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

Osteoclasts 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).

We 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.

The 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.

These 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.