Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis

Abstract

Our study aimed to determine the functional activity of different osteoclast progenitor (OCP) subpopulations and signals important for their migration to bone lesions, causing local and systemic bone resorption during the course of collagen-induced arthritis in C57BL/6 mice. Arthritis was induced with chicken type II collagen (CII), and assessed by clinical scoring and detection of anti-CII antibodies. We observed decreased trabecular bone volume of axial and appendicular skeleton by histomorphometry and micro-computed tomography as well as decreased bone formation and increased bone resorption rate in arthritic mice in vivo. In the affected joints, bone loss was accompanied with severe osteitis and bone marrow hypercellularity, coinciding with the areas of active osteoclasts and bone erosions. Flow cytometry analysis showed increased frequency of putative OCP cells (CD3 B220 NK1.1 CD11b CD117 CD115 for bone marrow and CD3 B220 NK1.1 CD11b CD115 Gr-1 for peripheral haematopoietic tissues), which exhibited enhanced differentiation potential in vitro. Moreover, the total CD11b population was expanded in arthritic mice as well as CD11b F4/80 macrophage, CD11b NK1.1 natural killer cell and CD11b CD11c myeloid dendritic cell populations in both bone marrow and peripheral blood. In addition, arthritic mice had increased expression of tumour necrosis factor-α, interleukin-6, CC chemokine ligand-2 (Ccl2) and Ccl5, with increased migration and differentiation of circulatory OCPs in response to CCL2 and, particularly, CCL5 signals. Our study characterized the frequency and functional properties of OCPs under inflammatory conditions associated with arthritis, which may help to clarify crucial molecular signals provided by immune cells to mediate systemically enhanced osteoresorption.

The Long Pentraxin 3 Plays a Role in Bone Turnover and Repair

“…Spleen cells, obtained by smashing the spleen tissue between a pair of frosted microscope slides, were cultured in 48-well plates at a density of 0.5 × 10 6 /well in 0.5 mL/well of α-MEM/10% FCS supplemented with 20 ng/mL M-CSF and 40 ng/mL RANKL. At day 5–9 of culture, tartrate-resistant acid phosphatase (TRAP) + multinucleated cells (≥3 nuclei/cell) were identified using a commercially available kit (Sigma-Aldrich) and counted by light microscopy ( 24 ). Four wells from two to three mice were done for each gender/genotype, and three independent experiments were performed.…”

“…For flow cytometric analysis, bone marrow and spleen cells were harvested as described previously in Ref. ( 24 ). Erythrocytes were lysed with red blood cell lysing buffer (Sigma-Aldrich), and single-cell suspensions were obtained by filtering through a 100-µm Nytex mesh.…”

“…Fluorescence-activated cell sorting (FACS) of osteoprogenitor cells was performed in a BD FACSAria I instrument. Cells were extracted from the periosteal layer and callus tissue and labeled as described previously ( 24 ). Briefly, tibias were dissected, soft tissue was removed, and bone marrow was flushed.…”

“…The distal metaphyses of femora and second lumbar vertebrae were scanned using a micro-computed tomography (μCT) system (1172 SkyScan, SkyScan, Kontich, Belgium) at 50 kV and 200 mA with a 0.5 aluminum filter using a detection pixel size of 4.3 µm ( 24 ). Images were captured every 0.7° through 180° [second lumbar (L2) vertebrae] and every 0.7° through 360° (femora) rotation.…”

Fas receptor induces apoptosis of synovial bone and cartilage progenitor populations and promotes bone loss in antigen‐induced arthritis

“…Cell sorting was performed with a BD FACSAria I (BD, Franklin Lakes, NJ, USA) instrument, as previously described (38). Labeled cells were acquired at a speed 2000-5000 cells/s with the following gating strategy: singlets were delineated from total cell population depicted on forward-vs. sidescatter plots, and exclusion of dead cells was based on DAPI dihydrochloride (MilliporeSigma) incorporation and subsequent dissection of populations of interest.…”