Fig. 2

Expanded osteoclast progenitor (OCP) population correlates with indicators of disease activity in rheumatoid arthritis (RA). a Peripheral blood mononuclear cells were analyzed by flow cytometry using the gating strategy for OCPs as nonlymphoid (CD3⁻CD19⁻CD56⁻) cells double-positive for monocyte markers CD11b and CD14, and they were further assessed for the expression of macrophage colony-stimulating factor receptor (CD115) and receptor activator of nuclear factor-κB (RANK). b Proportion of OCPs, and CD115⁺ and RANK⁺ subsets in peripheral blood of control (CTRL) subjects and patients with RA, as well as RA synovial fluid (SF) samples. Gates for the analysis were defined using unlabeled cells and "fluorescence minus one control". c Association of the proportion of circulating OCPs with plasma levels of rheumatoid factor (RF) and tumor necrosis factor-α (TNF-α), as well as association of the proportion of RANK⁺ OCPs with tender joint count (TJC). d Quantification, morphology, and bone-resorbing activity of osteoclasts (OCs) differentiated from sorted peripheral blood OCPs in RA and CTRL samples. Osteoclastogenic potential of circulating OCPs was evaluated in vitro by stimulating sorted CD3⁻CD19⁻CD56⁻CD11b⁺CD14⁺ cells with macrophage colony-stimulating factor (30 ng/ml) and RANK ligand (100 ng/ml). Multinuclear cells positive for tartrate-resistant acid phosphatase (TRAP) were counted as mature OCs. Bone-resorbing activity was confirmed for sorted cells by pit formation assay under the same culture conditions. TRAP-stained mature OCs and bovine cortical bone slices from CTRL and RA cultures were scanned using light microscopy at × 200 magnification. Red arrows indicate bone resortion pits, formed by active mature osteoclasts. Values are presented as medians (middle lines), with boxes representing IQR, whiskers representing 1.5 times the IQR, and squares or circles representing outliers. Group-to-group comparisons were performed using a nonparametric Mann-Whitney U test, whereas correlations were done using rank correlation and Spearman’s coefficient ρ. p Values <0.05 are shown