CCL11, a novel mediator of inflammatory bone resorption

Abstract

Normal bone homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts is perturbed by inflammation. In chronic inflammatory disease with disturbed bone remodelling, e.g. rheumatoid arthritis, patients show increased serum levels of the chemokine eotaxin-1 (CCL11). Herein, we demonstrate an inflammatory driven expression of CCL11 in bone tissue and a novel role of CCL11 in osteoclast migration and resorption. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that expression increased during inflammatory conditions. Osteoclasts did not express CCL11, but the high affinity receptor CCR3 was significantly upregulated during osteoclast differentiation and found to colocalise with CCL11. Exogenous CCL11 was internalised in osteoclast and stimulated the migration of pre-osteoclast and concomitant increase in bone resorption. Our data pinpoints that the CCL11/CCR3 pathway could be a new target for treatment of inflammatory bone resorption.

Figure 1

Injection of Pam2 stimulates osteoclast formation, bone resorption and increased CCL11 expression in mouse parietal bones. (a) Sections of paraffin embedded parietal bones from mice given subcutaneous injections of saline (left panel) and 50 μg Pam2 (right panel) stained with Safranin O and immunohistochemically for TRAP and CCL11 respectively. Arrows point few osteoblasts (OB) and osteoclasts (OC). (b) mRNA expression of TNF-α, IL-1β and CCL11 in homogenized parietal bones from mice subcutaneously injected with Pam2 (n = 4) compared to saline control (n = 5). Data expressed as means ± SEM.

Figure 2

CCL11 mRNA and protein expression in TNF-α and IL-1β stimulated mouse osteoblasts. (a) mRNA expression of CCL11 in non-stimulated and TNF-α or IL-1β stimulated osteoblasts after 6 h, 24 h and 48 h of incubation. (b) CCL11 release measured in medium cultured in absence and presence of TNF-α and IL-1β after 24 h and 48 h of incubation. Data are expressed as means ± SEM.

Figure 3

Osteoclasts do not express CCL11 but CCR3 mRNA and protein. (a) mRNA of CCL11 and the osteoclast associated enzyme Cathepsin K (left panel) and MCP-1 (right panel) analysed in BMM cultures stimulated with M-CSF alone or in presence of RANKL for 1, 2 and 3 days. (b) CCR2, CCR5 and CCR3 mRNA expression during RANKL-stimulated osteoclast differentiation. Data are expressed as means ± SEM. (c) Representative immunofluorescence staining of osteoclast culture (day 3) with CCR3 antibody in combination with F-actin to visualize actin filaments.

Figure 4

Co-localization of CCL11 and CCR3 receptor in osteoclasts of mouse parietal bones treated with PAM2. Representative immunofluorescence staining of parietal tissue section using CCL11 and CCR3 antibodies. Arrows point the layer of osteoblasts lining up on the bone surface (black). Lower panel demonstrates the higher magnification of squared osteoclast from image above.

Figure 5

CCL11 binding and effects on osteoclast migration and bone resorption. (a) Number of TRAP⁺ cells determined by a chemotaxis migration assay in presence of MCP-1 or CCL11. (b) Confocal section of living osteoclasts incubated for 30 min with Alexa Fluor®647 labelled rmCCL11. DIC images were overlaid with the fluorescent images to illustrate the morphology of the cells. Bottom and side projections show slice of the merged maximum projection at position indicated by yellow lines at 90° rotation. (c) BMM cultured on bone slices in absence or presence of rmCCL11 in medium supplemented with M-CSF (30 ng/ml) and RANKL (2 ng/ml). Total resorption pit area measured in both groups at day 5 and 6. Data are expressed as means ± SEM. Representative images of resorption pits on bone slices and TRAP⁺ osteoclasts in absence (upper panel) and presence (lower panel) of CCL11. Arrows point resorption pits.