Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor κB ligand (RANKL)

Abstract

Osteoclasts are bone-resorbing cells that are derived from haemopoietic precursors, including cells present in peripheral blood. The recent identification of RANKL [receptor activator of nuclear factor (NF)-kappaB ligand], a new member of the tumour necrosis factor ligand superfamily that has a key role in osteoclastogenesis, has allowed the in vitro generation of osteoclasts in the absence of cells of the stromal/osteoblast lineage. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with soluble RANKL and human macrophage colony-stimulating factor form osteoclasts. However, PBMC are heterogeneous, consisting of subsets of monocytes and lymphocytes as well as other blood cells. As the CD14 marker is strongly expressed on monocytes, the putative osteoclast precursor in peripheral blood, we have selected CD14(+) cells from PBMC to examine their osteoclastogenic potential and their expression of novel members of the tumour necrosis factor superfamily involved in osteoclastogenesis. Highly purified CD14(+) cells demonstrated mRNA expression of receptor activator of NF-kappaB, but no expression of RANKL or osteoprotegerin, whereas PBMC expressed mRNAs for all three factors. CD14(+) (but not CD14(-)) cells cultured on bone slices for 21 days with human macrophage colony-stimulating factor and soluble RANKL generated osteoclasts and showed extensive bone resorption. Similar numbers of osteoclasts were generated by 10(5) CD14(+) cells and 10(6) PBMC, but there was significantly less intra-assay variability with CD14(+) cells, suggesting the absence of stimulatory/inhibitory factors from these cultures. The ability of highly purified CD14(+) cells to generate osteoclasts will facilitate further characterization of the phenotype of circulating osteoclast precursors and cell interactions in osteoclastogenesis.