Background Oxidative stress and chronic inflammation impair white blood cells and endothelial cells activities in rheumatoid arthritis (RA) patients, which eventually result in the early atherogenesis and the cardiovascular disease (CVD) displayed by those patients. Anti-cyclic citrulinated protein antibodies (anti-CCP) are the most specific autoantibody markers in RA patients. However any study has evaluated their direct involvement in the atherogenic process.
Objectives 1) To assess the mitochondrial integrity of circulating leukocytes from RA patients, their overall oxidative status, and their relationship with chronic inflammation and early atherogenesis;
2) To investigate the involvement of anti-CCP antibodies in all that processes.
Methods Fifthy two RA patients and 30 healthy donors were recruited to the study. Cell surface tissue factor (TF), protease activated receptors (PARs) expression, peroxides and peroxinitrites expression levels, and mitochondrial membrane potential (MMP), were analyzed by flow cytometry in purified monocytes and neutrophils; glutathione peroxidase activity (GPx) was evaluated in cell lysates. Plasma nitric oxide (NO), and N-Tyr were further measured. Atherosclerosis/cardiovascular risk markers were also evaluated. The carotid-intimate media thickness (CIMT) was measured as a clinical surrogate parameter of atherosclerosis. In parallel in vitro studies, isolated monocytes or neutrophils were incubated with non-specific human IgG or with anti-CCP antibodies purified from pooled anti-CCP-positive RA sera.
Results Increased expression of TF and PAR2 was found in neutrophils from AR patients, with higher plasma levels of VEGF, tPA, MCP1, MIP1α, TNFα, IL-6, -8, -17A and -23. AR monocytes and neutrophils had higher peroxides and peroxynitrite levels, more depolarised mitochondria and lower GPx activity. Plasmatic NO was reduced in AR patients, with increased plasmatic N-Tyr levels. Both peroxide/peroxinytrite production in neutrophils and N-Tyr plasma levels showed positive correlations with TF and PAR2, and inflammatory markers (TNFα, IL17A and IL-23). Association studies showed direct relationship between positivity for anti-CCP and increased expression of IL-2 and IL-17A, and neutrophils MMP. A direct association was found between the presence of an increased CIMT and oxidative stress in neutrophils (peroxides and MMP), and proinflammatory molecules (IL-2, IL-6, IL-17 and IL-23).In vitro treatment of monocytes and neutrophils with anti-CCP promoted significant increase in peroxide production as well as on the percentage of cells with increased MMP. Moreover, elevated cell surface TF expression, as well as increased mRNA levels of MCP-1, IL-1β, IL-6, IL-8 and TNFαwas induced in monocytes by anti-CCP treatment.
Conclusions These observations indicate that anti-CCP antibodies act as direct inductors of inflammation and oxidative stress in monocytes and neutrophils of AR patients, possibly contributing to the accelerated atherosclerosis and the CVD found in that patients. Supported by JA0246/2009, P08-CVI-04234, PS09/01809, and Spanish foundation of Rheumatology (FER).
Disclosure of Interest None Declared
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