Lucie Andrés Cerezo

Vaspin and omentin: new adipokines differentially regulated at the site of inflammation in rheumatoid arthritis

Scientific interest in adipose tissue-derived peptides has increased dramatically in recent years (1). Several mediators known as adipo(cyto)kines were first associated with the pathophysiology of obesity-related complications; however a significant role for adipokines such as leptin, adiponectin, resistin and visfatin in regulating immune responses and inflammation has recently been discovered (1, 2). Several reports (3-6) have already demonstrated association of these adipokines with the severity of rheumatoid arthritis (RA). Vaspin, a member of the serine protease inhibitor family, and omentin (also known as intelectin) were recently identified in adipose tissue (7, 8). Vaspin is an adipokine with insulin-sensitizing effects that has been suggested to be a compensatory mediator for abrogating obesity and its inflammatory complications (7). Expression of the omentin gene was demonstrated in omental adipose tissue of patients with Crohn’s disease, suggesting that it may be implicated in chronic inflammatory diseases (8). The aim of the present report was to compare local concentrations of vaspin and omentin in synovial fluid of RA patients with those in osteoarthritis (OA) patients and to characterize their potential association with the severity of the disease. Synovial fluid was obtained during therapeutic arthrocentesis from 33 patients with RA and 33 patients with knee OA. The disease activity of RA patients was assessed by DAS28. C-reactive protein (CRP), IgM-rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) were routinely analyzed from peripheral blood obtained at the time of arthrocentesis. Characteristics of the patients are given in Table 1. Vaspin (AdipoGen Inc. Korea) and omentin (Apotech Corporation) were analyzed in synovial fluid by ELISA assays. Ethical approval was obtained from the local Ethics Committee and all patients provided informed consent. Statistical analysis was performed using GraphPad Prism 5.0 software. To meet a normal distribution, vaspin and omentin concentrations were naturaly logarithmically (log)-transformed. Differences between two independent parameters were determined by Kruskal-Wallis or T-test. The Spearman test was used for correlation of parameters. As shown in figure 1, the mean (SD) levels of vaspin were significantly higher in the synovial fluid of RA patients than in OA patients (-2.439±1.226 vs. -3.366±1.318 (log) pg/ml; p=0.003), but interestingly the levels of omentin were significantly lower in the synovial fluid of RA patients compared to OA patients (1.491±0.948 vs. 1.964±0.902 (log) ng/ml; p=0.045). After log-transformation, synovial fluid vaspin, but not omentin, had a tendency to correlate with DAS28 (r=0.320, p=0.070) in RA patients. However, neither vaspin nor omentin correlated with serum CRP or leukocyte counts in synovial fluid. In addition, levels of synovial fluid omentin, but not vaspin, significantly correlated with serum ACPA (r=0.398, p=0.029) and IgM-RF (r=0.592, p<0.001). The levels of synovial fluid vaspin and omentin were not affected by body mass index (BMI) or age of the patients. The mean concentration of synovial fluid vaspin, but not omentin, was twice as high in female as in male patients, but possibly due to the low number of male patients in this study, it failed to reach statistical significance. Our data shows different levels of the new adipokines vaspin and omentin at the site of local inflammation. We demonstrate here for the first time elevated levels of vaspin and reduced levels of omentin in synovial fluid of patients with RA compared with those with OA.

Decreases in serum levels of S100A8/9 (calprotectin) correlate with improvements in total swollen joint count in patients with recent-onset rheumatoid arthritis

IntroductionThe aim of this study was to examine the serum levels of S100 proteins and to evaluate their role in patients with recent-onset rheumatoid arthritis (RA).MethodsSerum levels of S100A8/9 and S100A12 were analysed in 43 patients with recent-onset RA, both before and three months after the initiation of conventional treatment, as well as in 32 healthy individuals. Disease activity was assessed based on serum levels of C-reactive protein (CRP), the Disease Activity Score for 28 joints (DAS28) and the total number of swollen joints count for 66 joints (SJC).ResultsThe levels of serum S100A8/9 and S100A12 were significantly higher in patients with recent-onset RA compared to the levels in healthy individuals (P < 0.0001) and normalised after three months of treatment. Using age- and sex-adjusted analysis, S100A8/9 levels were correlated with CRP (r = 0.439, P < 0.01), DAS28 (r = 0.501, P = 0.002) and SJC (r = 0.443, P = 0.007), while S100A12 was less significantly correlated with these parameters. Higher levels of S100A8/9 at baseline predicted improvement in the levels of CRP and SJC over time. Moreover, decreases in serum S100A8/9 were associated with decreased serum levels of CRP (r = 0.459, P = 0.005) and improvements in SJC (r = 0.459, P = 0.005). In multiple linear regression analyses, decreases in S100A8/9 but not CRP were significant predictors for improvements in SJC (P = 0.001).ConclusionsThis study is the first to show normalisation of elevated S100 proteins in patients with recent-onset RA after the initiation of conventional treatment. Therefore, S100A8/9 might potentially be a predictive marker for improvement in the total number of swollen joints in patients in the early phase of RA.

The metastasis-associated protein S100A4 promotes the inflammatory response of mononuclear cells via the TLR4 signalling pathway in rheumatoid arthritis

OBJECTIVES S100A4 has been implicated in cancer and several inflammatory diseases, including RA. The aim of the present study was to determine whether S100A4 can stimulate proinflammatory cytokine production in mononuclear cells. METHODS Peripheral blood mononuclear cells (PBMCs) isolated from patients with RA were stimulated with S100A4, S100A8, S100A9 and S100A12. The production of IL-1β, IL-6 and TNF-α was measured by ELISA. Receptor for advanced glycation end products (RAGEs) and Toll-like receptor 4 (TLR4) signalling were examined. For signalling pathway blocking studies, inhibitors of myeloid differentiation primary response gene 88 (MyD88), nuclear factor kappa B (NF-κB) and the mitogen activated protein (MAP) kinases p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) were used. MAP kinase activation was evaluated by western blotting. RESULTS Stimulation of PBMCs with S100A4 significantly up-regulated IL-1β, IL-6 and TNF-α production compared with unstimulated cells (P < 0.001). Importantly, the production of these cytokines was markedly enhanced in response to S100A4 compared with S100A8 and S100A12; however, it was less pronounced compared with S100A9. Furthermore, enhanced production of proinflammatory cytokines in S100A4-stimulated PMBCs was at least partly mediated via TLR4, but not RAGEs, and by activation of the transcription factor NF-κB and the MAP kinases p38 and ERK1/2. CONCLUSION This is the first study to demonstrate that S100A4 can induce an inflammatory response mediated by TLR4 and by the activation of NF-κB and the kinases p38 and ERK1/2 in mononuclear cells from patients with RA. Therefore S100A4 may be a potential therapeutic target for immune-mediated diseases.

The level of serum visfatin (PBEF) is associated with total number of B cells in patients with rheumatoid arthritis and decreases following B cell depletion therapy.

OBJECTIVE Visfatin, also known as pre-B cell colony-enhancing factor, was recently characterized as a potent pro-inflammatory mediator in rheumatoid arthritis (RA). The aim of this study was to determine the effect of B cell depletion with rituximab on serum visfatin levels in patients with active RA. METHODS We evaluated 31 patients with RA starting rituximab therapy at baseline and after 16 and 24 weeks using disease activity score (DAS28). The control group consisted of 33 gender and age-matched healthy individuals. CD19(+) B cells were assessed by flow cytometry and serum levels of visfatin and B cell-activating factor of the TNF family (BAFF) were measured by ELISA at baseline and week 16. RESULTS Total number of B cells correlated positively with serum visfatin levels (rs=0.417, P=0.025) and negatively with serum BAFF levels (rs=-0.486, P=0.008) at baseline. Serum visfatin levels were significantly higher in patients with RA compared with healthy controls (P=0.026), and significantly decreased (P=0.010), while BAFF increased (P<0.001), and both proteins became negatively correlated following treatment with rituximab (rs=-0.438, P=0.017). Visfatin levels did not correlate with the disease activity, but lack of change in the serum visfatin levels between baseline and week 16 predicted worsening disease activity between weeks 16 and 24 (rs=0.452, P=0.014). CONCLUSION In patients with active RA, serum visfatin levels are related to the number of B cells rather than to disease activity and decrease in response to treatment with rituximab. Further studies are necessary to show if visfatin is a marker with predictive value for deterioration of RA.

The metastasis promoting protein S100A4 is increased in idiopathic inflammatory myopathies

OBJECTIVES The S100A4 protein is known as a metastasis promoting factor; however, its involvement in non-malignant diseases such as RA and psoriasis has been recently described. The aim of this study was to investigate the expression and possible role of S100A4 in idiopathic inflammatory myopathies. METHODS S100A4 protein expression was detected by immunohistochemistry in muscle tissue from control individuals (n = 11) and patients with PM and DM (n = 8/6). IF staining was used to co-localize S100A4 with selected cells. Cytokine expression and protein synthesis in S100A4-treated cells were analysed by RT-PCR and ELISA. RESULTS S100A4 protein was significantly up-regulated in muscle tissue of patients with inflammatory myopathies compared with control individuals and was associated particularly with the presence of mononuclear infiltrates. Only few regenerating muscle fibres in PM/DM expressed S100A4. Then we analysed the effect of S100A4 on human myocytes and peripheral blood mononuclear cells (PBMCs). Although S100A4 did not affect myocytes, stimulation of PBMCs with S100A4 significantly induced the expression and synthesis of TNF-α, IL-1β and IL-6, but not of IFN-α. We showed that S100A4 is not directly involved in perforin/granzyme B-induced apoptosis and that it does not modulate the expression of Bax and Bcl2 mRNA in myocytes and PBMCs. CONCLUSION Increased expression of S100A4 in inflamed muscle tissue highlights its potential role in the pathogenesis of inflammatory myopathies. S100A4 may act as a cytokine-like factor indirectly promoting muscle fibre damage by stimulating mononuclear cells to increase the synthesis of pro-inflammatory cytokines.

S100A4 amplifies TGF-β-induced fibroblast activation in systemic sclerosis

Objectives S100A4 is a calcium binding protein with regulatory functions in cell homeostasis, proliferation and differentiation that has been shown to promote cancer progression and metastasis. In the present study, we evaluated the role of S100A4 in fibroblast activation in systemic sclerosis (SSc). Methods The expression of S100A4 was analysed in human samples, murine models of SSc and in cultured fibroblasts by real-time PCR, immunohistochemistry and western blot. The functional role of S100A4 was evaluated using siRNA, overexpression, recombinant protein and S100A4 knockout (S100A4−/−) mice. Transforming growth factor β (TGF-β) signalling was assessed by reporter assays, staining for phosphorylated Smad2/3 and analyses of target genes. Results The expression of S100A4 was increased in SSc skin and in experimental fibrosis in a TGF-β/Smad-dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, knockdown of S100A4 reduced the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4−/− mice were protected from bleomycin-induced skin fibrosis with reduced dermal thickening, decreased hydroxyproline content and lower myofibroblast counts. Deficiency of S100A4 also ameliorated fibrosis in the tight-skin-1 (Tsk-1) mouse model. Conclusions We characterised S100A4 as a downstream mediator of the stimulatory effects of TGF-β on fibroblasts in SSc. TGF-β induces the expression of S100A4 to stimulate the release of collagen in SSc fibroblasts and induce fibrosis. Since S100A4 is essentially required for the pro-fibrotic effects of TGF-β and neutralising antibodies against S100A4 are currently evaluated, S100A4 might be a candidate for novel antifibrotic therapies.

Resistin in idiopathic inflammatory myopathies

IntroductionThe purpose of this study was to evaluate and compare the serum levels and local expression of resistin in patients with idiopathic inflammatory myopathies to controls, and to determine the relationship between resistin levels, inflammation and disease activity.MethodsSerum resistin levels were determined in 42 patients with inflammatory myopathies and 27 healthy controls. The association among resistin levels, inflammation, global disease activity and muscle strength was examined. The expression of resistin in muscle tissues from patients with inflammatory myopathies and healthy controls was evaluated. Gene expression and protein release from resistin-stimulated muscle and mononuclear cells were assessed.ResultsIn patients with inflammatory myopathies, the serum levels of resistin were significantly higher than those observed in controls (8.53 ± 6.84 vs. 4.54 ± 1.08 ng/ml, P < 0.0001) and correlated with C-reactive protein (CRP) levels (r = 0.328, P = 0.044) and myositis disease activity assessment visual analogue scales (MYOACT) (r = 0.382, P = 0.026). Stronger association was observed between the levels of serum resistin and CRP levels (r = 0.717, P = 0.037) as well as MYOACT (r = 0.798, P = 0.007), and there was a trend towards correlation between serum resistin and myoglobin levels (r = 0.650, P = 0.067) in anti-Jo-1 positive patients. Furthermore, in patients with dermatomyositis, serum resistin levels significantly correlated with MYOACT (r = 0.667, P = 0.001), creatine kinase (r = 0.739, P = 0.001) and myoglobin levels (r = 0.791, P = 0.0003) and showed a trend towards correlation with CRP levels (r = 0.447, P = 0.067). Resistin expression in muscle tissue was significantly higher in patients with inflammatory myopathies compared to controls, and resistin induced the expression of interleukins (IL)-1β and IL-6 and monocyte chemoattractant protein (MCP)-1 in mononuclear cells but not in myocytes.ConclusionsThe results of this study indicate that higher levels of serum resistin are associated with inflammation, higher global disease activity index and muscle injury in patients with myositis-specific anti-Jo-1 antibody and patients with dermatomyositis. Furthermore, up-regulation of resistin in muscle tissue and resistin-induced synthesis of pro-inflammatory cytokines in mononuclear cells suggest a potential role for resistin in the pathogenesis of inflammatory myopathies.

Progranulin Is Associated with Disease Activity in Patients with Rheumatoid Arthritis

Objective. Progranulin (PGRN) is implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to assess the relationship between PGRN and disease activity in RA. Methods. PGRN levels were evaluated in patients with RA (n = 47) and OA (n = 42) and healthy controls (n = 41). Immunohistochemical analysis of PGRN in synovial tissues was performed. The association between PGRN and C-reactive protein (CRP), disease activity score (DAS28-CRP), and health assessment questionnaire (HAQ) was studied. Results. Circulating PGRN was elevated in patients with RA and OA compared to healthy controls (227.1 ± 100.2 and 221.5 ± 102.5 versus 128.1 ± 34.7 ng/mL; P < 0.001). Synovial fluid levels of PGRN were higher in patients with RA compared to OA (384.5 ± 275.3 versus 241.4 ± 165.2 ng/mL; P = 0.002). PGRN expression was significantly upregulated in the synovial tissue of RA patients particularly in the inflammatory infiltrates. Serum PGRN levels correlated with DAS28 (r = 0.327, P = 0.049) and HAQ score (r = 0.323, P = 0.032), while synovial fluid PGRN correlated only with HAQ (r = 0.310, P = 0.043) in patients with RA. PGRN levels were not associated with CRP or autoantibodies. Conclusions. This study demonstrates increased PGRN expression at local sites of inflammation and association between PGRN levels, disease activity, and functional impairment in patients with RA.

Adipokine profile is modulated in subcutaneous adipose tissue by TNFα inhibitors in patients with rheumatoid arthritis

A protective effect of obesity showing that increased adiposity protects against radiographic joint damage in patients with rheumatoid arthritis (RA) has been reported.1 2 Recently, adipokines were suggested as a molecular link explaining this paradoxical association.3,–,5 The levels of serum adiponectin and visfatin have been shown to be associated with increased, radiographic joint damage in RA, while leptin is associated with reduced radiographic joint damage.4 5 Therefore, we tested whether tumour necrosis factor α (TNFα) inhibitor therapy, which prevents joint damage in majority of patients with RA, may be associated with a change in the local production of adipokines in subcutaneous adipose tissue (SAT). SAT samples were obtained from the same abdominal region by aspiration with a bioptic needle in nine patients with RA prior to and 6 months after the treatment with etanercept. Adipose tissue samples were …

The level of fatty acid-binding protein 4, a novel adipokine, is increased in rheumatoid arthritis and correlates with serum cholesterol levels

OBJECTIVE To assess the expression of the novel adipokine Fatty Acid Binding Protein-4 (FABP4) in synovial tissues, serum and the synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to study the relationships among FABP4, disease activity and metabolic status. METHODS FABP4 levels were measured in the serum and synovial fluid of 40 patients with RA and 40 control patients with OA. The disease activity score (DAS28), C-reactive protein (CRP) levels and serum lipids were assessed in patients with RA. Immunohistochemical analysis and confocal microscopy were used to study the expression and cell-specific distribution of FABP4 in synovial tissues. RESULTS The age, sex and body mass index (BMI) adjusted levels of FABP4 were significantly higher in the serum (p=0.001) and synovial fluid (p=0.005) of patients with RA when compared to OA patients. FABP4 levels were higher in females than in males and correlated positively with body mass index (BMI) in patients with RA. Independent of confounders, FABP4 levels correlated with total cholesterol and LDL cholesterol in patients with RA, but not in OA patients. FABP4 levels were not affected by disease activity. Furthermore, the increased expression of FABP4 that was otherwise restricted to synovial fibroblasts, macrophages and B-cells was noted in RA patients at levels higher than that observed in OA patients. CONCLUSIONS The observed elevation of FABP4 levels in RA patients and the positive correlation of the adipokine to cholesterol suggest that FABP4 may represent a potential link between RA and the increased risk of atherosclerotic changes.