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Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Inflammatory Stimuli Up-Regulate Secretion of the Soluble GM-CSF Receptor in Human Monocytes: Evidence for Ectodomain Shedding of the Cell Surface GM-CSF Receptor Subunit¹

Jay M. Prevost^2,*, Jennifer L. Pelley^2,*,, Weibin Zhu^*, Gianni E. D’Egidio^*, Paul P. Beaudry, Carin Pihl^*, Graham G. Neely, Emmanuel Claret^¶, John Wijdenes^¶ and Christopher B. Brown^3,*,||

^* Cancer Biology Research Group, Southern Alberta Cancer Research Center, Departments of Biochemistry and Molecular Biology, Surgery, and Medical Science, University of Calgary, Calgary, Alberta, Canada; ^¶ Diaclone Research, Besancon, France; and ^|| Alberta Bone Marrow and Stem Cell Transplant Program

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Soluble GM-CSF receptor subunit (sGMR) is a soluble isoform of the GMR that is believed to arise exclusively through alternative splicing of the GMR gene product. The sGMR mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMR. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMR in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMR arose exclusively through alternative splicing of the GMR gene product, or whether it could also be generated through ectodomain shedding of GMR, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMR (Ba/F3.GMR). The Ba/F3.GMR cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMR-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMR (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMR. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMR by monocytes, suggesting that shedding of GMR by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMR is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMR secretion, and that sGMR arises not only through alternative splicing but also through ectodomain shedding of cell surface GMR.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
GM-CSF is a multifunctional cytokine that mediates its hematopoietic and inflammatory activities (1) through a membrane-spanning cell surface receptor that consists of a ligand-binding subunit, GM-CSF receptor subunit (GMR)⁴ (2), and a signal-transducing subunit, c (3). GMR is a 378-aa protein that consists of a 298-aa extracellular domain, a 26-aa membrane-spanning domain, and a 54-aa cytoplasmic domain (2). GMR is also expressed as an alternatively spliced soluble isoform, soluble GMR (sGMR). sGMR arises through alternative splicing of the parental GMR gene product (4, 5, 6, 7). Exclusion of the exon that encodes the transmembrane domain of GMR results in translation of a protein that is missing both the membrane-spanning domain of GMR and the cytoplasmic domain, but that retains the 295-aa GMR ectodomain. In addition, the alternative splicing event generates a frameshift in the coding sequence that results in the addition of 16-aa to the carboxy terminus of the sGMR protein.

sGMR has been cloned and expressed as a recombinant protein (5, 6, 7). Recombinant sGMR is a heavily glycosylated 60-kDa protein that binds GM-CSF in solution with the same affinity as cell surface GMR (K_d = 2–10 nM) (2, 7). However, unlike cell surface GMR, which binds GM-CSF then associates with c, an exogenous source of sGMR added to c-expressing cells will not associate with c, whether in the presence or absence of GM-CSF (8, 9). Instead, sGMR antagonizes the function of GM-CSF in vitro by binding to it and preventing it from interacting with the cell surface GMR/c complex (7). sGMR has also been shown to inhibit the proliferation of GM-CSF-dependent cell lines (5, 6, 10) and to inhibit bone marrow colony formation (7).

The alternatively spliced GMR message is expressed by a variety of cell types, including human placental tissue (7), myeloid leukemic cell lines (5, 6, 11), bone marrow progenitors, monocyte/macrophages, and synovial fibroblasts (6). Less is known about the expression of the alternatively spliced sGMR protein. A soluble GM-CSF binding protein was identified in medium conditioned by a human choriocarcinoma cell line (JEG-3) (12), but no immunological or biochemical data were presented as to whether this protein was equivalent to alternatively spliced sGMR. However, a 60-kDa soluble GM-CSF binding protein was recently identified in medium conditioned by human myeloid leukemic cell lines (U937, THP-1, and HL-60), by normal human granulocytes, and in normal human plasma (13). This protein migrated as a 60-kDa band by SDS-PAGE, was recognized by a mAb that was raised against the ectodomain of GMR, and bound GM-CSF with the same affinity as recombinant alternatively spliced sGMR, suggesting that these two proteins were equivalent.

We were interested in determining whether normal human monocytes, which express both cell surface GMR and the mRNA for sGMR, could also secrete the alternatively spliced sGMR protein. We were also interested in determining whether the secretion of alternatively spliced sGMR by monocytes could be regulated by GM-CSF and other stimuli. Using an ELISA that recognized all soluble isoforms of GMR, we found that monocytes but not lymphocytes could constitutively secrete a sGMR-like protein. We also found that stimulation of monocytes with GM-CSF and mediators such as LPS, PMA, and the calcium ionophore A23187 rapidly up-regulates the secretion of this sGMR-like protein. Importantly, this protein did not arise exclusively through the alternative splicing of GMR, but was also derived through ectodomain shedding of cell surface GMR, demonstrating for the first time that sGMR can arise through both alternative splicing and proteolytic cleavage of cell surface GMR.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokines, reagents, and cell culture

Recombinant human GM-CSF (a gift of Cangene, Mississauga, ON, Canada) and G-CSF (Amgen Canada, Mississauga, ON, Canada) were reconstituted in sterile PBS. LPS (Escherichia coli serotype 0111:B4; Sigma-Aldrich, St. Louis, MO) was reconstituted in PBS. PMA (Sigma-Aldrich) and A23187 (Calbiochem, La Jolla, CA) were solubilized in DMSO (Sigma-Aldrich) and diluted in PBS. The broad-spectrum hydroxamic acid-based inhibitor of metalloprotease activity (BB94; British Biotechnology, Oxford, U.K.) was solubilized in DMSO and used directly. Recombinant alternatively spliced sGMR was purified from medium conditioned by a baby hamster kidney fibroblast cell line engineered to express the alternatively spliced sGMR cDNA (BHK.sGMR) as previously described (7). Similarly, a recombinant isoform of sGMR that was missing the unique 16-aa C-terminal domain of alternatively spliced sGMR was purified from medium conditioned by the BHK.ECD cell line (9, 14). Unless otherwise indicated, experiments were performed in sterile 1.5-ml Eppendorf tubes in HEPES-modified RPMI 1640 (Sigma-Aldrich) containing 1% penicillin/streptomycin (Life Technologies, Rockville, MD) and supplemented with 10% heat-inactivated FBS (Sigma-Aldrich) (complete medium) in a 37°C incubator containing 5% CO₂.

Cell isolation

Blood was collected from healthy volunteers by venipuncture after informed consent. Blood was collected into vacuum containers containing sodium heparin (Vacutainer; BD Biosciences, Mountain View, CA). PBMCs were isolated by density centrifugation of diluted blood over a cushion of Ficoll (Ficoll-Paque Plus; Pharmacia, Peapack, NJ) followed by osmotic lysis of residual erythrocytes. PBMC viability was typically 98% and contaminating granulocytes accounted for <3% of cells as assessed by light scatter and flow cytometry. For lymphocyte isolation, PBMCs were depleted of monocytes by plating them on polystyrene petri dishes. The nonadherent cells were decanted and the procedure was repeated a total of three times. The lymphocyte preparations contained <1% CD14⁺ cells as assessed by flow cytometry. Monocytes were isolated from whole blood using a negative selection procedure (RosetteSep Monocyte Isolation Kit; StemCell Technologies, Vancouver, British Columbia, Canada) and density centrifugation over Ficoll. The enriched mononuclear cell layer typically consisted of 75–85% CD14⁺ monocytes.

Flow cytometry

The percentage of monocytes in either the PBMC preparations or the monocyte-enriched/depleted preparations was determined by CD14 staining and FACS. Briefly, 10⁶ cells were stained for 15 min on ice with 0.5 µg of a FITC-labeled mouse anti-human CD14 mAb or an IgG2a-FITC isotype control mAb (BD PharMingen, San Diego, CA). The cells were washed and fixed in PBS containing 1% PBS-buffered formalin. Data were acquired using a FACStation flow cytometer (BD Biosciences) and were analyzed using Flowjo (Treestar, San Carlos, CA). The expression of GMR on the cell surface was determined in a similar manner using 1 µg of a mouse anti-human GMR mAb (8G6; a gift of Dr. A. Lopez, Hansen Center for Cancer Research, Adelaide, Australia) or an equivalent murine IgG1 isotype control (Sigma-Aldrich), followed by staining with 0.5 µg of a PE-labeled F(ab')₂ goat anti-mouse Ab (Calbiochem).

Detection of total sGMR protein by ELISA

Supernatants from cell culture experiments were screened by ELISA for the presence of all sGMR protein (sCD116 ELISA; Diaclone Research, Besancon, France). The sCD116 ELISA uses a capture mAb raised against the ectodomain of GMR (SCO6), whereas the detection mAb (SCO4) was also raised against the ectdomain of GMR. Because of this, the sCD116 ELISA is not specific for alternatively spliced sGMR but instead recognizes any sGMR isoform that shares the common ectodomain of GMR. Linear regression analysis of ELISA data was performed using Graph Pad (Prism, San Diego, CA). All cultures were performed in duplicate and were measured again in duplicate by ELISA. Experiments were repeated with cells from an individual donor or from different donors, as indicated.

Engineering of the pBabePuro3/GMR retroviral construct

The cDNA for GMR was generated by PCR from the previously described -gt11/GMR clone (7) with primers designed to amplify the entire coding sequence. The blunt-end PCR product was cloned into the pCR-Script Amp SK⁺ cloning vector (Stratagene, La Jolla, CA). The full sequence of the cDNA was confirmed by sequencing and was subsequently subcloned into the XhoI-ClaI sites of the retrovirus expression vector pBabePuro3 (gift of Dr. S. Robbins).

Retroviral infection of the Ba/F3 cell line

The murine IL-3-dependent pro-B cell line Ba/F3 was kindly provided by Dr. K. Kaushansky (University of Washington, Seattle, WA) and was maintained in RPMI 1640 medium (Life Technologies) with 10% FBS supplemented with 1 ng/ml murine IL-3 (BD PharMingen). The 2 ecotropic retroviral packaging cell line was maintained in DMEM (Life Technologies) plus 10% FBS. Retroviral infection was performed using stably transfected 2 packaging cells as follows: 2 cells were transfected with pBabePuro3/GMR using Effectene Reagent (Qiagen, Valencia, CA) and stable transfectants were selected with 3 µg/ml puromycin. These retrovirus-producing 2 cells were grown to subconfluence (5 x 10⁶ cells/10-cm dish), and 10 ml of fresh medium was added. The viral supernatant was harvested 18 h later and filtered through a 0.45-µm membrane. Six microliters of the supernatant was applied to 5 x 10⁵ cells in a 10-cm dish containing 6 ml of fresh medium and 8 µg/ml Polybrene. The infected cells were grown for 48 h and then were selected in medium containing 4 µg/ml puromycin. Cell surface expression of GMR on the Ba/F3.GMR cell line, or lack thereof on the Ba/F3 cell line, was confirmed by flow cytometry.

GM-CSF ligand-affinity chromatography

GM-CSF binding proteins were isolated from medium conditioned by the Ba/F3 cell line (240 ml) or the Ba/F3.GMR cell line (120 ml) using affinity chromatography as previously described (7, 8, 9, 13, 14, 15). Briefly, cell-conditioned medium was passed over a Sepharose 4B column (Pharmacia) that had been coupled to recombinant human GM-CSF. The column was washed extensively with PBS and the adsorbed proteins were eluted with a 0.1 M glycine buffer (pH 2.5). The eluted fractions were immediately neutralized with 1 M Tris buffer. The neutralized fractions were volume reduced to 5 µl by centrifugal filtration (Ultrafree, 5 kDa; Millipore, Bedford, MA) and were analyzed by SDS-PAGE and Western blotting.

SDS-PAGE and Western blotting

The concentrated eluents were boiled in an equal volume of 2x SDS-PAGE loading buffer in the presence of the reducing agent DTT. The solubilized and denatured proteins were electrophoresed on 8% SDS polyacrylamide minigels and transferred to polyvinylidene fluoride membranes (BioTrace PVDF; Pall Corporation, Ann Arbor, MI). The blots were blocked in 5% skim milk in TBST and were probed with a mouse mAb raised against the ectodomain of GMR (8D10; a gift of Dr. A. Lopez) followed by an HRP-conjugated rabbit anti-mouse IgG secondary Ab (Bio/Can Scientific, Mississauga, Ontario, Canada). Proteins on the blots were visualized by ECL (Amersham Life Sciences, Oakville, Ontario, Canada) and exposure to x-ray film.

Preparation of rabbit splicing-specific antiserum against the 16-aa C-terminal tail of alternatively spliced sGMR

Peptide corresponding to the predicted 16-aa sequence of the alternatively spliced sGMR C-terminal tail (LGYSGCSRQFHRSKTN) was synthesized and coupled to the carrier protein KLH by the Peptide Synthesis Core Facility of the University of Calgary. Female New Zealand white rabbits were immunized by bilateral i.m. injection with a total of 50 µg of KLH-conjugated peptide in CFA (200 µl total volume). Animals were boosted every 3–4 wk with an identical dose of peptide in IFA. A total of five injections were administered before the initial test bleed. An affinity column was prepared by covalently attaching non-KLH-coupled 16-aa peptide to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow resin (Pharmacia), according to the manufacturer’s instructions. The rabbit antiserum was then affinity purified by passage first over a Sepharose 4B sham column and then passage over the peptide-conjugated affinity column. Affinity-purified Ab was then eluted from the column using a 0.1 M glycine buffer (pH 2.5) and then immediately neutralized with 1 M Tris.

Statistics

Data from representative and replicate experiments are presented as the mean ± SEM, whereas the significance of the differences between groups was determined by paired or unpaired t test, as applicable to the assay condition, with p < 0.05 being deemed a significant change.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
sGMR is secreted constitutively by monocytes but not by lymphocytes

Mature human monocytes express GMR on their cell surface whereas lymphocytes do not (16, 17, 18), suggesting that monocytes but not lymphocytes might also secrete sGMR. To test this hypothesis, we first confirmed that monocytes could express GMR on their cell surface. PBMCs were stained with an anti-CD14-FITC mAb and an anti-GMR mAb (8G6) or with an IgG1 isotype control and were analyzed by flow cytometry. The majority of the CD14⁺ monocytes, but none of the CD14^- lymphocytes, stained positively with the anti-GMR Ab but not with the IgG1 isotype control, confirming that monocytes but not lymphocytes express GMR on their cell surface (Fig. 1A). To determine whether monocytes could also secrete the sGMR protein, we incubated PBMCs from healthy donors at a density of 10⁶/ml (equivalent to 3 x 10⁵ monocytes/ml) for 24 h in complete medium. The conditioned medium was harvested and assayed for the presence of sGMR by sCD116 ELISA (Diaclone Research). PBMCs from 16 different donors secreted an average of 165 ± 26 pg/ml of sGMR during the 24-h culture period (Fig. 1B). Monocytes were then isolated from PBMCs (3 x 10⁵/ml monocytes) and cultured for 24 h. The monocyte-enriched population secreted 136 ± 58 pg/ml of sGMR during the 24-h culture period, which is consistent with the concentration of sGMR that was secreted by the unfractionated PBMCs, suggesting that it was the monocytes and not the lymphocytes that were secreting sGMR. To confirm this, PBMC preparations were depleted of monocytes by adherence to plastic and were cultured for 24 h. Lymphocytes cultured at 10⁷/ml, a log-fold higher concentration than was used with the unfractionated PBMCs, showed no detectable secretion of sGMR, demonstrating that it is monocytes and not lymphocytes that secrete sGMR.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. sGMR is secreted constitutively by monocytes but not by lymphocytes. A, PBMCs were stained with a FITC-labeled mouse anti-human CD14 mAb and a mouse anti-human GMR mAb (8G6) or an isotype-matched IgG1, followed by a PE-labeled F(ab')2 goat anti-mouse Ab. The cells were analyzed for the cell surface expression of CD14 and GMR by flow cytometry. B, PBMCs (106/ml, n = 16 donors), monocytes (3 x 105/ml, n = 3 donors), or lymphocytes (107/ml, n = 5 donors) were cultured in duplicate for 24 h in complete medium. The supernatants were harvested and screened for the presence of sGMR by sCD116 ELISA. Values shown are the mean ± SEM (ns, not statistically different). PBMCs (106/ml) (C) and monocytes (3 x 105/ml) (D) were cultured in duplicate for 0–96 h and the cell supernatants were screened every 24 h for the presence of sGMR by ELISA. Shown are the means ± SEM. * p < 0.05, as compared with the previous time point; representative of n = 3 individual experiments.

Induction of sGMR secretion by GM-CSF, LPS, PMA, and A23187

GM-CSF, LPS, PMA, and A23187 alter the ability of monocytes and neutrophils to bind GM-CSF on their cell surface, suggesting a role for them in regulating the expression of cell surface GMR (19, 20, 21, 22, 23). Because sGMR arises via alternative splicing of the GMR transcript, it was possible that these stimuli might also regulate the secretion of sGMR. To test this hypothesis, we incubated PBMCs from individual donors with increasing concentrations of GM-CSF, LPS, PMA, or A23187 and analyzed the conditioned medium for the presence of sGMR. GM-CSF increased the secretion of sGMR in a dose-dependent manner, with half-maximal activity occurring at 262 pg/ml (Fig. 2A). LPS also up-regulated sGMR secretion in a dose-dependent manner with 1.5 ng/ml inducing a half-maximal response (Fig. 2B). PMA and A23187 also induced a dose-dependent increase in sGMR secretion with half-maximal induction occurring with 8 ng/ml PMA and 1 ng/ml A23187 (Fig. 2, C and D, respectively). DMSO, used at a maximal dilution of 1/1000 (0.1%) in the dose response experiments, had no effect on sGMR secretion by monocytes (data not shown). These results demonstrate that GM-CSF and other mediators increase the secretion of sGMR by monocytes in a dose-dependent manner.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Induction of sGMR secretion by GM-CSF, LPS, PMA, and A23187. PBMCs were incubated for 1 h with increasing concentrations (log-fold dilutions) of GM-CSF (A), LPS (B), PMA (C), or A23187 (D). The conditioned medium was harvested and screened for the presence of sGMR by ELISA. The results are expressed as the level of induction of sGMR secretion (pg/ml) above the unstimulated control. Shown are the means ± SEM for one representative of n = 2 individual experiments (*, p < 0.05, as compared with unstimulated control). E, PBMCs were incubated for 10, 20, or 30 min with or without 100 ng/ml PMA, and the conditioned medium was screened for the presence of sGMR using the sCD116 ELISA. Shown are the mean ± SEM of data obtained from five independent experiments using five different donors (*, p < 0.05, meaning that there is a significant difference between the PMA-stimulated and unstimulated control at this time point).

Assay development for the specific detection of alternatively spliced sGMR

sGMR was believed to arise exclusively through alternative splicing of the GMR gene product (4, 5, 6, 7). However, the rapid secretion of sGMR from monocytes in response to stimulation with PMA (Fig. 2E) suggested that sGMR may arise in part through a mechanism other than de novo translation of the alternatively spliced mRNA. Because available Abs were not specific for the alternatively spliced sGMR protein but instead recognized the shared ectodomain of GMR, we endeavored to produce an Ab that was specific for the alternatively spliced sGMR isoform. To this end, we immunized rabbits with a 16-aa peptide that corresponded to the unique C-terminal tail of the alternatively spliced sGMR protein. The rabbit antiserum was purified by affinity chromatography using the 16-aa peptide as a ligand. The specificity of the purified antiserum (splicing-specific antiserum for alternatively spliced sGMR was then tested by SDS-PAGE and immunoblotting. Both recombinant alternatively spliced sGMR protein, which contains the 16-aa C-terminal domain, and recombinant sGMR lacking the 16-aa C-terminal tail (non-alternatively spliced) were recognized by a mAb (8D10) that was raised against the shared ectodomain of GMR (Fig. 3A). Neither the alternatively spliced nor the non-alternatively spliced recombinant sGMR proteins were recognized using rabbit preimmune serum, and only the alternatively spliced sGMR protein was recognized with the splicing-specific antiserum (Fig. 3A). Furthermore, preincubating the splicing-specific antiserum with an excess of the 16-aa peptide Ag (splicing specific + peptide) completely inhibited the ability of the splicing-specific antiserum to recognize alternatively spliced sGMR. Taken together, these results demonstrate that the rabbit splicing-specific antiserum specifically recognizes only the alternatively spliced sGMR isoform, which contains the unique 16-aa C-terminal domain.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Production of rabbit antiserum against the 16-aa C-terminal domain of alternatively spliced sGMR. A, Recombinant sGMR protein containing the 16-aa C-terminal domain (A, alternatively spliced) or a recombinant version of sGMR that is missing the 16-aa epitope (NA, non-alternatively spliced) was fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for immunoblotting. The blots were probed with a mAb raised against the shared domain of cell surface GMR (8D10), rabbit preimmune serum, affinity-purified alternatively spliced sGMR rabbit antiserum (splicing-specific antiserum), or rabbit antiserum that was preincubated with an excess of the 16-aa peptide used as Ag (splicing specific + peptide). B, 1 ng/ml purified recombinant alternatively spliced (A) or non-alternatively spliced (NA) sGMR protein was loaded onto a SCO6-coated ELISA plate. Total sGMR protein was detected using the biotinylated mAb SCO4, whereas alternatively spliced sGMR protein was detected using biotinylated splicing-specific antiserum.

sGMR is released by stimulated monocytes through both alternative splicing and proteolytic cleavage

Because the presence of alternatively spliced sGMR mRNA in monocytes had previously been demonstrated (6), we anticipated that the sGMR protein being produced by monocytes would represent the alternatively spliced isoform. However, the rapid up-regulation of sGMR secretion by PBMCs in response to stimulation led us to hypothesize that monocytes might also secrete a sGMR variant generated through ectodomain shedding. With the development of the splicing-specific ELISA, we were now able to test this hypothesis. Because most known sheddases are metalloproteases (24), we decided to treat unstimulated and stimulated monocytes with the broad-spectrum metalloprotease inhibitor BB94 (batimastat) and to look at the effect of this inhibitor on the secretion of total and alternatively spliced sGMR by monocytes. To this end, we pretreated isolated PBMCs (from n = 7 individual donors) for 2 h at 37°C with 100 µM of either the metalloprotease inhibitor BB94 or the appropriate vehicle control (1% DMSO), pelleted the cells, and resuspended them in fresh medium containing either 1% DMSO or 100 µM BB94. Cell viability was assessed by trypan blue dye exclusion, and no significant difference between the viability of the DMSO- or BB94-treated cells was noted (data not shown). The PBMCs were then either left unstimulated or stimulated for an additional hour using concentrations of PMA, A23187, LPS, or GM-CSF, which were predicted to induce maximal sGMR secretion based on the dose response curves in Fig. 2, A–D. This conditioned medium was then collected and screened in parallel on three different ELISAs: the Diaclone sCD116 ELISA was used to quantitate total sGMR levels (Fig. 4A), the splicing-specific ELISA was used to look at levels of only the alternatively spliced sGMR (Fig. 4B), and the samples were also screened on an IL-8 ELISA. Importantly, treatment of PBMCs with 100 µM BB94 led to a significant decrease in the amount of total sGMR secreted constitutively (p = 0.02) and in response to PMA (p = 0.004), A23187 (p = 0.001), and LPS (p < 0.001), but it did not have a significant effect on the amounts of secreted alternatively spliced sGMR (Fig. 4B) or IL-8 (data not shown). The lack of effect on either alternatively spliced sGMR or IL-8 suggests that BB94 does not have a nonspecific effect on cell viability or protein secretion by PBMCs. This implies that a metalloprotease-mediated cleavage of GMR from the surface of human monocytes can lead to the generation of a shed sGMR variant.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. BB94 inhibits secretion of total sGMR but not alternatively spliced sGMR by PBMCs. PBMCs from healthy donors were resuspended in complete medium at a concentration of 1–2 x 107 cells/ml and were incubated for 2 h at 37°C in the presence of either 1% (v/v) DMSO (vehicle control) or 100 µM BB94. Cells were then pelleted by centrifugation and resuspended at a concentration of 107/ml in fresh medium containing either DMSO or BB94. PBMCs were then incubated for an additional hour in the absence of stimulus (control) or in the presence of 100 ng/ml PMA, 1 µg/ml A23187, 1 µg/ml LPS, or 100 ng/ml GM-CSF. Cells were pelleted and conditioned medium was collected and then screened by ELISA to quantitate either the amount of total sGMR using the Diaclone sCD116 ELISA (A) or the amount of alternatively spliced sGMR using the splicing-specific ELISA described in this paper (B). Pooled results from n = 7 individual donors are presented as percent change compared with the unstimulated 1% DMSO vehicle control. *, p < 0.05, comparing DMSO control- and BB94-treated conditions; ns, not statistically different.

Up-regulated secretion of both alternatively spliced and total sGMR in response to stimulation

In Fig. 4, one can also compare the effect of the various stimuli (PMA, A23187, LPS, and GM-CSF) on the regulated secretion of either the alternatively spliced or shed sGMR variants. When looking at the production of total sGMR in Fig. 4A, we note that stimulation of DMSO control PBMCs led to a significant up-regulation of total sGMR secretion (vs unstimulated cells) in the case of PMA (p = 0.02), A23187 (p = 0.03), and LPS (p = 0.05), whereas treatment with GM-CSF did not. Importantly, pretreatment of PBMCs with BB94 appears to have abrogated the ability of these cells to up-regulate secretion of total sGMR in response to LPS. Therefore, there was no significant difference between the amount of total sGMR secreted by the BB94-treated unstimulated control and LPS-treated cells (Fig. 4A). In contrast, production of total sGMR by the PMA- and A23187-stimulated BB94-treated PBMCs remained significantly up-regulated vs the unstimulated control (p = 0.01 and p = 0.02, respectively). In contrast, when looking at the production of alternatively spliced sGMR by monocytes (Fig. 4B), we note that alternatively spliced soluble receptor levels were also induced in response to both PMA (p = 0.03) and A23187 (p = 0.003), but not in response to either LPS or GM-CSF.

Ectodomain shedding of GMR

The observed inhibition of sGMR secretion by PBMCs in response to treatment with a metalloprotease inhibitor strongly suggested that a sGMR variant could arise through ectodomain shedding. To confirm that shedding of cell surface GMR could occur, we engineered a murine pro-B cell line (Ba/F3) to express the cDNA for human transmembrane GMR (Ba/F3.GMR). Because this is a murine cell line transfected with human cDNA, the Ba/F3.GMR cell line was incapable of secreting human alternatively spliced sGMR.

Expression of GMR on the cell surface of the Ba/F3.GMR cell line, but not on the surface of the parental Ba/F3 cell line, was confirmed by flow cytometry using the anti-GMR mAb 8G6 or an IgG1 isotype control (Fig. 5A). The Ba/F3 and the Ba/F3.GMR cell lines were then cultured at a density of 10⁷/ml in complete medium for 2 h. The cell-conditioned medium was harvested and screened for the presence of total sGMR protein by sCD116 ELISA. There was no detectable sGMR in the Ba/F3 conditioned medium, but the Ba/F3.GMR cell line secreted 157 ± 13 pg/ml sGMR (Fig. 5B), demonstrating that the ectodomain of GMR could be shed from the cell surface. We were also interested in determining whether the ectodomain shedding of sGMR from the Ba/F3.GMR cell line could also be induced using the stimuli shown previously to up-regulate sGMR secretion by monocytes. The Ba/F3 cell line is not LPS-responsive (25) and lacks the _c subunit of the human GM-CSF receptor. Therefore, we looked only at the effect of PMA and A23187 on the shedding of sGMR from the Ba/F3.GMR cell line. Neither stimulus appeared to have an effect on the shedding of sGMR from the surface of these cells (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Ectodomain shedding of GMR from a GMR-expressing recombinant cell line. A, Ba/F3 and Ba/F3.GMR cells were stained with a mouse anti-human GMR mAb (8D10) or an isotype-matched IgG1, followed by PE-labeled F(ab')2 goat anti-mouse Ab. The cells were analyzed for the cell surface expression of GMR by flow cytometry. B, The Ba/F3 and the Ba/F3.GMR cell lines were cultured in duplicate at 107/ml in complete medium containing 1 ng/ml murine IL-3 for 2 h. The conditioned medium was harvested and screened for the presence of sGMR by ELISA. Shown is the mean ± SEM of three independent experiments (*, p < 0.05). C, Ba/F3.GMR cells were incubated at 37°C for 2 h at a concentration of 107/ml in complete medium supplemented with murine IL-3 in the presence of 1% DMSO (vehicle control) or 100 µm BB94, and then the cells were pelleted and resuspended in fresh medium containing DMSO or BB94 and incubated in triplicate for an additional hour. The conditioned medium was then screened for the presence of the shed sGMR variant using the Diaclone sCD116 ELISA. Results shown are the mean ± SEM (*, p < 0.05). D and E, Ba/F3 (240 ml) or Ba/F3.GMR (120 ml) conditioned medium was passed over a GM-CSF-Sepharose 4B affinity column. The column was washed extensively with PBS and the adsorbed proteins were eluted with a 0.1 M glycine buffer (pH 2.5). The eluted fractions were pH neutralized, volume reduced, and fractionated by SDS-PAGE, along with recombinant alternatively spliced sGMR. The blot was probed using a mouse anti-human GMR mAb (8D10) (D) and then was stripped and reprobed with the alternatively spliced sGMR splicing-specific antiserum (E).

To determine the molecular mass of the constitutively shed sGMR protein, we attempted to purify the sGMR-like protein out of medium conditioned by the Ba/F3.GMR cell line. To this end, Ba/F3 or Ba/F3.GMR conditioned medium was passed over a GM-CSF-Sepharose 4B ligand affinity column and the adsorbed proteins were analyzed by SDS-PAGE and Western blotting. No sGMR-like proteins were purified from the Ba/F3 cell-conditioned medium; however, a distinct protein band was present in the Ba/F3.GMR column eluent (Fig. 5D). This 60-kDa protein was the same size as recombinant alternatively spliced sGMR and was specifically recognized by the anti-GMR mAb (8D10) (Fig. 5D), but not by the anti-alternatively spliced sGMR rabbit splicing-specific antiserum (Fig. 5E), demonstrating that the sGMR protein released from the Ba/F3.GMR cells arose via shedding of cell surface GMR. Importantly, the fact that we were able to enrich this shed sGMR variant using a GM-CSF ligand affinity column suggests that the shed sGMR protein retained the ability to specifically bind to GM-CSF.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The alternatively spliced sGMR mRNA has been identified in many cell types that express GMR, including human placental tissue (4, 7), myeloid leukemic cell lines (5, 11), monocytes, macrophages, bone marrow progenitors, and synovial fibroblasts (6). However, there is little information available about which primary human cells secrete the protein. In this paper, we demonstrate that monocytes constitutively secrete sGMR but lymphocytes do not (Fig. 1). More importantly, we also demonstrate that secretion of sGMR by monocytes can be rapidly up-regulated by GM-CSF, LPS, PMA, and A23187 (Fig. 2). We further describe the production of a rabbit antiserum that specifically recognizes the 16-aa tail of the predicted alternatively spliced sGMR protein (Fig. 3), and using this new "splicing-specific" antiserum in an ELISA, we demonstrate for the first time that monocytes do indeed secrete the predicted alternatively spliced sGMR protein (Fig. 4B). We also demonstrate that the secretion of total sGMR (Fig. 4A), but not of alternatively spliced sGMR (Fig. 4B) or IL-8 (data not shown), can be inhibited using the broad-spectrum metalloprotease inhibitor BB94. Together, these results show for the first time that monocytes secrete sGMR protein that represents a mixed population of alternatively spliced and proteolytically cleaved species.

Monocytes but not lymphocytes express GMR on their cell surface (Fig. 1A), suggesting that monocytes might also secrete sGMR. PBMCs and an equivalent number of purified monocytes secreted similar concentrations of sGMR in vitro, whereas lymphocytes secreted none (Fig. 1B). Secretion of sGMR by human monocytes is consistent with previous findings that demonstrated expression of the alternatively spliced sGMR mRNA in human monocytes (6) and secretion of a sGMR protein by myeloid leukemic cell lines (13). The inability of lymphocytes to secrete sGMR is consistent with the lack of sGMR mRNA in lymphocytes (11) and the absence of GMR expression on the cell surface of normal human lymphocytes (Fig. 1A). Together, these results show that normal human monocytes but not lymphocytes can secrete a sGMR protein.

In the absence of stimulation, human monocytes secreted sGMR for up to 4 days in culture (Fig. 1, C and D), suggesting that monocytes constitutively secrete sGMR. Other soluble cytokine receptors, such as sTNFR-p55/75 (26) and sIL-6R, are also constitutively secreted in vitro by normal human monocytes and monocyte-like cell lines (27, 28). Furthermore, sIL-1RII (29, 30), along with sTNFR-p55 (31), sIL-6R (32), and sGMR (13), are also constitutively present in normal human plasma, suggesting that sGMR may also be constitutively secreted in vivo. The reason for the constitutive secretion of sGMR is unclear. It is possible that sGMR is constitutively secreted to modulate the activity of GM-CSF during homeostasis. However, GM-CSF, like most other cytokines, is secreted in a tightly regulated manner and does not normally circulate in human plasma in the absence of inflammation or disease. Elucidation of the role of sGMR during homeostasis will have to await further investigation.

Stimulation of monocytes with GM-CSF, LPS, PMA, and A23187 up-regulated the secretion of sGMR in a dose-dependent manner (Fig. 2, A–D), demonstrating that sGMR secretion could be regulated by its cognate ligand, proinflammatory stimuli (LPS), and heterogeneous chemical stimuli (PMA and A23187). DMSO, used at a maximum dilution of 1/1000 (0.1%) in the dose response experiments, had no effect on sGMR secretion (data not shown). Together, these results demonstrate that stimulation of monocytes can increase the secretion of sGMR.

The regulation of secretion of a soluble cytokine receptor by its cognate ligand has been demonstrated in vitro and in vivo for TNF- (33, 34, 35), where stimulation of cells with TNF- or i.v. injection of recombinant TNF- into mice leads to the rapid up-regulation of sTNFR-p55/p75 secretion. Although the signal-transduction pathways responsible for ligand-induced secretion of soluble receptors are unclear, it is likely that secretion is initiated after ligand-induced phosphorylation of the cognate cell surface receptor complex. This suggests that soluble cytokine receptors such as sGMR and sTNFR-p55/p75 may play a critical role in modulating the activity of their respective cytokines during inflammation and hematopoiesis. It is interesting to note that recombinant sGMR anatagonizes GM-CSF activity in vitro, preventing GM-CSF-induced cell proliferation (5, 10), bone marrow colony formation (7), and neutrophil functional activity (J.M.P. and C.B.B., unpublished data), suggesting that sGMR secretion in response to GM-CSF stimulation may indeed occur to moderate the activity of GM-CSF during inflammation and/or hematopoiesis.

Proinflammatory mediators such as LPS, along with chemical stimuli such as PMA and A23187 that work through divergent signaling pathways that do not necessarily result in phosphorylation of c, also up-regulated monocyte secretion of sGMR (Fig. 2), demonstrating a direct role for inflammatory mediators in regulating sGMR secretion. LPS, PMA, and A23187 have also been shown to up-regulate the secretion of other soluble cytokine receptors such as sIL-1RII (29, 36), sIL-6R (27, 28), and sTNFR-p55/p75 (27), highlighting a common role for inflammatory mediators in regulating the secretion of not only proinflammatory cytokines, but also their respective soluble cytokine receptors.

The rapid release of sGMR from monocytes in response to stimulation with PMA (Fig. 2E) made us wonder whether sGMR may in fact arise through proteolytic cleavage of cell surface GMR rather than, or in addition to, alternative splicing of the GMR gene product. To this end, we generated rabbit antiserum that specifically recognized the 16-aa C terminus of alternatively spliced sGMR, allowing us to differentiate between sGMR that arose via alternative splicing and sGMR that arose via another mechanism (Fig. 3A). Using this reagent, we developed a splicing-specific ELISA that allowed us to demonstrate conclusively for the first time that monocytes could secrete alternatively spliced sGMR protein (Fig. 4B). However, it also became clear that there was an additional sGMR variant being produced by monocytes. Because metalloprotease-mediated cleavage of cell surface receptors is a common mechanism through which other soluble receptors arise (24, 37, 38), we wondered whether our non-alternatively spliced sGMR protein may also have arisen via metalloprotease-mediated ectodomain shedding of cell surface GMR. Using a broad-spectrum metallprotease inhibitor (BB94), we demonstrated that the amount of total sGMR protein released by monocytes was reduced in the presence of BB94 (vs a 1% DMSO vehicle control) (Fig. 4A). Importantly, release of the alternatively spliced sGMR protein (Fig. 4B) or of IL-8 (data not shown) from PBMC cultures was not inhibited by BB94, demonstrating that the observed inhibition of the non-alternatively spliced sGMR protein production was not due to a nonspecific down-regulation of protein secretion. There was no difference in the viability of the 1% DMSO vehicle control and BB94-treated PBMCs after 2 h. However, the presence of 1% DMSO in PBMC cultures did significantly reduce the amount of sGMR secreted by PBMCs as compared with PBMCs in the absence of DMSO.

We had expected that the sGMR produced constitutively by monocytes would be the product of alternative splicing and that shedding would account for the additional sGMR protein produced in response to stimulation, as has been previously demonstrated for sIL-6R in THP-1 cells (28). Using our new splicing-specific ELISA, we have now demonstrated that alternatively spliced sGMR can be secreted constitutively by monocytes (Fig. 4B). However, both PMA and A23187 treatment led to a statistically significant up-regulation of the alternatively spliced sGMR variant (Fig. 4B), which suggests that additional alternatively spliced sGMR can be inducibly secreted by monocytes upon exposure to chemical stimuli. We conclude that alternatively spliced sGMR can be produced by monocytes both constitutively and in response to stimulation.

Similarly, shedding of GMR from the surface of monocytes also appears to occur constitutively, because BB94 treatment of unstimulated cells led to a decrease in secretion of total sGMR. At present, because of a background signal observed when analyzing PBMC-conditioned medium on the splicing-specific ELISA, the sCD116 and splicing-specific ELISAs can be used to measure relative levels of total and alternatively spliced sGMR, but cannot yet be used to reliably quantitate the amount of shed sGMR being produced by monocytes. It is therefore somewhat more difficult to make conclusions about whether the shedding of GMR from the surface of monocytes is also a regulated process. However, we would argue that because LPS up-regulates the secretion of total sGMR in the absence of BB94 (p = 0.05) but not when BB94 is present, this implies that LPS might be specifically up-regulating the shedding of sGMR. This conclusion is further supported by the observation that LPS did not significantly up-regulate the amount of alternatively spliced sGMR produced by monocytes (Fig. 4B). Therefore, it appears that LPS may be acting to induce total sGMR protein production by specifically up-regulating the proteolytic activity of the relevant sheddase. As mentioned previously, PMA and A23187 both led to the up-regulated secretion of both total and alternatively spliced sGMR by monocytes. On the basis of the data presented in Fig. 4A, we would suggest that the degree of the up-regulated secretion in response to PMA and A23187 seems to be reduced by BB94 treatment. Thus, PMA and A23187 may up-regulate the shedding of GMR from the surface of monocytes in addition to inducing the secretion of alternatively spliced sGMR. However, we are cautious about making conclusions as to the effect of PMA and A23187 on shedding of sGMR until we are able to specifically quantitate the amount of shed sGMR being produced by monocytes or are able to directly measure the effect of PMA and A23187 on the activity of the relevant sheddase.

The ability of GMR to be shed from the cell surface was confirmed using a murine pro-B cell line that we engineered to express cell surface GMR but not alternatively spliced sGMR (Ba/F3.GMR; Fig. 5A). A sGMR-like protein was detected by ELISA in medium conditioned by the Ba/F3.GMR cell line but not from the parental Ba/F3 cells (Fig. 5B) or pBabe vector control cells (data not shown). Importantly, treatment of Ba/F3.GMR cells with BB94, but not with the DMSO vehicle control, led to a complete inhibition of sGMR by these cells (Fig. 5C), confirming that the sGMR released by these cells is generated by proteolytic cleavage rather than by vesicle budding or nonspecific shearing of the receptor from the cell surface.

The sGMR variant was then purified from Ba/F3.GMR-conditioned medium using a GM-CSF-Sepharose affinity column, which indicates that this shed sGMR protein retains the ability to bind specifically to GM-CSF. This was not surprising because we have previously demonstrated that purified recombinant GMR extracellular domain binds to GM-CSF with an affinity comparable to the interaction between GM-CSF and alternatively spliced sGMR (9, 14). Furthermore, the 60-kDa shed Ba/F3.GMR variant was recognized specifically by a mAb raised against the ectodomain of GMR (Fig. 5D), but not by our splicing-specific antiserum (Fig. 5E). The similar electropheretic mobility of the shed and alternatively spliced isoforms (Fig. 5D) is, at least in part, accounted for by the heterogeneous glycosylation of the extracellular domain of GMR (7) that would blur the differences in the length of the primary sequence of the two isoforms. The similar mobility may also provide a hint as to the cleavage site of the shed GMR. The apparent 60-kDa molecular mass of the shed sGMR suggests that the cleavage site lies very close to the membrane. If we assume that the WSSWS motif conserved among cytokine receptors and spanning residues 305–310 in GMR is essential for the presentation and integrity of GMR, then the cleavage may occur between residue 311 and the putative membrane entrance at residue 320. This would predict a relatively small difference between the number of amino acids in either the shed or alternatively spliced receptor variants. Importantly, the fact that the shed and alternatively spliced species are indistinguishable by SDS-PAGE likely explains why we did not detect the second sGMR species when we previously purified sGMR from normal human plasma (13). Determination of the exact cleavage site will await further study.

In conclusion, we have demonstrated that a soluble GM-CSF receptor is secreted constitutively by normal human monocytes but not by lymphocytes, and that GM-CSF and other inflammatory mediators can rapidly up-regulate its secretion. We have developed an Ab that specifically recognizes the predicted alternatively spliced sGMR protein and have used it in an ELISA to demonstrate that monocytes do in fact secrete alternatively spliced sGMR. Furthermore, we have demonstrated for the first time that the ectodomain of cell surface GMR can be shed in vitro and that shedding is mediated in part through the activity of metalloproteases. This dual mechanism of soluble receptor production has also been seen for other cytokine receptors such as sIL-6R, sIL-4R, and the growth hormone receptor (reviewed in Ref. 39). It will be of interest to determine whether the alternatively spliced and shed sGMR proteins have similar or unique biological functions in mediating GM-CSF-induced inflammation and/or hematopoiesis.

	Acknowledgments

 
We thank Laurie Robertson of the Alberta Cancer Board/University of Calgary Flow Cytometry Facility, the staff of the Alberta Cancer Board/University of Calgary Hybridoma Facility, and Diane Teoh for excellent technical assistance. We would also like to thank Dr. Angel Lopez for mAbs 8G6 and 8D10, Dr. Ken Kaushansky for the Ba/F3 cell line, and Dr. Stephen Robbins for thoughtful discussions during this project and in the preparation of this manuscript.

	Footnotes

 
¹ This work was supported by grants from the Canadian Institutes of Health Research and the Arthritis Society of Canada (to C.B.B.). J.L.P. is the recipient of a studentship award from the Canadian Institutes of Health Research.

² J.M.P. and J.L.P. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Christopher B. Brown, Departments of Medicine and Oncology, University of Calgary, Heritage Medical Research Building, Room 311, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail address: cbrown{at}ucalgary.ca

⁴ Abbreviations used in this paper: GMR, GM-CSF receptor subunit; s, soluble.

Received for publication January 16, 2002. Accepted for publication September 17, 2002.

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