Anticyclic Citrullinated Protein Antibodies Are Implicated in the Development of Cardiovascular Disease in Rheumatoid Arthritis Nuria Barbarroja,* Carlos Pérez-Sanchez,* Patricia Ruiz-Limon, Carmen Castro-Villegas, Maria Angeles Aguirre, Rosario Carretero, Pedro Segui, Yolanda Jimenez-Gomez, Manuela Sanna, Antonio Rodriguez-Ariza, Eduardo Collantes-Estevez, Alejandro Escudero,* Chary López-Pedrera* Objective—Previous studies have suggested a relationship between anticyclic citrullinated protein (CCP) levels and development of cardiovascular disease in rheumatoid arthritis (RA). However, a limited number of studies have demonstrated an involvement of anti-CCPs in those processes. This study was aimed to define the specific role of these auto-antibodies in the pro-oxidative, inflammatory, and proatherogenic profile observed in leukocytes from RA patients. Approach and Results—Seventy-five RA patients and 31 healthy donors were enrolled. Carotid intima media thickness was evaluated as atherosclerosis marker. Several procoagulant and inflammatory factors, leukocyte activation, and oxidative stress markers were analyzed in plasma and leukocyte subsets. Anti-CCPs were purified from plasma of RA patients, and in vitro treatment of healthy leukocytes was conducted. High titers of anti-CCPs were associated to altered expression of prothrombotic and inflammatory markers, high oxidative stress, and pathological carotid intima media thickness in RA patients. Notably, gene expression analysis showed that lymphocytes were major players in altered inflammatory profile, monocytes were responsible for the protrombotic and atherogenic status, and neutrophils mainly displayed a prooxidative feature. In vitro treatment with purified anti-CCPs fully recapitulated that pathogenic profile, promoting the activation of leukocytes. Conclusions—Anti-CCPs are key players in the inflammatory and proatherogenic status of RA patients. The effects are specific of the immune cell targeted, promoting overexpression of thrombotic, inflammatory, and pro-oxidative markers in monocytes; pro-oxidative status in neutrophils; and proinflammatory profile in lymphocytes. Targeting these autoantibodies would be an excellent strategy to prevent the development of cardiovascular disease in RA. (Arterioscler Thromb Vasc Biol. 2014;34:2706-2716.) Key Words: cardiovascular disease ◼ inflammation ◼ leukocytes ◼ rheumatoid arthritis
R
heumatoid arthritis (RA) is a disorder comprising a complex onset mechanism and many associated complications. In particular, cardiovascular disease (CVD) significantly contributes to morbidity and mortality in these patients, causing the 39% to 50% of deaths,1 and atherosclerosis at early stage of the disease is considered a possible preclinical manifestation. In fact, the risk of CVD events, such as myocardial infarction, is increased in the 2 years preceding formal diagnosis of RA,2 and once the disease is diagnosed, the risks of carotid plaques and CVD events increase with the duration of RA.3 Thus, the strong relationship
between atherosclerosis and RA has led some authors to include atherosclerosis among the extra-articular manifestations of the disease.4 The mechanisms responsible for the premature atherosclerosis in RA are not well understood, but traditional risk factors alone are not fully responsible and a role of inflammation in this process has been suggested. Considering that RA is a chronic inflammatory disease and the pathogenic role of inflammation in the atherosclerosis,5 it is likely that inflammatory mediators might be causal in the accelerated atherosclerosis observed in RA.
Received on: April 10, 2014; final version accepted on: September 11, 2014. From the Rheumatology Service (N.B., C.P.-S., P.R.-L., C.C.-V., M.A.A., R.C., Y.J.-G., M.S., A.R.-A., E.C.-E., A.E., C.L.-P.) and Radiology Service (P.S.), Maimonides Institute for Research in Biomedicine of Cordoba (IMIBIC)/Reina Sofia University Hospital, Cordoba, Spain; and Department of Biomedical Sciences and Centre of Excellence for Biotechnology, Development and Biodiversity Research, University of Sassari, Italy (M.S.). *These authors contributed equally to this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304475/-/DC1. Correspondence to Nuria Barbarroja, PhD, Unidad de Investigación, Hospital Reina Sofia, Avda. Menendez Pidal s/n, E-14006 Córdoba, Spain. E-mail [email protected] © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
2706
DOI: 10.1161/ATVBAHA.114.304475
Barbarroja et al Anti-CCPs Induce Cardiovascular Disease in RA 2707
Nonstandard Abbreviations and Acronyms CCP CIMT CRP CVD DAS MCP-1 MIP-1α RA RF TF Th TNFα Treg
cyclic citrullinated protein carotid intima media thickness C reactive protein cardiovascular disease disease activity score monocyte chemotactic protein-1 macrophage inflammatory protein-1 alpha rheumatoid arthritis rheumatoid factor tissue factor T helper tumor necrosis factor-α T regulatory
Oxidative stress is another process frequently altered in RA, which also contributes to atherosclerosis, being associated with a poorer prognosis. Neutrophils are thought to be responsible for the elevated levels of reactive oxygen species found in plasma and synovial fluid of RA patients, thus contributing to the joint tissue damage within synovial membrane.6 In addition, a positive correlation has recently been shown between neutrophils reactive oxygen species levels in blood and synovial fluid with disease activity score (DAS)-28, C reactive protein (CRP), and anticyclic citrullinated protein (CCP) levels.7 However, monocytes also seem to contribute to the elevated oxidative stress in blood of RA patients through the production of superoxide anion.8 Many different cell components can be considered as key elements in the long course of RA, including blood white cells, such as lymphocytes, monocytes, and neutrophils. Activation of these cells leads to the production of cytokines and mediators of inflammation, oxidative stress, and atherosclerosis. In this regard, monocytes/macrophages are central players in inflammation and have been found to be activated in RA through the release of cytokines9 and massive infiltration in inflammatory sites, such as synovial membranes.10 In addition, monocytes/macrophages have been associated with bone erosion in RA, owing to their excessive differentiation into osteoclasts.11 Because of its role in RA, several monocyte-targeted therapies are being developed. Lymphocytes are the most studied cells mediating the pathogenesis of RA, which in fact is considered a T helper 1 (Th1) cell–driven disorder with a Th1/Th2 imbalance toward higher Th1 levels compared with healthy donors.12 Th1 releases inflammatory molecules, such as interferon-γ, interleukin (IL)-2, and tumor necrosis factor-α (TNFα), that prevent CD4+T cells from differentiating into Th2 cells. Moreover, Th17 has lately been associated with RA pathogenesis,13 mainly producing IL-17A, IL-6, and TNFα, cytokines that are increased in serum of RA patients and are responsible for the activation of other cells. On the other hand, regulatory T cells (Treg) also participate in the course of this disease. They play an important role in immunosuppression through
cell–cell interaction and are found significantly decreased at advanced stage, and their function has been shown impaired in RA.12 Thus, it has been proposed that the development and progression of RA is caused by the imbalance of Th1/Th2 and Th17/Treg cells. Neutrophils are the most abundant cells in the synovial fluid of affected joints and at the pannus/cartilage interface, where tissue damage occurs. They promote damage through the induction of oxidative stress, enzyme release, and expression of proinflammatory cytokines. However, the role of neutrophils in RA pathogenesis is not fully understood.14 Among the auto-antibodies detected in RA, the anti-CCPs are highly specific for this disease15 because IgM rheumatoid factor (RF) is also present in an elevated percentage of patients with several chronic infections. Moreover, atherosclerosis status in RA is independent of RF levels.16 In contrast, detection of anti-CCP antibodies has become a useful diagnostic tool, particularly at early stage.17 Thus, the new 2010 RA classification criteria included detection of anti-CCPs as a key item for diagnosing the disease.18 High levels of anti-CCPs have been suggested to predict an adverse cardiovascular profile in RA patients. However, that hypothesis just arises from association studies in various cohorts, and no study has still demonstrated a direct effect of anti-CCPs in all those processes. Here we demonstrate the direct involvement of anti-CCPs, purified from RA patients, in the induction of a pro-oxidative, proinflammatory, and atherogenic status in different leukocyte subsets. In addition, specific effects on macrophage activation and survival were also demonstrated.
Materials and Methods Material and methods are available in the online-only Data Supplement.
Results Clinical and Analytic Details of RA Patients Clinical details of RA patients and healthy donors included in this study are stated in Table 1. A 50.66% of RA patients was positive for RF, whereas 65.33% presented anti-CCP antibodies. The patients included in the present study had a low–moderate disease activity with a DAS28 of 3.49±0.17, considering moderate level of disease DAS28 values from 2.4 to 3.7 and low level DAS28
R
heumatoid arthritis (RA) is a disorder comprising a complex onset mechanism and many associated complications. In particular, cardiovascular disease (CVD) significantly contributes to morbidity and mortality in these patients, causing the 39% to 50% of deaths,1 and atherosclerosis at early stage of the disease is considered a possible preclinical manifestation. In fact, the risk of CVD events, such as myocardial infarction, is increased in the 2 years preceding formal diagnosis of RA,2 and once the disease is diagnosed, the risks of carotid plaques and CVD events increase with the duration of RA.3 Thus, the strong relationship
between atherosclerosis and RA has led some authors to include atherosclerosis among the extra-articular manifestations of the disease.4 The mechanisms responsible for the premature atherosclerosis in RA are not well understood, but traditional risk factors alone are not fully responsible and a role of inflammation in this process has been suggested. Considering that RA is a chronic inflammatory disease and the pathogenic role of inflammation in the atherosclerosis,5 it is likely that inflammatory mediators might be causal in the accelerated atherosclerosis observed in RA.
Received on: April 10, 2014; final version accepted on: September 11, 2014. From the Rheumatology Service (N.B., C.P.-S., P.R.-L., C.C.-V., M.A.A., R.C., Y.J.-G., M.S., A.R.-A., E.C.-E., A.E., C.L.-P.) and Radiology Service (P.S.), Maimonides Institute for Research in Biomedicine of Cordoba (IMIBIC)/Reina Sofia University Hospital, Cordoba, Spain; and Department of Biomedical Sciences and Centre of Excellence for Biotechnology, Development and Biodiversity Research, University of Sassari, Italy (M.S.). *These authors contributed equally to this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304475/-/DC1. Correspondence to Nuria Barbarroja, PhD, Unidad de Investigación, Hospital Reina Sofia, Avda. Menendez Pidal s/n, E-14006 Córdoba, Spain. E-mail [email protected] © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
2706
DOI: 10.1161/ATVBAHA.114.304475
Barbarroja et al Anti-CCPs Induce Cardiovascular Disease in RA 2707
Nonstandard Abbreviations and Acronyms CCP CIMT CRP CVD DAS MCP-1 MIP-1α RA RF TF Th TNFα Treg
cyclic citrullinated protein carotid intima media thickness C reactive protein cardiovascular disease disease activity score monocyte chemotactic protein-1 macrophage inflammatory protein-1 alpha rheumatoid arthritis rheumatoid factor tissue factor T helper tumor necrosis factor-α T regulatory
Oxidative stress is another process frequently altered in RA, which also contributes to atherosclerosis, being associated with a poorer prognosis. Neutrophils are thought to be responsible for the elevated levels of reactive oxygen species found in plasma and synovial fluid of RA patients, thus contributing to the joint tissue damage within synovial membrane.6 In addition, a positive correlation has recently been shown between neutrophils reactive oxygen species levels in blood and synovial fluid with disease activity score (DAS)-28, C reactive protein (CRP), and anticyclic citrullinated protein (CCP) levels.7 However, monocytes also seem to contribute to the elevated oxidative stress in blood of RA patients through the production of superoxide anion.8 Many different cell components can be considered as key elements in the long course of RA, including blood white cells, such as lymphocytes, monocytes, and neutrophils. Activation of these cells leads to the production of cytokines and mediators of inflammation, oxidative stress, and atherosclerosis. In this regard, monocytes/macrophages are central players in inflammation and have been found to be activated in RA through the release of cytokines9 and massive infiltration in inflammatory sites, such as synovial membranes.10 In addition, monocytes/macrophages have been associated with bone erosion in RA, owing to their excessive differentiation into osteoclasts.11 Because of its role in RA, several monocyte-targeted therapies are being developed. Lymphocytes are the most studied cells mediating the pathogenesis of RA, which in fact is considered a T helper 1 (Th1) cell–driven disorder with a Th1/Th2 imbalance toward higher Th1 levels compared with healthy donors.12 Th1 releases inflammatory molecules, such as interferon-γ, interleukin (IL)-2, and tumor necrosis factor-α (TNFα), that prevent CD4+T cells from differentiating into Th2 cells. Moreover, Th17 has lately been associated with RA pathogenesis,13 mainly producing IL-17A, IL-6, and TNFα, cytokines that are increased in serum of RA patients and are responsible for the activation of other cells. On the other hand, regulatory T cells (Treg) also participate in the course of this disease. They play an important role in immunosuppression through
cell–cell interaction and are found significantly decreased at advanced stage, and their function has been shown impaired in RA.12 Thus, it has been proposed that the development and progression of RA is caused by the imbalance of Th1/Th2 and Th17/Treg cells. Neutrophils are the most abundant cells in the synovial fluid of affected joints and at the pannus/cartilage interface, where tissue damage occurs. They promote damage through the induction of oxidative stress, enzyme release, and expression of proinflammatory cytokines. However, the role of neutrophils in RA pathogenesis is not fully understood.14 Among the auto-antibodies detected in RA, the anti-CCPs are highly specific for this disease15 because IgM rheumatoid factor (RF) is also present in an elevated percentage of patients with several chronic infections. Moreover, atherosclerosis status in RA is independent of RF levels.16 In contrast, detection of anti-CCP antibodies has become a useful diagnostic tool, particularly at early stage.17 Thus, the new 2010 RA classification criteria included detection of anti-CCPs as a key item for diagnosing the disease.18 High levels of anti-CCPs have been suggested to predict an adverse cardiovascular profile in RA patients. However, that hypothesis just arises from association studies in various cohorts, and no study has still demonstrated a direct effect of anti-CCPs in all those processes. Here we demonstrate the direct involvement of anti-CCPs, purified from RA patients, in the induction of a pro-oxidative, proinflammatory, and atherogenic status in different leukocyte subsets. In addition, specific effects on macrophage activation and survival were also demonstrated.
Materials and Methods Material and methods are available in the online-only Data Supplement.
Results Clinical and Analytic Details of RA Patients Clinical details of RA patients and healthy donors included in this study are stated in Table 1. A 50.66% of RA patients was positive for RF, whereas 65.33% presented anti-CCP antibodies. The patients included in the present study had a low–moderate disease activity with a DAS28 of 3.49±0.17, considering moderate level of disease DAS28 values from 2.4 to 3.7 and low level DAS28
