Background/Purpose: Carboxypeptidase B2 (CPB2) is well established to play an anti-fibrinolytic role by removing C-terminal lysine residues from fibrin, thereby preventing its cleavage by plasmin. CPB2 also removes arginine/lysine residues from other proinflammatory substrates such as C5a, and SDF-1a. Previously we demonstrated that CPB2 plays a major role to down regulate synovial inflammation in collagen induced arthritis by down-regulating C5a (Song et al, 2011 JCI). However, its mechanism to prevent bone erosion in RA is unknown. We investigate the molecular mechanism how CPB2 prevents bone erosion.

Methods: LPS induced osteoclast (OC) activation model was performed by injection of LPS on cpb2^-/- and cpb2^+/+ mice. On day 8, femur was collected for microCT and histology. OC precursor (OCP) population in bone marrow was evaluated by FACS. OC differentiation was evaluated by TRAP staining on OCP ex vivo culture. OCP migration was measured by transwell migration assay with C5a and SDF-1a with or without CPB2 treatment. C5a receptor expression in bone marrow was measured by FACS.

Results: As compared to wild-type (WT) mice, CPB2 deficient mice exhibited significantly more severe osteoclast activation measured by microCT after LPS injection (bone volume/total volume cpb2^-/- 4.3±0.3 vs cpb2^+/+ 7.7±0.3, p< 0.01). There is increased OCP population in bone marrow of cpb2^-/- mice (Gr1 low, CD11b high population; cpb2^-/- 40% vs cpb2^+/+ 25.2%). There are more TRAP positive multinuclear cells from bone marrow of cpb2^-/- mice as compared to cpb2^+/+ mice (130.6 ±4.2 vs 107.3±3.5, p<0.01). OCP chemotaxis was decreased by CPB treatment on C5a and SDF-1a. C5a receptor expression is increased in CPB2 deficient mice.

Conclusion: These results suggest that CPB2 plays a critical role in osteoclast migration and activation in inflammation and that this effect could be in part mediated by its down-regulation of C5a and SDF-1a. CPB2 deficient mice demonstrate more osteoclast activation in response to LPS challenge. We also demonstrated that SDF-1a, a known chemotactic factor for OCP, is a novel substrate of CPB2. Studies are underway to investigate the role of CPB2 on osteoclast activation in human rheumatoid arthritis.