title: RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.
abstract: RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool.
  
  
  BACKGROUND
  The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs.
  
  
  METHODS
  Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions.
  
  
  RESULTS
  RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK-RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45(+)CD14(+)alphaVbeta3(+)c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45(+)alphaVbeta3(+)TRACP(+) OCs. Importantly, sorted CD14(+)RANK(high) PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANK(mid) or RANK(low) cells.
  
  
  CONCLUSIONS
  These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity.
authors.affiliation: Department of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Atkins
authors.forename: Panagiota
authors.initials: P
authors.lastname: Kostakis
authors.forename: Cristina
authors.initials: C
authors.lastname: Vincent
authors.forename: Amanda N
authors.initials: AN
authors.lastname: Farrugia
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Houchins
authors.forename: David M
authors.initials: DM
authors.lastname: Findlay
authors.forename: Andreas
authors.initials: A
authors.lastname: Evdokiou
authors.forename: Andrew C W
authors.initials: AC
authors.lastname: Zannettino
mesh_terms: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood* / Q000378:metabolism*; D019070:Cell Lineage / Q000276:immunology; D018212:Giant Cell Tumor of Bone / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000097:blood* / Q000378:metabolism*; D009000:Monocytes / Q000276:immunology; D010010:Osteoclasts / Q000378:metabolism*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D002352:Carrier Proteins; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human