Thrombin-activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis.
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 alpha4beta1 and alpha9beta1 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 alpha 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 alpha4beta1, whereas OPN-L did not.


CONCLUSIONS
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.
Sharif
Shadi A
SA
Stanford University School of Medicine, Stanford, California, CA 94305, USA.
Du
Xiaoyan
X
Myles
Timothy
T
Song
Jason J
JJ
Price
Elizabeth
E
Lee
David M
DM
Goodman
Stuart B
SB
Nagashima
Mariko
M
Morser
John
J
Robinson
William H
WH
Leung
Lawrence L K
LL
D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D017209:Apoptosis / Q000502:physiology; D015535:Arthritis, Psoriatic / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D043424:Carboxypeptidase B / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism* / Q000473:pathology*; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D053495:Osteopontin / Q000276:immunology / Q000378:metabolism*; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology*; D013917:Thrombin / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D053495:Osteopontin; D043424:Carboxypeptidase B; D013917:Thrombin