Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Thrombin-Activatable Carboxypeptidase B (CPB) Plays a Central Role in Down-Regulating Inflammatory Responses in Rheumatoid Arthritis by Cleaving C5a.
Song3, Jason J., Hwang3, Inyong, Wang4, Tiffany, Lee1, Annette T., Bridges5, S. Louis, Gregersen1, Peter K., Leung4, Lawrence
Thrombin-activatable CPB is known to play an anti-fibrinolytic role by removing C-terminal lysine residues from fibrin, thereby preventing the cleavage of fibrin by plasmin. Recently, C3a, C5a, thrombin-cleaved osteopontin, and bradykinin have been identified as additional substrates of CPB. CPB removes arginine residues from the C-terminus of these inflammatory mediators, thereby altering their biological functions. Here we investigate the molecular mechanisms underlying the anti-inflammatory role of CPB in rheumatoid arthritis (RA).
We evaluated arthritis in CPB-/- mice, osteopontin-/- mice, C5-/- mice, and bradykinin B2 receptor-/- mice after injection of anti-collagen antibody. Arthritis was assessed visually and by caliper measurement of paw thickness daily for 10 days after injections. To determine the effect of CPB on C5a-mediated chemotaxis in vivo, we injected intact C5a or CPB cleaved C5a into mouse peritoneum and assessed neutrophil migration. To study the role of CPB in human RA, CPB and C5a levels were measured in synovial fluids derived from RA and osteoarthritis (OA) patients using commercial ELISA kits. To investigate the role of CPB genetic variants in RA, we genotyped two nonsynonymous CPB2 SNPs (rs1926447 and rs3742264) and evaluated the association with modified Sharp score (MSS) in the SONORA cohort (n=109, 70% female, 100% Caucasian) and the CLEAR Registry (n=118, 100 % female, 100% African-American).
We found that anti-collagen antibody-induced arthritis was much more severe in CPB-/- mice than in wild-type mice. In contrast, C5-/- mice were protected from arthritis, while bradykinin B2 receptor-/- mice and osteopontin-/- mice developed arthritis equivalent in severity to that in wild-type mice. In vivo studies showed that C5a chemotactic effect is decreased with CPB incubation (peritoneal neutrophil of intact C5a vs CPB cleaved C5a; 15.66% vs 5.14%, p<0.01). We found that levels of CPB and C5a are increased in RA synovial fluid compared to OA synovial fluid (CPB 644.2 vs 374.1%, p<0.0001; total C5a 24.0 vs 9.6 ng/ml, p <0.05). CPB level is correlated with C5a level (p<0.01, r=0.41). CPB SNP rs1926447 is associated with radiologic progression of RA: (MSS for CT/TT vs CC; SONORA (year 2), 4.5 vs 8.4, p=0.03; CLEAR I (year 3), 1.8 vs 5.9 p<0.01).
These results suggest that thrombin-activatable CPB plays a critical role in down-regulating inflammatory responses in anti-collagen antibody-induced arthritis, and that CPB exerts its anti-inflammatory effects by cleaving, and thereby inactivating, C5a. We also demonstrate that CPB is elevated in RA synovial fluid, and is associated with synovial C5a level. Our SNP analysis results suggest that CPB variant 1064T (minor allele of rs1926447) that has been reported to produce a CPB protein with a two-fold longer half-life, results in less radiographic damage in Caucasians and African-Americans with RA, further suggesting that CPB plays a central role in down-regulating inflammatory responses in RA.
To cite this abstract, please use the following information:
Song, Jason J., Hwang, Inyong, Wang, Tiffany, Lee, Annette T., Bridges, S. Louis, Gregersen, Peter K., et al; Thrombin-Activatable Carboxypeptidase B (CPB) Plays a Central Role in Down-Regulating Inflammatory Responses in Rheumatoid Arthritis by Cleaving C5a. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1386