Serum levels of soluble receptor for advanced glycation end products and of S100 proteins are associated with inflammatory, autoantibody, and classical risk markers of joint and vascular damage in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to bone and cartilage destruction and extra-articular complications, including atherosclerotic vascular disease and premature mortality [1]. The receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of RA through its ability to amplify inflammatory pathways [2, 3]. A member of the immunoglobulin superfamily of cell surface receptors, RAGE binds advanced glycation end products (AGEs), which are non-enzymatically glycated or oxidized proteins, lipids and nucleic acids formed under conditions of oxidative stress and hyperglycemia (reviewed in [4]). In addition to these, RAGE binds some proinflammatory ligands, including members of the S100/calgranulin family, and high mobility group box chromosomal protein 1 (HMGB-1), which is implicated in cell signaling by synergizing with DNA CpG motifs [5, 6]. Several RAGE ligands are characteristically overexpressed in RA and psoriatic arthritis, compared to healthy controls [7‐9]. S100A8/A9 (calprotectin) and S100A12 (calgranulin C, EN-RAGE) levels are significantly elevated in serum and synovial fluid from RA patients compared to healthy normal donors [3, 10]. S100A8/A9 levels are also higher in supernatants of cultured RA synoviocytes than of osteoarthritis synoviocytes [11].

Soluble C-truncated RAGE (sRAGE) lacks the transmembrane and cytosolic domains of the full-length receptor and can prevent proinflammatory effects of RAGE signaling by acting as a decoy [12‐14]. For example, in a collagen-induced arthritis (CIA) murine model, treatment with murine sRAGE significantly reduced joint inflammation and destruction [15]. Serum or plasma levels of sRAGE from patients with RA, hypertension or metabolic syndrome were lower than those in healthy subjects [16‐18], suggesting that sRAGE levels may identify those RA patients exposed to high levels of RAGE ligands. A gain-of-function Gly82Ser polymorphism in the RAGE gene (RAGE 82Ser) occurs more frequently in RA patients than in healthy controls [19]. Monocytes expressing the RAGE 82Ser allele activated a stronger inflammatory response to S100A12 in vitro [15]. Although this might be predicted to contribute to enhanced proinflammatory mechanisms in RA, we found no evidence that patients with the RAGE 82S allele had higher levels of inflammation, or a greater likelihood of complicating cardiovascular (CV) events [19].

Most S100 proteins have a mass between 9 and 14 kDa, and are characterized by two calcium binding sites of the EF-hand type (helix-loop-helix) [20]. S100A8 and S100A9, generally functioning as the S100A8/A9 heterocomplex, and S100A12 are implicated in non-infectious chronic inflammatory diseases such as RA, psoriasis and inflammatory bowel disease [21‐25]. Longitudinal and cross-sectional studies suggest a relationship between S100A12 and RA disease activity [26‐28]. The S100A12 gene is rapidly upregulated in human monocytoid cells and blood monocytes by tumor necrosis factor (TNF) and lipopolysaccharide (LPS), suggesting its production in response to proinflammatory signals in RA [10, 25]. S100A12 is a potent monocyte chemoattractant and activates mast cells, which are important effector cells in RA and atherosclerosis [25, 29, 30]. S100A12 is also proposed to promote proinflammatory activities by binding and activating RAGE [31]. However, these studies were established using a murine model, and since it was later shown that mice have no S100A12 in their genome [20], alternate receptors are implicated [25]. In addition, recombinant S100 ligands may contain contaminating endotoxin, and their effects may not always be fully RAGE dependent [32].

S100A8 and S100A9 regulate leukocyte migration and adhesion [33]. The S100A8/A9 complex has antimicrobial effects, transports arachidonic acid to endothelial cells, and activates expression of endothelial cell adhesion molecules [11, 34, 35]. Although the receptor for S100A8/A9 complex is still unknown, RAGE has been implicated in some circumstances [36]. Murine S100A8 stimulates proatherogenic activity, such as uptake of low-density lipoprotein (LDL), in macrophages. S100A8 is a key target of oxidation by peroxide, hypochlorite and nitric oxide [37, 38]. Furthermore, S100A9 and S100A12 are implicated in vascular damage, whereas sRAGE is associated with vascular protection in atherosclerosis [30, 39‐41].

The relationship between S100 protein levels and vascular disease or risk factors in RA patients has not been examined to date. We measured serum levels of S100A8, S100A9 heterocomplexes, S100A12 and sRAGE in a previously characterized cohort of established RA patients to identify their possible relationship to joint and vascular damage and risk factors in RA patients [19]. We report associations of each protein with both joint and vascular disease and their risk factors.

We found associations of sRAGE and S100 proteins with clinical inflammatory factors, complications, and CV risk factors in established RA patients. S100 A8, A9 and A12 were all elevated in serum from patients with established RA relative to healthy controls, and their levels were correlated in RA patients. By contrast, serum sRAGE levels did not differ in healthy controls and patients with established RA on treatment. Although a previous study reported reduced levels of sRAGE in RA compared to healthy control sera [17], it seems likely that we observed similar levels because of good control of inflammation in the RA group. In support of this, elevated serum sRAGE levels were generally associated with a more favorable vascular risk profile in our RA cohort, and potentially associated with concomitant reduction in proinflammatory and/or pro-atherogenic RAGE ligand binding.

Despite the generally negative association of sRAGE with vascular risk factors, the single factor that showed a reverse trend in multivariate models was serum HDL. Serum HDL levels were also negatively associated with serum sRAGE among diabetic subjects with CV disease [51]. In contrast, no association with serum HDL and sRAGE was found in patients with essential hypertension [16]. In spite of its known protective role, HDL can become proinflammatory [52‐54], and inflammatory HDL may increase the risk of atherosclerosis in SLE and RA patients [55, 56]. Moreover, HDL function, which is partly independent of HDL concentration, may be a more critical determinant of the atheroprotective capacity of HDL [57].

The positive association of S100A9 with S100A8 and S100A12 suggests that these proteins may be co-regulated in RA. This is supported by a previous study of S100 proteins in RA patients [58], and is in keeping with the high S100 gene expression profiles reported in RA [59]. Despite the positive associations between of S100A8, A9 and A12 levels, only S100A12 and S100A8 were associated with RA autoantibodies including ACPA and RF. Presence of ACPA or RF predicts a more aggressive RA disease course, including joint erosion and destruction [60]. Although S100A8/A9 from macrophages in RA patients amplified proinflammatory cytokine production in one study [11], the properties of serum S100A8/A9 are still debated. S100A8/A9 expression is seen in macrophages at the cartilage-pannus junction in RA, and expression of S100A8 significantly increased in macrophages in RA patients treated with high dose glucocorticoids compared to pre-treatment samples [61, 62]. Interestingly, glucocorticoids amplify LPS-induced S100A8 transcription in macrophages in an interleukin 10-dependent manner. Since we found S100A9 levels were associated with body mass index, it will be of interest to explore the relationship of this protein with endogenous or exogenous glucocorticoids. Recently, S100A9 or S100A8/A9 were reported to promote de-differentiation of dendritic cells and macrophages to myeloid suppressor cells in a tumor-bearing mouse model, suggesting anti-inflammatory effects of S100A9 which may reduce antigen-specific priming, for example, of cytotoxic T cell responses [63]. In support of an anti-inflammatory role for S100A9, S100A8 induced TNF in murine bone marrow cells through TLR4 signaling, and S100A9 negated this activity [64]. We found S100A9 to be associated with dystrophic calcification [39], which may be of relevance to atherosclerotic disease, and warrants future investigation in RA. Thus, the S100A8/A9 complex might have anti-inflammatory properties, or may be related to repair function in damaged or inflamed joints and vessels. It is also plausible that S100A8/A9 has variable effects depending on the presence of other disease factors or treatments. Finally, our assay measured S100A9, whether monomeric or heterocomplexed with S100A8. The ratio of S100A8:A9 may also play a role, given that the heterocomplex can have functions distinct from each protein alone.

In patients with Kawasaki disease, or with chronic hyperglycemia, serum levels of S100A12 were inversely associated with serum levels of sRAGE [65, 66]. Although the inverse correlation between sRAGE and S100A12 did not achieve statistical significance in the current study, the associations we found suggest opposing effects on RA severity. S100A12 has potent inflammatory effects. In chronic inflammatory arthritis, S100A12 is expressed by infiltrating granulocytes and by synovial macrophages, is a potent monocyte chemoattractant and activates mast cells to sequester them in inflammatory lesions [25, 29].

Our analysis indicates that sRAGE and S100 proteins are associated not only with RA inflammation and autoantibody production, but also with the recruitment of classical vascular risk factors to end-organ damage. This association with vascular risk supports previous reports of low sRAGE and high S100A8/A9 and S100A12 levels in patients with type 1 and type 2 diabetes, and essential hypertension [16, 66‐68]. These data support evidence from clinical studies of atherosclerosis, suggesting that the roles of classical risk factors and inflammation are difficult to separate in RA [69]. As we observed here, increasing sRAGE levels are associated with a favorable vascular risk profile, potentially associated with concomitant reduction in proinflammatory and/or pro-atherogenic RAGE ligand binding [70, 71].

Finally, we observed a novel association of low sRAGE levels with presence of the RAGE 82Ser polymorphism, which is found more frequently in RA patients [15, 19]. It is conceivable that this, or other linked polymorphisms in the RAGE gene affect splicing of the C-truncated, endogenously secreted form of the receptor, or susceptibility to cell surface RAGE cleavage by matrix metalloproteinases [72], thus altering the ratio of soluble to membrane RAGE.

Several studies have been published which demonstrate the role of sRAGE, S100A8/A9 and S100A12 in the long-term development of vascular disease. These include a negative association between sRAGE levels and coronary artery disease in non-diabetic men [41], prediction of unstable plaque by S100A8/A9 levels in acute coronary syndromes [73], and of accelerated atherosclerosis by high levels of S100A12 in hemodialysis patients [74]. However, this is the first time such an association has been shown with CVD and RA.

sRAGE and S100 proteins were associated with RA inflammatory factors and autoantibody production, and with the recruitment of classical vascular risk factors to end-organ damage. Consistent with its role as a RAGE decoy molecule, sRAGE had opposing effects to S100A12 and S100A8 in RA. Our data suggest that RAGE may mediate a key pathway coordinating conventional risk factors in the inflammatory RA setting for co-development of joint and vascular disease. Prospective studies will be of interest to determine the utility of these proteins as prognostic biomarkers of joint and vascular damage.