Development of a method for the identification of receptor activator of nuclear factor-κB+ populations in vivo

Abstract

ObjectivesOsteoclasts are induced by macrophage colony-stimulating factor-1 (CSF-1) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL). Monocyte/macrophage lineages are thought to be osteoclast precursors; however, such cells have not been fully characterized owing to a lack of tools for their identification. Osteoclast precursors express colony-stimulating factor-1 receptor (CSF-1R) and RANK. However, the capacity of conventional methods using anti-RANK antibodies to detect RANK+ cells by flow cytometry is insufficient. Here, we developed a high-sensitivity method for detecting RANK+ cells using biotinylated recombinant glutathione S-transferase-RANKL (GST-RANKL-biotin). MethodsWe sorted sub-populations of mouse bone marrow (BM) or peripheral blood (PB) cells using GST-RANKL-biotin, anti-CSF1R, and anti-B220 antibodies and induced osteoclastogenesis in vitro. ResultsThe frequency of the RANK+ population in BM detected by GST-RANKL-biotin was significantly higher than that detected by anti-RANK antibodies. Although RANK+ cells were detected in both the B220+ and B220− populations, the macrophage lineage was present only in B220−. Unexpectedly, a significantly higher number of osteoclasts was induced in RANK−CSF-1R+ cells than in RANK+CSF-1R+ cells contained in the B220− population. In contrast, the PB-derived B220−RANK+CSF-1R+ population contained a significantly higher frequency of osteoclast precursors than the B220−RANK−CSF-1R+ population. ConclusionsThese results suggest that GST-RANKL-biotin is useful for the detection of RANK+ cells and that RANK and CSF-1R may be helpful indicators of osteoclast precursors in PB.