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

Abstract

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.

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.

Figure 1. Characterization of specific antibodies against OPN-FL, OPN-R and OPN-L and development of specific ELISAs

Recombinant human OPN-FL was digested with thrombin (IIa) or thrombin (IIa)/CPB to produce OPN-R and OPN-L respectively. A. Western blot analysis of human OPN-FL, OPN-R and OPN-L with the specific anti-peptide antibodies anti-OPN, anti-OPN-R and anti-OPN-L. OPN-R (SLAYGLR) and OPN-L (SVVYGL) peptides were used for competitive inhibition of antibody binding. B, ELISAs for OPN-FL (commercial), OPN-R and OPN-L ELISA. C. Competition of substituted peptides for binding of anti-OPN-R and anti-OPN-L to immobilized purified OPN-R and OPN-L respectively. Inhibition of binding by the cognate unsubstituted peptide was defined as 100%.

Figure 2. Determination of OPN-FL, OPN-R and OPN-L levels in synovial fluid of patients with osteoarthritis, psoriatic arthritis and rheumatoid arthritis by ELISA

A, Osteoarthritis (OA) n = 18, psoriatic arthritis (PsA) n = 10, rheumatoid arthritis (RA) n = 26. Bars are median values of each disease group. *P < 0.003 by Wilcoxon rank test. B, The ratio of cleaved OPN levels (OPN-R plus OPN-L) to total OPN (sum of the three different OPN forms) for RA, OA and PsA. *P < 0.003.

Figure 3. Identification of thrombin-activatable pCPB and its activity in fibroblast-like synoviocytes

A, Detection of pCPB in OA and RA synovial tissue lysates from two patients each by a monoclonal anti-pCPB antibody in Western blot. B, Immunofluoresence cell labeling of pCPB, thrombomodulin and OPN in FLS. In anti-pCPB panel, green color indicates pCPB and blue color stands for nuclear labeling by DAPI. C, Thrombomodulin expressed on FLS enhanced pCPB activation by thrombin. pCPB activation was blocked in the presence of anti-thrombomodulin antibody. Results represent the mean ± SEM (n = 6). D, Generation of endogenous OPN-R and OPN-L by incubation of thrombin with FLS, with or without exogenous pCPB. Thrombin was used at either 1 U/mL or 10 U/mL; OPN-R or OPN-L in FLS supernatant was measured by direct ELISA as described in Methods. Potato carboxypeptidase inhibitor (CPI) is a specific inhibitor of CPB. Results represent mean ± SEM (n = 6) P < 0.05.

Figure 4. Detection of OPN-FL and OPN-R in RA synovium

Immunostaining of RA synovial tissue: A, H&E stain. B, anti-OPN (10A16). C, anti-OPN-R. D, anti-OPN-R following in situ treatment with thrombin, magnification 400X.

Figure 5. OPN protected neutrophils from apoptosis and fibroblast-like synoviocytes binding to OPN was enhanced in OPN-R but not in OPN-L

A, Effect of recombinant WT OPN-FL, OPN-R, OPN-L and their RAA substituted counterparts (RAA-FL, RAA-R, and RAA-L) on neutrophil apoptosis. Each data point represents mean ± SEM (n = 3), and the results are representative of one of three separate experiments. B, FLS adhesion to immobilized WT and RGD-substituted OPN-FL, OPN-R, OPN-L. Data are presented as mean ± SEM (n = 8), and results are representative of one of four separate experiments. C, Schematic model of OPN-R and OPN-L in RA: While OPN and pCPB within the inflammatory joint space can be derived from plasma, synoviocytes also produce and release these molecules locally. Following initial insult, thrombin is generated and cleaves OPN to OPN-R, which enhances tissue inflammation. 27 On the other hand, thrombin also binds to thrombomodulin (TM) on the synovial cell surface, which then activates pCPB locally to CPB and converts OPN-R to OPN-L, thereby dampening inflammation.