Thrombin‐activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis

SA Sharif, X Du, T Myles, JJ Song… - … : Official Journal of …, 2009 - Wiley Online Library
SA Sharif, X Du, T Myles, JJ Song, E Price, DM Lee, SB Goodman, M Nagashima, J Morser
Arthritis & Rheumatism: Official Journal of the American College …, 2009Wiley Online Library
Objective Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in
the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in
OPN‐R and exposing the cryptic C‐terminal α4β1 and α9β1 integrin–binding motif
(SVVYGLR). Thrombin‐activatable carboxypeptidase B (CPB), also called thrombin‐
activatable fibrinolysis inhibitor, removes the C‐terminal arginine from OPN‐R, generating
OPN‐L and abrogating its enhanced cell binding. We undertook this study to investigate the …
Objective
Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in OPN‐R and exposing the cryptic C‐terminal α4β1 and α9β1 integrin–binding motif (SVVYGLR). Thrombin‐activatable carboxypeptidase B (CPB), also called thrombin‐activatable fibrinolysis inhibitor, removes the C‐terminal arginine from OPN‐R, generating OPN‐L and abrogating its enhanced cell binding. We undertook this study to investigate the roles of OPN‐R and OPN‐L in synoviocyte adhesion, which contributes to the formation of invasive pannus, and in neutrophil survival, which affects inflammatory infiltrates in RA.
Methods
Using specifically developed enzyme‐linked immunosorbent assays, we tested the synovial fluid of patients with RA, osteoarthritis (OA), and psoriatic arthritis (PsA) to determine OPN‐R, OPN‐L, and full‐length OPN (OPN‐FL) levels.
Results
Elevated levels of OPN‐R and OPN‐L were found in synovial fluid samples from RA patients, but not in samples from OA or PsA patients. Increased levels of OPN‐R and OPN‐L correlated with increased levels of multiple inflammatory cytokines, including tumor necrosis factor α and interleukin‐6. Immunohistochemical analyses revealed robust expression of OPN‐FL, but only minimal expression of OPN‐R, in RA synovium, suggesting that cleaved OPN is released into synovial fluid. In cellular assays, OPN‐FL, and to a lesser extent OPN‐R and OPN‐L, had an antiapoptotic effect on neutrophils. OPN‐R augmented RA fibroblast‐like synoviocyte binding mediated by SVVYGLR binding to α4β1, whereas OPN‐L did not.
Conclusion
Thrombin activation of OPN (resulting in OPN‐R) and its subsequent inactivation by thrombin‐activatable CPB (generating OPN‐L) occurs locally within inflamed joints in RA. Our data suggest that thrombin‐activatable CPB plays a central homeostatic role in RA by regulating neutrophil viability and reducing synoviocyte adhesion.
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