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

Osteoclasts are bone-resorbing cells that are derived from haemopoietic pre cursors, including cells present in peripheral blood. The recent identifica tion of RANKL [receptor activator of nuclear factor (NF)-kappa B 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 osteocl asts in the absence of cells of the stromal/osteoblast lineage. Human perip heral blood mononuclear cells (PBMC) cultured in vitro with soluble RANKL a nd human macrophage colony-stimulating factor form osteoclasts. However, PB MC are heterogeneous, consisting of subsets of monocytes and lymphocytes as well as other blood cells. As the CD14 marker is strongly expressed on mon ocytes, the putative osteoclast precursor in peripheral blood, we have sele cted CD14(+) cells from PBMC to examine their osteoclastogenic potential an d their expression of novel members of the tumour necrosis factor superfami ly involved in osteoclastogenesis. Highly purified CD14(+) cells demonstrat ed mRNA expression of receptor activator of NF-kappa B, but no expression o f RANKL or osteoprotegerin, whereas PBMC expressed mRNAs for all three fact ors. CD14(+) (but not CD14(-)) cells cultured on bone slices for 21 days wi th human macrophage colony-stimulating factor and soluble RANKL generated o steoclasts and showed extensive bone resorption. Similar numbers of osteocl asts were generated by 10(5) CD14(+) cells and 10(6) PBMC, but there was si gnificantly 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 f urther characterization of the phenotype of circulating osteoclast precurso rs and cell interactions in osteoclastogenesis.