fractions
were pooled, dialyzed against distilled H20, lyophilized,
resuspended in 500 µL PBS and studied as described. Shown are
representative results of n = 3 separate preparations. The starting
volume of supernatant for the illustrated results was 300 mL. Panel A:
25 µL sample from each cell line was used in 125I-GM-CSF
solution phase binding assays in the absence or presence of a large
excess of unlabeled GM-CSF. Specifically bound radioactivity (spec
bound, cpm) was calculated by subtracting the precipitated
radioactivity in the absence of unlabeled GM-CSF from that in the
presence of unlabeled GM-CSF. Bars represent the mean and SEM of
duplicate experiments. Panel B: 30 µL sample from each cell line was
subjected to 10.5% SDS PAGE under reducing conditions and transferred
to PVDF membrane. Immunoblotting was performed with anti-GMR
antibody 8G6. Positive control (+ve) was ligand affinity purified
recombinant solGMR. The position of the molecular weight markers is
shown in kilodaltons.
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To further characterize the soluble GM-CSF binding moiety, saturation
binding experiments were performed on the ligand affinity purified
supernatants from each of the cell lines. Analysis of the binding data
(Figure 2) illustrates that the soluble
GM-CSF receptor produced by each of the tmGMR-positive cell lines
demonstrates single site binding characteristics and Table
1 shows that the affinity is very similar
to that which we have previously documented for recombinant solGMR
(Kd = 2-3 nmol).6 However Table 1 also shows that in all
the samples examined the molar quantity of soluble receptors in the
binding assay was extremely small ranging from 0.1 to 1.0 nmol.
Remembering that the initial volume of supernatant of each of the cell
lines was reduced approximately 1000-fold and allowing for losses
during chromatography, dialysis, and lyophylization, the initial
concentration of solGMR in the conditioned media was likely 100- to
1000-fold less (1.0-10 pmol).
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| Fig 2.
Binding characteristics of the soluble GM-CSF receptor
produced by human leukemic cell lines.
125I-GM-CSF saturation binding experiments were performed
on supernatant conditioned by the human leukemic cell lines shown.
Before analysis the supernatants were subjected to ligand affinity
column chromatography and eluted fractions were pooled, dialyzed
against distilled H2O lyophilized and resuspended in 500 µL PBS. Shown are Scatchard analysis and saturation binding curves
(inset) of representative experiments for U937 (A) (n = 6), THP-1 (B)
(n = 5), and HL60 (C) (n = 2) cells.
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Identification of a soluble GM-CSF receptor in plasma conditioned by
human hematopoietic cells
To examine a more physiologic potential source of solGMR in an
environment in which a very large number of human cells had conditioned
the supernatant, we examined plasma conditioned by peripheral blood
stem cell products collected for autologous and allogeneic stem cell
transplants. Five donors had undergone PBSC harvest by leukapheresis,
whereas 1 (patient 4) had been harvested by direct operative removal of
bone marrow from the posterior superior iliac crests of the pelvis.
1011 to 1012 nucleated hematopoietic cells had
conditioned each sample. As can be seen in panel A of Figure
3 GM-CSF specific binding was found in the supernatant from all 6 of the samples. Panel B of Figure 3 demonstrates that this corresponded to the presence of a band migrating with recombinant solGMR and recognized by anti-GMR monoclonal antibody 8G6 by Western analysis.
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| Fig 3.
The soluble GM-CSF receptor is found in plasma
conditioned by human hematopoietic progenitor cells.
Plasma conditioned by PBSC products was subjected to Con-A sepharose
and ligand affinity column chromatography. Pooled fractions from the
ligand affinity column were dialyzed against distilled H2O
lyophilized, and then resuspended in 500 µL PBS for analysis. Samples
1, 5, 6 = donors for autologous transplant. Samples 2, 3, 4 = donors for allogeneic transplant. Panel A: 25 µL sample from
each donor was used in 125I-GM-CSF solution phase binding
assays in the absence or presence of a large excess of unlabeled
GM-CSF. Specifically bound radioactivity (spec bound, cpm) was
calculated by subtracting the precipitated radioactivity in the absence
of unlabeled GM-CSF from that in the presence of unlabeled GM-CSF. Bars
represent the mean and SEM of duplicate experiments. Panel B: 30 µL
sample from each donor was subjected to 10.5% SDS PAGE under reducing
conditions and transferred to PVDF membrane. Immunoblotting was
performed with anti-GMR antibody 8G6. Positive control (+ve) was
ligand affinity purified recombinant solGMR. Negative control (-ve)
was sham ligand affinity column eluate. The position of the molecular
weight markers is shown in kilodaltons.
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To further characterize the soluble receptor produced in PBSC and bone
marrow products saturation binding experiments were performed. However,
the amount of soluble receptor produced by any 1 product was found to
be too small to allow the performance of accurate saturation binding
experiments. To circumvent this, the eluates from all 6 samples that
remained after initial analysis were pooled, volume reduced, and then
subjected to saturation binding experiments. With this manipulation, we
were able to demonstrate that the pooled samples contained a soluble
GM-CSF binding moiety with single site binding characteristics,
Kd = 7.02 ± 3.25 nmol, n = 3 (Figure
4). This is again quite close to the
dissociation constant we have established for recombinant solGMR
(2-3 nmol).6 However, even in the pooled, volume-reduced
sample the soluble receptor concentration only reached 5.2 nmol.
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| Fig 4.
Binding characteristics of the soluble GM-CSF receptor
identified in PBSC and bone marrow products.
Plasma conditioned by PBSC products was subjected to Con-A sepharose
and ligand affinity column chromatography and eluted fractions were
pooled, dialyzed against distilled H2O lyophilized, and
resuspended in 500 µL PBS. 150 µL of each of these samples was
pooled and volume reduced in a centrifugal filtration cartridge
(Ultrafree® Biomax-5K NMWL membranes, Millipore Corp, Bedford, MA).
125I-GM-CSF saturation binding experiments were performed
on these pooled samples. Shown are Scatchard analysis and saturation
binding curves (inset) of a representative experiment (n = 3).
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Highly purified human neutrophil preparations produce solGMR
To examine a more homogeneous physiologic cell source than the PBSC
products, fresh human neutrophils were obtained from normal volunteers
and were purified to > 95% homogeneity. The cell preps were
incubated overnight at 37°C in PBS pH 7.4 and the supernatants were
collected and processed as described previously. Material conditioned
by 1.1 × 109 neutrophils was analyzed. Figure
5 demonstrates that the neutrophil preparations produced soluble GM-CSF binding activity (panel A) and a
molecule that was recognized by the anti-GMR antibody 8G6 and that
comigrated with recombinant solGMR on SDS-PAGE (panel B).
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| Fig 5.
Highly purified preparations of human neutrophils produce
the soluble GM-CSF receptor.
110 mL of supernatant conditioned overnight by freshly isolated human
neutrophils (1 × 107 neutrophils/mL) were applied
to a GM-CSF ligand affinity column and eluted fractions were pooled,
dialyzed against distilled H2O lyophilized, and resuspended
in 500 µL PBS. Panel A: 25 µL of the pooled sample was used in
125I-GM-CSF solution phase binding assays in the absence or
presence of a large excess of unlabeled GM-CSF. Specifically bound
radioactivity (spec bound, cpm) was calculated by subtracting the
precipitated radioactivity in the absence of unlabeled GM-CSF from that
in the presence of unlabeled GM-CSF. Bars represent the mean and SEM of
duplicate experiments. Panel B: 30 µL of the pooled sample was
subjected to 10.5% SDS PAGE under reducing conditions and transferred
to PVDF membrane. Immunoblotting was performed with anti-GMR
antibody 8G6. Positive control (+ve) was ligand affinity purified
recombinant solGMR. Negative control (-ve) was sham ligand affinity
column eluate.
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Comparison of immunological and functional evidence for solGMR
We were disturbed by the lack of correlation between the binding
data and the band intensity by Western analysis when examining the
various samples for the presence of solGMR (Figures 1 and 3). To
clarify the validity of the data we examined the individual fractions
eluted from the ligand affinity column for each of the samples
described previously using both binding analysis and Western blot.
Figure 6 demonstrates that there was a
direct relationship between the amount of binding in each fraction and
the intensity of the band by Western analysis. The same direct
relationship was shown for each of the cell lines although, as
expected, in the K562 cell line no binding or immunologic signal was
seen in any fraction (data not shown). However, in the PBSC and
neutrophil samples in which we suspected the amount of receptor was
extremely low in the individual fractions, the degree of binding and
the intensity of the immunologic bands were so weak that no
reproducible linear relationship could be established.
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| Fig 6.
Relationship between receptor binding and immunologic
signal.
Approximately 500 mL of supernatant conditioned by the human leukemic
cell lines were subjected to a GM-CSF ligand affinity column and eluted
fractions were volume reduced in centrifugal filtration cartridges.
Panel A: 25 µL of each of the volume reduced fractions was used in
125I-GM-CSF solution phase binding assays in the absence or
presence of a large excess of unlabeled GM-CSF. Specifically bound
radioactivity (spec bound, cpm) was calculated by subtracting the
precipitated radioactivity in the absence of unlabeled GM-CSF from that
in the presence of unlabeled GM-CSF. Bars represent the mean and SEM of
duplicate experiments. Panel B: 30 µL of each of the volume reduced
fractions was subjected to 10.5% SDS PAGE under reducing conditions
and transferred to PVDF membrane. Immunoblotting was performed with
anti-GMR antibody 8G6. Positive control (+ve) was ligand affinity
purified recombinant solGMR. Shown are representative results for
the U937 cell line.
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Characterization of an immunometric assay for solGMR
To simplify the study of solGMR in vivo a human
solGMR-specific, ELISA was developed. Two noncross reactive
anti-GMR monoclonal antibodies (SCO4, SCO6) were used in a sandwich
technique. eGMR was used as the recombinant standard after stepwise
analysis. First, the signal strength of serial 2-fold dilutions of
recombinant IL-2 and eGMR/IL-2 were compared in a commercial IL-2
ELISA kit (Immnunotech Inc, Marseille, France) and the curves were
found to be parallel, suggesting the ELISA recognized both molecules equally. This was confirmed when amino acid analysis of eGMR/IL-2 revealed a very close concordance between the concentration determined by the IL-2 ELISA and by amino acid analysis (data not shown). Similar
parallel dilution curves were found when eGMR/IL-2, immunoaffinity purified eGMR and human plasma were analyzed in the
solGMR ELISA suggesting that all forms were recognized
equally. eGMR subsequently replaced eGMR/IL-2 as the standard in
the solGMR ELISA. Sensitivity, defined as the lowest
solGMR concentration significantly different from the zero standard
with a probability of 95%, was 5 pmol in serum, plasma, and tissue
culture media.
The assay showed no cross-reactivity with TNF (10 ng/mL), IL-1 (1 ng/mL), IL-1 (1 ng/mL), IL-2 (10 ng/mL), IL-3 (3 ng/mL), IL-4 (1 ng/mL), IL-5 (650 pg/mL), IL-6 (10 ng/mL), IL-10 (1 ng/mL), GM-CSF (10 ng/mL), or EPO (10 ng/mL). There was also no interference by GM-CSF (10 ng/mL) or by IL-3 (3 ng/mL), IL-5 (650 pg/mL) or EPO (10 ng/mL).
Intra-assay precision was determined by examining 3 samples 16 times
each on a single ELISA plate (Table 2).
Inter-assay precision was determined by examining 3 other samples
in duplicate in 8 different assays (Table 2).
The sample sources initially examined by receptor binding assay and
Western blot were reexamined with the solGMR ELISA. As shown in
Table 3, the ELISA confirmed the presence
of solGMR in media conditioned by tmGMR-positive human leukemic
cell lines, plasma conditioned by PBSC products, and supernatant
conditioned by freshly isolated human neutrophils.
SolGMR is present in normal human plasma
Plasma was collected from normal healthy volunteers and analyzed in
the solGMR ELISA to determine whether circulating levels of
solGMR could be detected. The plasma samples were introduced into
the ELISA without manipulation. As shown in Figure
7, the amount of solGMR in human plasma
follows a normal distribution with a mean concentration of
36 ± 17 pmol (95% CI = 10-85.5 pmol, n = 47, statistical
analysis was performed in MedCalc v4.16f.). To confirm the specificity
of the solGMR signal, plasma samples were incubated overnight at
4°C with recombinant human GM-CSF covalently linked to sepharose
beads (NHS-Sepharose 4 fast flow, Pharmacia, Uppsala, Sweden). The
plasma samples were subsequently centrifuged to remove the
GM-CSF-linked beads and the plasma analyzed in the solGMR ELISA. The
GM-CSF-linked beads successfully depleted the plasma samples of the
solGMR signal (data not shown).
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| Fig 7.
Distribution of plasma solGMR levels in healthy
donors.
Plasma solGMR levels were determined in 47 volunteers using the
solGMR ELISA. 50 µL unmanipulated plasma was applied to each of
duplicate wells in the 96-well format ELISA. Shown is the number of
donors whose circulating solGMR levels fell within each 5 pmol
range.
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Characterization of circulating solGMR
To further characterize the nature of the circulating moiety
detected by the solGMR ELISA in human plasma, 1000 mL of human plasma were first subjected to immunoaffinity chromatography with the
SCO4 anti-GMR antibody. Eluted fractions were evaluated by Western
analysis again with SCO4 (Figure 8A) and a
band was identified that had an identical electrophoretic mobility as
recombinant solGMR. To further characterize this molecule fraction 9 from the immunoaffinity column was volume reduced and subjected to gel
filtration chromatography. Collected fractions were analyzed by
solGMR ELISA. Figure 8B demonstrates that solGMR derived from
plasma has a molecular mass of 50 kd as determined by gel filtration.
Both the Western analysis and gel filtration gave values for the size
of solGMR in the circulation, which is in reasonable agreement with
the size of recombinant solGMR6 and with the solGMR
species detected by Western blot analysis of material conditioned by
human leukemic cell lines, PBSC products, and freshly isolated human
neutrophils.
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| Fig 8.
Characterization of solGMR derived from human plasma.
1 L plasma was applied to an anti-GMR immunoaffinity column and
eluted in 1 mL fractions. Panel A. 40 µL of each fraction was
subjected to 10.5% SDS PAGE under reducing conditions and transferred
to nitrocellulose. Immunoblotting was performed with biotinylated
anti-GMR antibody SCO4. The number of the fractions is shown above
the figure. The position of the molecular weight markers is shown in
kilodaltons. Panel B. Fraction 9 was volume reduced to 100 µL and
loaded on a Superdex 75 10/30 gel filtration column. Fractions were
collected at 1 minute intervals and analyzed in the solGMR ELISA.
The elution position of molecular weight markers is shown in
kilodaltons above the graph.
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Analysis of plasma from patients with hematologic malignancies
Plasma samples from 5 patients diagnosed with acute myelogenous
leukemia (AML) (kindly provided by Dr C. Chabannon, Institut Paoli
Calmette, Marseille, France) and 4 patients with multiple myeloma (MM)
(kindly provided by Dr E. Tartour, Institut Curie, Paris, France) were
analyzed in the solGMR ELISA. As shown in Table
4, circulating levels of solGMR were
elevated in 4 of the 5 cases of AML and in 1 patient with MM.
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Discussion |
The existence and cloning of the alternatively spliced mRNA that
encodes the soluble isoform of the GM-CSF receptor -subunit has been
documented for some time now by several groups,3,4,7 and
there are a number of reports describing the properties of the
recombinant solGMR molecule in vitro.6,7,12,17-19
However, there has been little formal evidence that the solGMR mRNA
supports the production of solGMR protein in vivo and, thus, the
physiologic or pathophysiologic relevance of solGMR has remained in
question. In this article, we provide experimental evidence that the
solGMR protein is produced by hematopoietic cells in vivo and that
levels of solGMR are altered in some patients with hematologic malignancies.
Expression of the cell surface GM-CSF receptor is most commonly
associated with hematopoietic cells of the myelomonocytic lineage,13,15,20-24 but has also been documented on
nonmyeloid hematopoietic cells25 and a variety of
nonhematopoietic cells.26-31 The truncated mRNA that
encodes solGMR has been documented in all cells that also bear
tmGMR, which have so far been examined.7,16 One would
therefore predict that solGMR protein would be a product of these
cells. The hypothesis that solGMR is expressed by tmGMR-positive hematopoietic cells is supported by our findings with human leukemic cell lines. U937, THP-1, and HL60 cells, all of which bear cell surface
GM-CSF receptors,13,15 secrete solGMR, whereas K562 cells, which are tmGMR-negative,14,16 could not be shown
to produce solGMR (Figures 1, 2 and Tables 1, 3). Plasma conditioned by PBSC products for autologous and allogeneic transplants also contains solGMR (Figures 3 and 4). The PBSC products are very rich
in GM-CSF receptor-bearing cells at many stages of differentiation but
are also very heterogeneous in their cellular constituents, so it
cannot be said with certainty what the cellular source of the solGMR
is in the PBSC products. The production of solGMR by preparations of
freshly isolated neutrophils (Figure 5) also seems to support the
hypothesis that solGMR is produced by tmGMR-positive cells. Human
neutrophils have a large population of cell surface GM-CSF receptors
compared with other physiologic cell sources.22 However,
the neutrophil preps, although > 95% pure, do contain a minor
population of mononuclear cells that have previously been shown to
contain the mRNA for solGMR.7 Thus, our data, although supportive of the ability of human neutrophils to produce solGMR, does not rule out that the contaminating peripheral blood mononuclear cells were responsible for some or all of the solGMR production in
the preparations. In any case, the data indicate that solGMR is
produced and secreted by hematopoietic cells and supports the notion
that production is limited to those cells that bear membrane anchored
GMR.
The data also reveal that solGMR is produced in very small amounts.
This fact has made its identification in vivo a challenge. Indeed, our
early attempts to identify solGMR in vivo with unmanipulated conditioned media were wholly unsuccessful. Only after enrichment and
purification steps were introduced were we able to make progress. Subsequent quantitation of solGMR by analysis of saturation binding assays reveals solGMR production levels in the picomolar
concentration ranges for all cellular sources examined. The development
of the solGMR ELISA has greatly simplified the identification and
quantitation of solGMR, and there seems to be good agreement between
the data derived by analysis of saturation binding assays and by the
ELISA. This is especially true for the human leukemic cell lines. The concordance between the binding analysis and the ELISA for the PBSC
products is not as close with the ELISA, suggesting a higher production
of solGMR than the binding analysis. However, the ELISA was
performed on unmanipulated plasma conditioned by the PBSC products,
whereas the binding assays were performed after ConA sepharose
chromatography, ligand affinity chromatography, and dialysis of the
eluates, followed by lyophylization. The discrepancy between the 2 methods of quantitation is therefore likely explained by solGMR
losses incurred by the multiple manipulations to allow enrichment
of solGMR before the saturation binding assays. We would suggest
that the PBSC solGMR concentrations derived from the ELISA are
likely the more accurate values for the true levels of solGMR in
plasma conditioned by the PBSC products.
The availability of the solGMR ELISA has afforded the opportunity to
establish a range of normal values for plasma levels of solGMR. Our
findings indicate that solGMR circulates in the blood of normal
subjects at concentrations of 36 ± 17 pmol with a range of 10 to
85.5 pmol (n = 47, Figure 7). Interpretation of the levels of
solGMR found in plasma conditioned by the PBSC products must keep in
mind this range of solGMR in normal plasma. Seven of the 8 PBSC
products examined by ELISA had solGMR levels within the normal range
for plasma. Thus, despite the fact the plasma had been conditioned ex
vivo for 3 to 8 hours by between 1010 and 1012
hematopoietic cells, there was little or no increase of solGMR above
normally circulating concentrations. This finding brings into question
whether this very large number of hematopoietic cells were producing
any significant amount of solGMR during the collection period and
begs the more general question of what cells are the source of the
solGMR detected in plasma. It is possible that the source of
circulating solGMR is not hematopoietic cells but other GM-CSF
receptor-bearing cells. For instance, vascular endothelial cells bear
GM-CSF receptors and respond to GM-CSF.26 The possibility
exists that such nonhematopoietic cells are the source of most of the
circulating solGMR, whereas hematopoietic cells produce solGMR
that is meant to be active only in localized microenvironments. Such a
juxtacrine model of activity for hematopoietic cells would require the
production of only small quantities of solGMR, in keeping with what
we have documented, but the model is entirely speculative. It is also possible that the conditions of PBSC mobilization inhibit the production of solGMR by the cells that collect during the
leukapheresis procedure.
The availability of the solGMR ELISA has greatly simplified certain
avenues of study of solGMR; however, the performance of ligand
affinity and gel filtration chromatography, saturation binding assays,
and Western blot analyses |
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Abstract
Introduction