Fig. 6

Stimulatory osteoclastogenic and chemotactic effects of chemokines on circulating osteoclast progenitors in rheumatoid arthritis. a Schematic presentation of experimental design. We evaluated chemokine osteoclastogenic and chemotactic effects on nonadherent cells harvested after overnight culturing of peripheral blood mononuclear cells (PBMCs) with 35 ng/ml macrophage colony-stimulating factor (M-CSF). For the osteoclastogenic assay, cells were further cultured with M-CSF (30 ng/ml) and receptor activator of nuclear factor-κB ligand (RANKL; 100 ng/ml), supplemented by CC chemokine ligand 2 (CCL2; 40 ng/ml), CCL5 (10 ng/ml), or CXC chemokine ligand 10 (CXCL10; 20 ng/ml). Multinuclear cells positive for tartrate-resistant acid phosphatase (TRAP) were counted as mature osteoclasts (OCs). For the migration assay, cells were first cultured for 2 days with M-CSF and RANKL, then transferred to a Transwell culture system, applying chemokine gradient (CCL2, CCL5, or CXCL10). Cells migrating through the Transwell membrane were enumerated by 4′,6-diamidino-2-phenylindole (DAPI) staining. Migrated cells were further differentiated by M-CSF and RANKL and assessed for the number of TRAP⁺ OCs. b Relative expression of chemokine receptors on osteoclast progenitors (CD3⁻CD19⁻CD56⁻CD11b⁺CD14⁺) in peripheral blood samples of patients with rheumatoid arthritis compared with the median value of control (healthy) subjects, as measured by flow cytometry. c Relative number of TRAP⁺ OCs generated by chemokine treatment compared with respective untreated osteoclastogenic culture (no chemokine). d Relative number of cells migrating by chemokine gradient through Transwell membrane compared with respective untreated migration assay (no chemokine). e Relative number of TRAP⁺ OCs generated from cells that had migrated to the lower chamber compared with respective untreated osteoclastogenic culture (no chemokine). Values are presented as medians (middle lines), with boxes representing IQR, whiskers representing 1.5 times the IQR, and squares or circles representing outliers. Red horizontal lines represent the relative value of the respective control. Group-to-group comparisons were performed using a nonparametric Mann-Whitney U test, and p values <0.05 are shown. CCR CC chemokine receptor, CXCR CXC chemokine receptor