Figure 3.

Properties of CD45⁺CD3^-B220^-NK1.1^-Ly6G^-CD11b^-/loCD115⁺CCR2^hi (CCR2^hi) and CD45⁺CD3^-B220^-NK1.1^-Ly6G^-CD11b^-/loCD115⁺CCR2^lo (CCR2^lo) osteoclast progenitor (OCP) subpopulations in B6 mice with collagen-induced arthritis (CIA). (A) Frequency of CCR2^hi and CCR2^lo OCPs in periarticular bone marrow (PBM) and spleen (SPL) in control (ctrl), complete Freund’s adjuvant treated mice (CFA), and arthritic mice (CIA). Representative dot plots are shown. (B) Representative flow cytometry data of the properties of CCR2^hi and CCR2^lo OCPs in arthritic PBM. Greater proportion of CCR2^hi cells is of lower SSC signal and CD11b low positive. Greater proportion of CCR2^lo cells shows higher SSC signal than CCR2^hi cells and is CD11b negative. Red gate—total CD115⁺ cells; dark blue gate—CCR2^hi subset; light blue gate—CCR2^lo subset. (C, D) Proliferative and osteoclastogenic potential of CCR2^hi (C) and CCR2^lo (D) subpopulations. Sorted cells were stimulated for 72 h and 96 h by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in culture plates at the same density (7,000 cells/well). Representative microphotographs of in vitro differentiated tartrate-resistant acid phosphatase (TRAP)⁺ osteoclast-like cells are presented (magnification 100×). Differentiated TRAP⁺ multinucleated osteoclast-like cells were counted per well (Oc.N.). For proliferation assay, sorted cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) before culturing for 2 days with RANKL and M-CSF, and flow cytometric analysis. (E) qPCR analysis of c-Fos, CD115/cFms, Rank, and Ccl2 gene expression levels in sorted CCR2^hi and CCR2^lo progenitor subpopulations after culturing with RANKL and M-CSF for 2 days, presented as relative quantity of RNA in ctrl, CFA, and CIA mice, and normalized to the average value of control group (ctrl avg). The experiments were repeated three times and pooled data are shown (n = 6–10 mice per group). Values are presented as medians (middle horizontal lines), boxes represent the interquartile range (IQR), whiskers represent 1.5 times the IQR. Statistically significant difference was determined at p < 0.05, Mann–Whitney U-test (C, D), Kruskal–Wallis test (p-value marked on the graph) with Conover post-hoc test for group-to-group comparisons (A, E); correction for multiple comparisons (E) was performed using Holm–Bonferroni method; line denotes significant difference between groups. CD115/cFms—colony stimulating factor 1 receptor (Csf1r), Rank—receptor activator of nuclear factor κB, LY—CD3/B220/NK1.1.