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.

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

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

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

eGMRα/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 eGMRα/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-GMRα antibodies were selected by the detection of immunoreactivity against microtiter wells coated with eGMRα/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-GMRα 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-GMRα 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-ε-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 solGMRα 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 × 107cells/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 solGMRα 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 αD-glucopyranoside. The eluate was diluted 3-fold and applied to the GM-CSF ligand affinity column.

Immunoaffinity chromatography.

Purification of eGMRα from media conditioned by eGMRα-transfected CHO cells and of solGMRα from 1 L of human serum was performed using immunoaffinity chromatography. The anti-GMRα 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-GMRα 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.

Human leukemic cell lines express sol

We hypothesized that hematopoietic cells, which expressed tmGMRα, would also express solGMRα. To test our hypothesis we collected supernatant conditioned by 3 human leukemic cell lines known to express tmα (HL60, U937, THP1) and 1 cell line that does not express tmα (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 solGMRα. Fractions corresponding to the elution pattern of recombinant solGMRα were thereafter pooled and volume reduced by dialysis and lyophylization. The samples were then analyzed for the presence of solGMRα. As shown in panel A of Figure1 the supernatants of each of the tmGMRα-positive cell lines were shown to contain a GM-CSF specific soluble binding moiety by solution phase receptor binding assays, whereas the tmGMRα-negative K562 cell line did not. As well, Western blot analysis of these samples with the use of an anti-GMRα 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-GMRα antibody and that comigrated with recombinant solGMRα on SDS-PAGE. The K562 cell line on the other hand showed no immunologic evidence for the production of solGMRα.

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

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

Close modal

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 tmGMRα-positive cell lines demonstrates single site binding characteristics and Table1 shows that the affinity is very similar to that which we have previously documented for recombinant solGMRα (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 solGMRα in the conditioned media was likely 100- to 1000-fold less (1.0-10 pmol).

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.

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.

Close modal

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

To examine a more physiologic potential source of solGMRα 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 Figure3 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 solGMRα and recognized by anti-GMRα monoclonal antibody 8G6 by Western analysis.

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-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Negative control (-ve) was sham ligand affinity column eluate. The position of the molecular weight markers is shown in kilodaltons.

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-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Negative control (-ve) was sham ligand affinity column eluate. The position of the molecular weight markers is shown in kilodaltons.

Close modal

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 (Figure4). This is again quite close to the dissociation constant we have established for recombinant solGMRα (2-3 nmol).6 However, even in the pooled, volume-reduced sample the soluble receptor concentration only reached 5.2 nmol.

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).

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).

Close modal

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. Figure5 demonstrates that the neutrophil preparations produced soluble GM-CSF binding activity (panel A) and a molecule that was recognized by the anti-GMRα antibody 8G6 and that comigrated with recombinant solGMRα on SDS-PAGE (panel B).

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 in125I-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-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Negative control (-ve) was sham ligand affinity column eluate.

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 in125I-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-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Negative control (-ve) was sham ligand affinity column eluate.

Close modal

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 solGMRα (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.

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 in125I-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-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Shown are representative results for the U937 cell line.

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 in125I-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-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Shown are representative results for the U937 cell line.

Close modal

Characterization of an immunometric assay for solGMR

To simplify the study of solGMRα in vivo a human solGMRα-specific, ELISA was developed. Two noncross reactive anti-GMRα monoclonal antibodies (SCO4, SCO6) were used in a sandwich technique. eGMRα was used as the recombinant standard after stepwise analysis. First, the signal strength of serial 2-fold dilutions of recombinant IL-2 and eGMRα/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 eGMRα/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 eGMRα/IL-2, immunoaffinity purified eGMRα and human plasma were analyzed in the solGMRα ELISA suggesting that all forms were recognized equally. eGMRα subsequently replaced eGMRα/IL-2 as the standard in the solGMRα ELISA. Sensitivity, defined as the lowest solGMRα 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 TNFα (10 ng/mL), IL-1α (1 ng/mL), IL-1β (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 solGMRα ELISA. As shown in Table 3, the ELISA confirmed the presence of solGMRα in media conditioned by tmGMRα-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 solGMRα ELISA to determine whether circulating levels of solGMRα could be detected. The plasma samples were introduced into the ELISA without manipulation. As shown in Figure7, the amount of solGMRα 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 solGMRα 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 solGMRα ELISA. The GM-CSF-linked beads successfully depleted the plasma samples of the solGMRα signal (data not shown).

Fig. 7.

Distribution of plasma solGMR levels in healthy donors.

Plasma solGMRα levels were determined in 47 volunteers using the solGMRα 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 solGMRα levels fell within each 5 pmol range.

Fig. 7.

Distribution of plasma solGMR levels in healthy donors.

Plasma solGMRα levels were determined in 47 volunteers using the solGMRα 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 solGMRα levels fell within each 5 pmol range.

Close modal

Characterization of circulating solGMR

To further characterize the nature of the circulating moiety detected by the solGMRα ELISA in human plasma, 1000 mL of human plasma were first subjected to immunoaffinity chromatography with the SCO4 anti-GMRα 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 solGMRα. 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 solGMRα ELISA. Figure 8B demonstrates that solGMRα 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 solGMRα in the circulation, which is in reasonable agreement with the size of recombinant solGMRα6 and with the solGMRα species detected by Western blot analysis of material conditioned by human leukemic cell lines, PBSC products, and freshly isolated human neutrophils.

Fig. 8.

Characterization of solGMR derived from human plasma.

1 L plasma was applied to an anti-GMRα 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-GMRα 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 solGMRα ELISA. The elution position of molecular weight markers is shown in kilodaltons above the graph.

Fig. 8.

Characterization of solGMR derived from human plasma.

1 L plasma was applied to an anti-GMRα 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-GMRα 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 solGMRα ELISA. The elution position of molecular weight markers is shown in kilodaltons above the graph.

Close modal

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 solGMRα ELISA. As shown in Table4, circulating levels of solGMRα were elevated in 4 of the 5 cases of AML and in 1 patient with MM.

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

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

The availability of the solGMRα ELISA has afforded the opportunity to establish a range of normal values for plasma levels of solGMRα. Our findings indicate that solGMRα 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 solGMRα found in plasma conditioned by the PBSC products must keep in mind this range of solGMRα in normal plasma. Seven of the 8 PBSC products examined by ELISA had solGMRα 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 1012hematopoietic cells, there was little or no increase of solGMRα above normally circulating concentrations. This finding brings into question whether this very large number of hematopoietic cells were producing any significant amount of solGMRα during the collection period and begs the more general question of what cells are the source of the solGMRα detected in plasma. It is possible that the source of circulating solGMRα 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 solGMRα, whereas hematopoietic cells produce solGMRα 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 solGMRα, 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 solGMRα by the cells that collect during the leukapheresis procedure.

The availability of the solGMRα ELISA has greatly simplified certain avenues of study of solGMRα; however, the performance of ligand affinity and gel filtration chromatography, saturation binding assays, and Western blot analyses has facilitated substantial characterization of the soluble GM-CSF receptor we have identified in vivo. SolGMRα found in vivo binds to and elutes from a GM-CSF ligand affinity column in the same manner as recombinant solGMRα. In addition, immunoaffinity purification of 1 L of human plasma, using an anti-GMRα antibody, leads to the isolation of a molecule that, on Western analysis with the same anti-GMRα antibody, demonstrates a very similar electrophoretic mobility to recombinant solGMRα (Figure8A). On gel filtration, this plasma derived solGMRα also displays the same size characteristics as recombinant solGMRα (Figure 8B). As well, saturation binding analysis demonstrates that naturally occurring and recombinant solGMRα have identical binding affinities for GM-CSF (Figures 2 and 4).

Examination of some of our data suggests that the story may not be completely told. For instance, there is some variation in the electrophoretic mobility of solGMRα from different sources. Comparison of binding data and corresponding Western blots in Figure 1and Figure 3 also shows that there can be discrepancies between the intensity of the solGMRα immunologic signal and the degree of binding when comparing different cellular sources of solGMRα. However, the linear relationship between immunologic signal strength and binding is preserved when the cellular sources are examined individually (Figure6). The reasons for these differences between cellular sources are unclear, but it suggests modification of solGMRα occurs in a cell specific manner and seems even to vary from one individual to another. Previous work has demonstrated glycosylation of solGMRα,6which gives rise to considerable size heterogeneity and which may contribute to differences in immunologic recognition, depending on the antibody in use. It is also possible that at least some of the solGMRα found in vivo arises by proteolytic cleavage of the membrane anchored GMRα rather than by secretion of the soluble product predicted by the alternatively spliced mRNA. This would lead to a very slightly smaller protein product, although not enough to account for all the observed size discrepancies. Previous work has documented that recombinant solGMRα can oligomerize in solution, but these studies have also shown that it is the monomeric form that binds ligand.17 Thus, some of the size discrepancies may be explained by oligomers of solGMRα, but currently available data would suggest that these forms are not responsible for the lack of concordance between band intensity and binding.

Although the experiments in this article provide evidence that solGMRα is a physiologic molecule in vivo, we also provide data that solGMRα plays a role in pathology. Elevated circulating levels of solGMRα were identified in a small series of patients with hematologic malignancies. Of 4 MM patients, 1 had elevated solGMRα levels but the value was only slightly above the upper limit of normal. On the other hand, 4 of the 5 AML patients had elevated levels, and in 3 of the patients the solGMRα value was considerably outside the normal range. No attempt was made in these experiments to document the exact cellular source of solGMRα in the patients, nor can we provide insight into the consequence of such elevated levels of solGMRα on disease progression. It would seem, however, that at least in a subset of patients with myeloid leukemia and perhaps other hematologic malignancies, solGMRα levels could serve as a marker of disease. It remains to be seen whether the molecule is actually contributing to the development or progression of the disease or is a response of nonleukemic cells to the presence of the disease. Further insight will require examination of a much larger and more comprehensive series of patients.

The potential for cytokine receptors to exist in soluble form has been known for some time now. For many of the cytokine receptors, the initial evidence for the production of soluble isoforms rested on the identification of variant mRNA that predicted the production of a soluble receptor product. Direct evidence of the existence of the soluble receptor at the protein level in vivo in humans exists for a smaller subset of the cytokine receptors. This includes the soluble isoforms of the Growth Hormone Receptor,32,33IL-2R,34 IL-6R,35 EPOR,36CNTFR,37 LIFR,38 gp13 0,39 and IL-5R (F. Montero-Julian, manuscript submitted). Our current article provides direct evidence that solGMRα is also produced in vivo in humans and that it is produced by cells of hematopoietic origin. In addition, we provide evidence that the soluble GM-CSF receptor has a role to play in both physiologic and pathophysiologic states. The challenge now becomes to understand the spectrum of physiologic and pathologic conditions in which solGMRα is altered and to gain insight into the mechanisms of the control of production of solGMRα at the protein level.

We wish to recognize the outstanding technical contributions of Carin Pihl and the secretarial support of Valerie Gilbertson. We also thank Dr Steve Robbins, University of Calgary, for supplying some of the cell line supernatants.

Supported by grants from the Medical Research Council of Canada and the Arthritis Society of Canada. F.S. is the recipient of an Alberta Cancer Board Studentship.

Reprints:Christopher B. Brown, Room 2880, Health Sciences Center, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta, Canada, T2N 4N1.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Heaney
ML
Golde
DW
Soluble cytokine receptors.
Blood.
87
1996
847
857
2
Heaney
ML
Golde
DW
Soluble receptors in human disease.
J Leukocyte Biol.
64
1998
135
146
3
Ashworth
A
Kraft
A
Cloning of a potentially soluble receptor for human GM-CSF.
Nucleic Acids Res.
18
1990
7178
4
Raines
MA
Liu
L
Quan
SG
Joe
V
DiPersio
JF
Golde
DW
Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.
Proc Natl Acad Sci U S A.
88
1991
8203
8207
5
Nakagawa
Y
Kosugi
H
Miyajima
A
Arai
K
Yokota
T
Structure of the gene encoding the alpha subunit of the human granulocyte-macrophage colony stimulating factor receptor. Implications for the evolution of the cytokine receptor superfamily.
J Biol Chem.
269
1994
10,905
10,912
6
Brown
CB
Beaudry
P
Laing
TD
Shoemaker
S
Kaushansky
K
In vitro characterization of the human recombinant soluble granulocyte-macrophage colony-stimulating factor receptor.
Blood.
85
1995
1488
1495
7
Williams
WV
VonFeldt
JM
Rosenbaum
H
Ugen
KE
Weiner
DB
Molecular cloning of a soluble form of the granulocyte-macrophage colony-stimulating factor receptor alpha chain from a myelomonocytic cell line. Expression, biologic activity, and preliminary analysis of transcript distribution.
Arthritis Rheum.
37
1994
1468
1478
8
Sasaki
K
Chiba
S
Mano
H
Yazaki
Y
Hirai
H
Identification of a soluble GM-CSF binding protein in the supernatant of a human choriocarcinoma cell line.
Biochem Biophys Res Commun.
183
1992
252
257
9
Russell
JA
Luider
J
Weaver
M
et al
Collection of progenitor cells for allogeneic transplantation from peripheral blood of normal donors.
Bone Marrow Transplant.
15
1995
111
115
10
Stewart
DA
Gyonyor
E
Paterson
AH
et al
High-dose melphalan +/- total body irradiation and autologous hematopoietic stem cell rescue for adult patients with Ewing's sarcoma or peripheral neuroectodermal tumor.
Bone Marrow Transplant.
18
1996
315
318
11
Woodman
RC
Reinhardt
PH
Kanwar
S
Johnston
FL
Kubes
P
Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels.
Blood.
82
1993
2188
2195
12
Murray
EW
Pihl
C
Morcos
A
Brown
CB
Ligand-independent cell surface expression of the human soluble granulocyte-macrophage colony-stimulating factor receptor alpha subunit depends on co-expression of the membrane-associated receptor beta subunit.
J Biol Chem.
271
1996
15,330
15,335
13
Byrne
PV
Human myeloid cells possessing high-affinity receptors for granulocyte-macrophage colony stimulating factor.
Leuk Res.
13
1989
117
126
14
Gasson
JC
Kaufman
SE
Weisbart
RH
Tomonaga
M
Golde
DW
High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells.
Proc Natl Acad Sci U S A.
83
1986
669
673
15
Park
LS
Waldron
PE
Friend
D
et al
Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness.
Blood.
74
1989
56
65
16
Heaney
ML
Vera
JC
Raines
MA
Golde
DW
Membrane-associated and soluble granulocyte/macrophage-colony-stimulating factor receptor alpha subunits are independently regulated in HL-60 cells.
Proc Natl Acad Sci U S A.
92
1995
2365
2369
17
Brown
CB
Pihl
CE
Murray
EW
Oligomerization of the soluble granulocyte-macrophage colony- stimulating factor receptor: identification of the functional ligand- binding species.
Cytokine.
9
1997
219
225
18
Murray
EW
Pihl
C
Robbins
SM
et al
The soluble granulocyte-macrophage colony-stimulating factor receptor's carboxyl-terminal domain mediates retention of the soluble receptor on the cell surface through interaction with the granulocyte-macrophage colony-stimulating factor receptor beta-subunit.
Biochemistry.
37
1998
14,113
14,120
19
Prevost JM, Farrell PJ, Iatrou K, Brown CB. Determinants of the functional interaction between the soluble GM-CSF receptor and the GM-CSF receptor α-subunit. Cytokine. 2000 (in press).
20
Wognum
AW
Westerman
Y
Visser
TP
Wagemaker
G
Distribution of receptors for granulocyte-macrophage colony-stimulating factor on immature CD34+ bone marrow cells, differentiating monomyeloid progenitors, and mature blood cell subsets.
Blood.
84
1994
764
774
21
Lanza
F
Castagnari
B
Rigolin
G
et al
Flow cytometry measurement of GM-CSF receptors in acute leukemic blasts, and normal hemopoietic cells.
Leukemia.
11
1997
1700
1710
22
DiPersio
JF
Hedvat
C
Ford
CF
Golde
DW
Gasson
JC
Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.
J Biol Chem.
266
1991
279
286
23
Santiago-Schwarz
F
Divaris
N
Kay
C
Carsons
SE
Mechanisms of tumor necrosis factor-granulocyte-macrophage colony-stimulating factor-induced dendritic cell development.
Blood.
82
1993
3019
3028
24
Emile
JF
Fraitag
S
Andry
P
et al
Expression of GM-CSF receptor by Langerhans' cell histiocytosis cells.
Virchows Arch.
427
1995
125
129
25
Till
KJ
Burthem
J
Lopez
A
Cawley
JC
Granulocyte-macrophage colony-stimulating factor receptor: stage-specific expression and function on late B cells.
Blood.
88
1996
479
486
26
Colotta
F
Bussolino
F
Polentarutti
N
et al
Differential expression of the common beta and specific alpha chains of the receptors for GM-CSF, IL-3, and IL-5 in endothelial cells.
Exp Cell Res.
206
1993
311
317
27
Baldwin
GC
Gasson
JC
Kaufman
SE
et al
Nonhematopoietic tumor cells express functional GM-CSF receptors.
Blood.
73
1989
1033
1037
28
Baldwin
GC
Golde
DW
Widhopf
GF
Economou
J
Gasson
JC
Identification and characterization of a low-affinity granulocyte-macrophage colony-stimulating factor receptor on primary and cultured human melanoma cells.
Blood.
78
1991
609
29
Zhao
Y
Rong
H
Chegini
N
Expression and selective cellular localization of granulocyte- macrophage colony-stimulating factor (GM-CSF) and GM-CSF alpha and beta receptor messenger ribonucleic acid and protein in human ovarian tissue.
Biol Reprod.
53
1995
923
930
30
Rokhlin
OW
Griebling
TL
Karassina
NV
Raines
MA
Cohen
MB
Human prostate carcinoma cell lines secrete GM-CSF and express GM-CSF- receptor on their cell surface.
Anticancer Res.
16
1996
557
563
31
Rivas
CI
Vera
JC
Delgado-Lopez
F
et al
Expression of granulocyte-macrophage colony-stimulating factor receptors in human prostate cancer.
Blood.
91
1998
1037
1043
32
Baumann
G
Stolar
MW
Amburn
K
Barsano
CP
DeVries
BC
A specific growth hormone-binding protein in human plasma: initial characterization.
J Clin Endocrinol Metab.
62
1986
134
141
33
Herington
AC
Ymer
S
Stevenson
J
Identification and characterization of specific binding proteins for growth hormone in normal human sera.
J Clin Invest.
77
1986
1817
1823
34
Rubin
LA
Kurman
CC
Fritz
ME
et al
Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro.
J Immunol.
135
1985
3172
3177
35
Honda
M
Yamamoto
S
Cheng
M
et al
Human soluble IL-6 receptor: its detection and enhanced release by HIV infection.
J Immunol.
148
1992
2175
2180
36
Baynes
RD
Reddy
GK
Shih
YJ
Skikne
BS
Cook
JD
Serum form of the erythropoietin receptor identified by a sequence-specific peptide antibody.
Blood.
82
1993
2088
2095
37
Davis
S
Aldrich
TH
Ip
NY
et al
Released form of CNTF receptor alpha component as a soluble mediator of CNTF responses.
Science.
259
1993
1736
1739
38
Zhang
JG
Zhang
Y
Owczarek
CM
et al
Identification and characterization of two distinct truncated forms of gp130 and a soluble form of leukemia inhibitory factor receptor alpha-chain in normal human urine and plasma.
J Biol Chem.
273
1998
10,798
10,805
39
Narazaki
M
Yasukawa
K
Saito
T
et al
Soluble forms of the interleukin-6 signal-transducing receptor component gp130 in human serum possessing a potential to inhibit signals through membrane-anchored gp130.
Blood.
82
1993
1120
1126
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