Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Inflammatory Stimuli Up-Regulate Secretion of the Soluble GM-CSF Receptor in Human This information is current as Monocytes: Evidence for Ectodomain of September 25, 2021. Shedding of the Cell Surface GM-CSF Receptor α Subunit Jay M. Prevost, Jennifer L. Pelley, Weibin Zhu, Gianni E. D'Egidio, Paul P. Beaudry, Carin Pihl, Graham G. Neely, Downloaded from Emmanuel Claret, John Wijdenes and Christopher B. Brown J Immunol 2002; 169:5679-5688; ; doi: 10.4049/jimmunol.169.10.5679 http://www.jimmunol.org/content/169/10/5679 http://www.jimmunol.org/ References This article cites 39 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/169/10/5679.full#ref-list-1 Why The JI? Submit online. by guest on September 25, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology 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 3 ʈ Christopher B. Brown * Downloaded from 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 http://www.jimmunol.org/ 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␣ by guest on September 25, 2021 arises not only through alternative splicing but also through ectodomain shedding of cell surface GMR␣. The Journal of Im- munology, 2002, 169: 5679–5688. M-CSF is a multifunctional cytokine that mediates its GMR␣ is also expressed as an alternatively spliced soluble iso- hematopoietic and inflammatory activities (1) through a form, soluble GMR␣ (sGMR␣). sGMR␣ arises through alternative membrane-spanning cell surface receptor that consists of splicing of the parental GMR␣ gene product (4–7). Exclusion of G 4 a ligand-binding subunit, GM-CSF receptor ␣ subunit (GMR␣) the exon that encodes the transmembrane domain of GMR␣ results (2), and a signal-transducing subunit, ␤c (3). GMR␣ is a 378-aa in translation of a protein that is missing both the membrane-span- protein that consists of a 298-aa extracellular domain, a 26-aa ning domain of GMR␣ and the cytoplasmic domain, but that re- membrane-spanning domain, and a 54-aa cytoplasmic domain (2). tains 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 *Cancer Biology Research Group, Southern Alberta Cancer Research Center, De- sGMR␣ protein. partments of †Biochemistry and Molecular Biology, ‡Surgery, and §Medical Science, sGMR␣ has been cloned and expressed as a recombinant protein University of Calgary, Calgary, Alberta, Canada; ¶Diaclone Research, Besancon, ʈ ␣ France; and Alberta Bone Marrow and Stem Cell Transplant Program (5–7). Recombinant sGMR is a heavily glycosylated 60-kDa pro- Received for publication January 16, 2002. Accepted for publication September tein that binds GM-CSF in solution with the same affinity as cell ␣ ϭ 17, 2002. surface GMR (Kd 2–10 nM) (2, 7). However, unlike cell sur- The costs of publication of this article were defrayed in part by the payment of page face GMR␣, which binds GM-CSF then associates with ␤c, an charges. This article must therefore be hereby marked advertisement in accordance exogenous source of sGMR␣ added to ␤c-expressing cells will not with 18 U.S.C. Section 1734 solely to indicate this fact. associate with ␤c, whether in the presence or absence of GM-CSF 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 (8, 9). Instead, sGMR␣ antagonizes the function of GM-CSF in award from the Canadian Institutes of Health Research. vitro by binding to it and preventing it from interacting with the 2 J.M.P. and J.L.P. contributed equally to this work. cell surface GMR␣/␤c complex (7). sGMR␣ has also been shown 3 Address correspondence and reprint requests to Dr. Christopher B. Brown, Depart- to inhibit the proliferation of GM-CSF-dependent cell lines (5, 6, ments of Medicine and Oncology, University of Calgary, Heritage Medical Research Building, Room 311, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. 10) and to inhibit bone marrow colony formation (7). E-mail address: [email protected] The alternatively spliced GMR␣ message is expressed by a va- 4 Abbreviations used in this paper: GMR␣, GM-CSF receptor ␣ subunit; s, soluble. riety of cell types, including human placental tissue (7), myeloid Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 5680 ECTODOMAIN SHEDDING OF GMR␣ IN MONOCYTES leukemic cell lines (5, 6, 11), bone marrow progenitors, monocyte/ sity centrifugation over Ficoll. The enriched mononuclear cell layer typi- ϩ macrophages, and synovial fibroblasts (6). Less is known about the cally consisted of 75–85% CD14 monocytes. ␣ expression of the alternatively spliced sGMR protein. A soluble Flow cytometry GM-CSF binding protein was identified in medium conditioned by a human choriocarcinoma cell line (JEG-3) (12), but no immuno- The percentage of monocytes in either the PBMC preparations or the monocyte-enriched/depleted preparations was determined by CD14 stain- logical or biochemical data were presented as to whether this pro- ing and FACS. Briefly, 106 cells were stained for 15 min on ice with 0.5 tein was equivalent to alternatively spliced sGMR␣. However, a ␮g of a FITC-labeled mouse anti-human CD14 mAb or an IgG2a-FITC 60-kDa soluble GM-CSF binding protein was recently identified in isotype control mAb (BD PharMingen, San Diego, CA). The cells were medium conditioned by human myeloid leukemic cell lines (U937, washed and fixed in PBS containing 1% PBS-buffered formalin. Data were THP-1, and HL-60), by normal human granulocytes, and in normal acquired using a FACStation flow cytometer (BD Biosciences) and were analyzed using Flowjo (Treestar, San Carlos, CA). The expression of human plasma (13). This protein migrated as a 60-kDa band by GMR␣ on the cell surface was determined in a similar manner using 1 ␮g SDS-PAGE, was recognized by a mAb that was raised against the of a mouse anti-human GMR␣ mAb (8G6; a gift of Dr. A. Lopez, Hansen ectodomain of GMR␣, and bound GM-CSF with the same affinity Center for Cancer Research, Adelaide, Australia) or an equivalent murine ␮ as recombinant alternatively spliced sGMR␣, suggesting that these IgG1 isotype control (Sigma-Aldrich), followed by staining with 0.5 gof a PE-labeled F(abЈ) goat anti-mouse Ab (Calbiochem). two proteins were equivalent. 2 We were interested in determining whether normal human Detection of total sGMR␣ protein by ELISA ␣ monocytes, which express both cell surface GMR and the mRNA Supernatants from cell culture experiments were screened by ELISA for for sGMR␣, could also secrete the alternatively spliced sGMR␣ the presence of all sGMR␣ protein (sCD116 ELISA; Diaclone Research, protein. We were also interested in determining whether the se- Besancon, France). The sCD116 ELISA uses a capture mAb raised against Downloaded from cretion of alternatively spliced sGMR␣ by monocytes could be the ectodomain of GMR␣ (SCO6), whereas the detection mAb (SCO4) was also raised against the ectdomain of GMR␣. Because of this, the sCD116 regulated by GM-CSF and other stimuli. Using an ELISA that ␣ ␣ ELISA is not specific for alternatively spliced sGMR but instead recog- recognized all soluble isoforms of GMR , we found that mono- nizes any sGMR␣ isoform that shares the common ectodomain of GMR␣.