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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
Top
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgments
References

On the basis of the finding of alternatively spliced mRNAs, the alpha -subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMRalpha ) and a soluble form (solGMRalpha ). However, only limited data has been available to support that the solGMRalpha protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMRalpha would have the potential to also produce solGMRalpha . To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMRalpha (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMRalpha -specific band by Western blot, whereas a tmGMRalpha -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 solGMRalpha . The solGMRalpha protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMRalpha . A human solGMRalpha ELISA was developed that confirmed the presence of solGMRalpha in supernatant conditioned by the tmGMRalpha -positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMRalpha in normal human plasma (36 ± 17 pmol) and provided data suggesting that plasma solGMRalpha levels can be elevated in acute myeloid leukemias. (Blood. 2000;95:461-469)

© 2000 by The American Society of Hematology.


    Introduction
Top
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgments
References

A common theme amongst the cytokine receptors is the existence of soluble isoforms1,2 mainly but not exclusively comprising the low affinity, ligand-specific "alpha -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 alpha -subunit of the GM-CSF receptor is 1 such cytokine receptor that is thought to exist both in a membrane-anchored (tmGMRalpha ) and in a soluble form (solGMRalpha ).3,4 Current evidence for the existence of solGMRalpha rests most solidly on the finding of a truncated mRNA species in all cells so far examined that also produce the full length tmGMRalpha mRNA and express tmGMRalpha on their surface. The solGMRalpha 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 solGMRalpha remain exactly the same as the extracellular domain of tmGMRalpha ; however, the deletion and subsequent frameshift predicts the replacement of the transmembrane and cytoplasmic domains of tmGMRalpha with a unique 16 amino acid "tail" on solGMRalpha . Recombinant solGMRalpha 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 solGMRalpha , 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 solGMRalpha was not produced by hematopoietic cells. However, in this article, we provide direct evidence for the production of solGMRalpha by hematopoietic cell lines and physiologic hematopoietic cells and show that this molecule demonstrates the characteristics of the recombinant solGMRalpha . We also demonstrate that solGMRalpha is a normal plasma constituent whose levels can be altered in some cases of acute leukemia.


    Materials and methods
Top
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgments
References

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 beta -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 GMRalpha (eGMRalpha , 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. eGMRalpha was also shuttled into a pBluescript vector containing a cDNA encoding human IL-2 (kindly provided by Dr M. Boneville, Nantes) such that eGMRalpha was upstream of IL-2, in-frame and separated by a sequence encoding the dipeptide ala-gly. The eGMRalpha /IL-2 sequence was subsequently cloned into pKCR. The pKCR inserts were confirmed by sequencing.

Ten micrograms of the pKCR-eGMRalpha and pKCR-eGMRalpha /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 eGMRalpha /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 eGMRalpha was initially established by Western blot of media conditioned by candidate clones using anti-GMRalpha antibody SCO4 (see below).

eGMRalpha /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-GMRalpha antibodies

BALB/C mice (Iffa Credo, Les Oncines, France) were immunized intraperitoneally twice, at 3 weekly intervals, with 5 µg of recombinant eGMRalpha /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-GMRalpha antibodies were selected by the detection of immunoreactivity against microtiter wells coated with eGMRalpha /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-GMRalpha 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-GMRalpha 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-epsilon -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 solGMRalpha 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 GMRalpha

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 solGMRalpha 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 alpha D-glucopyranoside. The eluate was diluted 3-fold and applied to the GM-CSF ligand affinity column.

Immunoaffinity chromatography. Purification of eGMRalpha from media conditioned by eGMRalpha -transfected CHO cells and of solGMRalpha from 1 L of human serum was performed using immunoaffinity chromatography. The anti-GMRalpha 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-GMRalpha 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
Top
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgments
References

Human leukemic cell lines express solalpha

We hypothesized that hematopoietic cells, which expressed tmGMRalpha , would also express solGMRalpha . To test our hypothesis we collected supernatant conditioned by 3 human leukemic cell lines known to express tmalpha (HL60, U937, THP1) and 1 cell line that does not express tmalpha (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 solGMRalpha . Fractions corresponding to the elution pattern of recombinant solGMRalpha were thereafter pooled and volume reduced by dialysis and lyophylization. The samples were then analyzed for the presence of solGMRalpha . As shown in panel A of Figure 1 the supernatants of each of the tmGMRalpha -positive cell lines were shown to contain a GM-CSF specific soluble binding moiety by solution phase receptor binding assays, whereas the tmGMRalpha -negative K562 cell line did not. As well, Western blot analysis of these samples with the use of an anti-GMRalpha 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-GMRalpha antibody and that comigrated with recombinant solGMRalpha on SDS-PAGE. The K562 cell line on the other hand showed no immunologic evidence for the production of solGMRalpha .


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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-GMRalpha antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRalpha . The position of the molecular weight markers is shown in kilodaltons.

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 tmGMRalpha -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 solGMRalpha (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 solGMRalpha 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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Table 1. Binding characteristics of solGMRalpha produced by human leukemic cell lines

Identification of a soluble GM-CSF receptor in plasma conditioned by human hematopoietic cells

To examine a more physiologic potential source of solGMRalpha 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 solGMRalpha and recognized by anti-GMRalpha 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-GMRalpha antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRalpha . Negative control (-ve) was sham ligand affinity column eluate. The position of the molecular weight markers is shown in kilodaltons.

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 solGMRalpha (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).

Highly purified human neutrophil preparations produce solGMRalpha

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-GMRalpha antibody 8G6 and that comigrated with recombinant solGMRalpha 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-GMRalpha antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRalpha . Negative control (-ve) was sham ligand affinity column eluate.

Comparison of immunological and functional evidence for solGMRalpha

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 solGMRalpha  (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-GMRalpha antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRalpha . Shown are representative results for the U937 cell line.

Characterization of an immunometric assay for solGMRalpha

To simplify the study of solGMRalpha in vivo a human solGMRalpha -specific, ELISA was developed. Two noncross reactive anti-GMRalpha monoclonal antibodies (SCO4, SCO6) were used in a sandwich technique. eGMRalpha was used as the recombinant standard after stepwise analysis. First, the signal strength of serial 2-fold dilutions of recombinant IL-2 and eGMRalpha /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 eGMRalpha /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 eGMRalpha /IL-2, immunoaffinity purified eGMRalpha and human plasma were analyzed in the solGMRalpha ELISA suggesting that all forms were recognized equally. eGMRalpha subsequently replaced eGMRalpha /IL-2 as the standard in the solGMRalpha ELISA. Sensitivity, defined as the lowest solGMRalpha 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 TNFalpha (10 ng/mL), IL-1alpha (1 ng/mL), IL-1beta (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).

                              
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Table 2. Intra- and inter-assay precision

The sample sources initially examined by receptor binding assay and Western blot were reexamined with the solGMRalpha ELISA. As shown in Table 3, the ELISA confirmed the presence of solGMRalpha in media conditioned by tmGMRalpha -positive human leukemic cell lines, plasma conditioned by PBSC products, and supernatant conditioned by freshly isolated human neutrophils.

                              
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Table 3. ELISA determination of solGMRalpha concentration

SolGMRalpha is present in normal human plasma

Plasma was collected from normal healthy volunteers and analyzed in the solGMRalpha ELISA to determine whether circulating levels of solGMRalpha could be detected. The plasma samples were introduced into the ELISA without manipulation. As shown in Figure 7, the amount of solGMRalpha 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 solGMRalpha 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 solGMRalpha ELISA. The GM-CSF-linked beads successfully depleted the plasma samples of the solGMRalpha signal (data not shown).


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Fig 7. Distribution of plasma solGMRalpha levels in healthy donors. Plasma solGMRalpha levels were determined in 47 volunteers using the solGMRalpha 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 solGMRalpha levels fell within each 5 pmol range.

Characterization of circulating solGMRalpha

To further characterize the nature of the circulating moiety detected by the solGMRalpha ELISA in human plasma, 1000 mL of human plasma were first subjected to immunoaffinity chromatography with the SCO4 anti-GMRalpha 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 solGMRalpha . 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 solGMRalpha ELISA. Figure 8B demonstrates that solGMRalpha 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 solGMRalpha in the circulation, which is in reasonable agreement with the size of recombinant solGMRalpha 6 and with the solGMRalpha 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 solGMRalpha derived from human plasma. 1 L plasma was applied to an anti-GMRalpha 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-GMRalpha 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 solGMRalpha ELISA. The elution position of molecular weight markers is shown in kilodaltons above the graph.

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 solGMRalpha ELISA. As shown in Table 4, circulating levels of solGMRalpha were elevated in 4 of the 5 cases of AML and in 1 patient with MM.

                              
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Table 4. Plasma solGMRalpha levels in hematologic malignancies


    Discussion
Top
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgments
References

The existence and cloning of the alternatively spliced mRNA that encodes the soluble isoform of the GM-CSF receptor alpha -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 solGMRalpha molecule in vitro.6,7,12,17-19 However, there has been little formal evidence that the solGMRalpha mRNA supports the production of solGMRalpha protein in vivo and, thus, the physiologic or pathophysiologic relevance of solGMRalpha has remained in question. In this article, we provide experimental evidence that the solGMRalpha protein is produced by hematopoietic cells in vivo and that levels of solGMRalpha 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 solGMRalpha has been documented in all cells that also bear tmGMRalpha , which have so far been examined.7,16 One would therefore predict that solGMRalpha protein would be a product of these cells. The hypothesis that solGMRalpha is expressed by tmGMRalpha -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 solGMRalpha , whereas K562 cells, which are tmGMRalpha -negative,14,16 could not be shown to produce solGMRalpha (Figures 1, 2 and Tables 1, 3). Plasma conditioned by PBSC products for autologous and allogeneic transplants also contains solGMRalpha (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 solGMRalpha is in the PBSC products. The production of solGMRalpha by preparations of freshly isolated neutrophils (Figure 5) also seems to support the hypothesis that solGMRalpha is produced by tmGMRalpha -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 solGMRalpha .7 Thus, our data, although supportive of the ability of human neutrophils to produce solGMRalpha , does not rule out that the contaminating peripheral blood mononuclear cells were responsible for some or all of the solGMRalpha production in the preparations. In any case, the data indicate that solGMRalpha is produced and secreted by hematopoietic cells and supports the notion that production is limited to those cells that bear membrane anchored GMRalpha .

The data also reveal that solGMRalpha is produced in very small amounts. This fact has made its identification in vivo a challenge. Indeed, our early attempts to identify solGMRalpha 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 solGMRalpha by analysis of saturation binding assays reveals solGMRalpha production levels in the picomolar concentration ranges for all cellular sources examined. The development of the solGMRalpha ELISA has greatly simplified the identification and quantitation of solGMRalpha , 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 solGMRalpha 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 solGMRalpha losses incurred by the multiple manipulations to allow enrichment of solGMRalpha before the saturation binding assays. We would suggest that the PBSC solGMRalpha concentrations derived from the ELISA are likely the more accurate values for the true levels of solGMRalpha in plasma conditioned by the PBSC products.

The availability of the solGMRalpha ELISA has afforded the opportunity to establish a range of normal values for plasma levels of solGMRalpha . Our findings indicate that solGMRalpha 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 solGMRalpha found in plasma conditioned by the PBSC products must keep in mind this range of solGMRalpha in normal plasma. Seven of the 8 PBSC products examined by ELISA had solGMRalpha 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 solGMRalpha above normally circulating concentrations. This finding brings into question whether this very large number of hematopoietic cells were producing any significant amount of solGMRalpha during the collection period and begs the more general question of what cells are the source of the solGMRalpha detected in plasma. It is possible that the source of circulating solGMRalpha 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 solGMRalpha , whereas hematopoietic cells produce solGMRalpha 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 solGMRalpha , 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 solGMRalpha by the cells that collect during the leukapheresis procedure.

The availability of the solGMRalpha ELISA has greatly simplified certain avenues of study of solGMRalpha ; however, the performance of ligand affinity and gel filtration chromatography, saturation binding assays, and Western blot analyses