Background Osteitis, replacement of bone marrow adipocytes with inflammatory cells, is an early sign of inflammation in rheumatoid arthritis (RA), seen as bone marrow edema by magnetic resonance imaging. Osteoclast progenitors (OCPs), normally present within circulating monocytes, are presumed to be increasingly attracted to inflamed joints in RA, where their enhanced activation and differentiation leads to excessive osteoresorption.
Objectives Our goal was to identify attraction signals important for homing of OCPs to the sites of osteitis. Therefore, we analyzed changes in immune cell populations as well as OCP chemokine receptor profile and migration potential in circulation, affected joints and periarticular bone in collagen induced arthritis (CIA) as a mouse model of RA.
Methods After obtaining Ethical approval, C57BL/6 mice were immunized with chicken type II collagen in complete Freund's adjuvant and evaluated for CIA development by clinical and histological scoring and anti-collagen antibody detection. Subchondral bone loss and osteitis were assessed by histology and histomorphometry. Distal tibial bone marrow, peripheral blood (PBL) and collagenase-digested tarsometatarseal joints were analyzed by flow-cytometry for the expression of hematopoietic markers and chemokine receptors (CD3, B220, NK1.1, CD11b, CD115, Gr1, CCR2, CCR5, CCR9). Transwell system was used to assess migration potential of M-CSF/RANKL stimulated PBL cells toward CCL2 or CCL5 gradient in vitro. For migration tracking, labeled PBL cells were transferred to recipient CIA mice and assessed by in vivo fluorescence imaging.
Results Immunized mice developed CIA with the maximum incidence of 60%. Arthritic areas showed histological presence of osteitis and significant subchondral bone loss (BV/TV 46.8±13.4% in control vs 32.1±8.6% in CIA) with increased number of mature osteoclasts (1.4±1.1 in control vs 5.4±4.7 in CIA). Flow-cytometry analysis revealed significant expansion of several populations in CIA, including lymphoid-negative CD11b+/Gr-1+ OCP subset within affected joints (24±4.1% in control vs 49±12.7% in CIA); lymphoid-negative CD11b+CD115+ OCP subset in PBL (4.6±0.7% in control vs 8.8±3.3% in CIA) and periarticular bone marrow (9.6±1.1% in control vs 21.9±3.4% in CIA). PBL OCP populations expressing chemokine receptors were reduced in arthritis (CCR2+ 32.1±4.3% in control vs 24.7±5.1% in CIA; CCR5+ 23.3±4.5% in control vs 18.1±3.7% in CIA; CCR9+ 5.9±3.7% in control vs 0.4±0.1% in CIA). In vitro migration of PBL cells toward chemotactic gradients was significantly enhanced in CIA (11.5 (IQR 11–13.25) in control vs 20.5 (IQR 20–25) in CIA for CCL2; 12.5 (IQR 9–18.5) in control vs 33.5 (IQR 23–35.75) in CIA for CCL5). Fluorescently-labeled PBL cells were efficiently attracted to tarsometatarseal joints 48 hours after in vivo transfer in mice with CIA.
Conclusions In fully developed CIA OCP populations are highly induced, with the reduction of circulating OCP subpopulations expressing chemokine receptors, possibly indicating their increased migration and homing to bone surfaces of the inflamed joints. Therapeutic blocking of chemokine signals may therefore be a promising approach to reduce osteoresorption in RA.
Acknowledgement This work was fully supported by Croatian Science Foundation (project nr. 5699)
Disclosure of Interest None declared