G. Santoboni

Characterization of circulating endothelial microparticles and endothelial progenitor cells in primary Sjögren’s syndrome: new markers of chronic endothelial damage?

OBJECTIVE Chronic autoimmune diseases are associated with increased risk of cardiovascular death. Endothelial dysfunction represents the first stage of subclinical atherosclerosis and multiple factors contribute to endothelial injury. Among these, an altered balance between endothelial microparticle (EMP) release and endothelial progenitor cell (EPC) generation promotes endothelial dysfunction. The role of EMPs and EPCs in promoting endothelial damage in primary SS (pSS) has never been investigated. Our aim was to evaluate the role of EMPs and EPCs as markers of endothelial damage in pSS and their correlation with disease clinical and immunological features. METHODS Circulating EMPs (CD31(+)/CD42(-)), true EPCs (CD34(+)/KDR(+)/CD133(+)) and mature EPCs (CD34(+)/KDR(+)/CD133(-)) were quantified by FACS analysis in 34 pSS patients and 18 age- and sex-matched controls. Correlation between EMP and EPC levels and parameters of disease activity and damage, clinical features and markers of immunological dysfunction was performed. RESULTS Patients displayed higher EMP numbers with respect to healthy controls [HCs; mean 450 n/μl (S.D. 155) vs 231 (110), P < 0.0001]. EPC and mature EPC levels were higher in patients compared with HCs [mean 226 n/ml (S.D. 181) vs 69 (53), P < 0.001 and 166 (161) vs 36 (32), P < 0.0001, respectively). EMP levels directly correlated with disease duration from symptoms and diagnosis (ρ = 0.5, P < 0.01). Early EPCs inversely correlated with disease duration from symptoms (ρ = -0.5, P < 0.01) and diagnosis (ρ = -0.4, P < 0.05). CONCLUSION This is the first demonstration of chronic endothelial fragmentation characterizing pSS. The reparative potentiality of the endothelial layer appears to be preserved in the earliest stages of disease. During the course of the disease, progressive exhaustion of the precursor endothelial pool may be hypothesized, leading to defective vascular layer restoration and endothelial dysfunction.

Atherosclerotic vascular damage and rheumatoid arthritis: a complex but intriguing link

Rheumatoid arthritis is a chronic inflammatory disease characterized by a reduced life expectancy mainly due to cardiovascular disease. In long-standing disease, it has been widely demonstrated that both traditional cardiovascular risk and disease-related factors, including chronic inflammation and immune-mediated mechanisms, play a key role in accelerating atherosclerotic damage of the arterial wall. The short- and long-term effects of immunosuppressive treatment on cardiovascular disease outcome is, however, uncertain and a multidisciplinary approach appears to represent the best management of cardiovascular risk in these patients.

Beneficial Cardiovascular Effects of Low-dose Glucocorticoid Therapy in Inflammatory Rheumatic Diseases

To the Editor: We read with great interest the report by Mazzantini, et al 1. That retrospective analysis of a large cohort of patients with polymyalgia rheumatica (PMR) demonstrated that duration or cumulative dose of longterm low-dose glucocorticoid (GC) therapy was significantly associated with higher risk of arterial hypertension and acute myocardial infarction. However, in a multivariate analysis adjusted for traditional cardiovascular (CV) risk factors, arterial hypertension was confirmed as the only adverse effect significantly associated with treatment duration. It is thought that longterm GC treatment, especially at high dose, may indirectly increase the risk of CV disease through its well-recognized effect on traditional CV risk factors, including arterial hypertension, dyslipidemia, hyperglycemia, and obesity2. As summarized in Table 1, GC have been associated with higher incidence of subclinical atherosclerosis, and their detrimental effects on the CV system seem to occur at a preclinical phase. Although results are not uniform, duration of treatment exposure and higher cumulative … Address correspondence to Dr. Gerli; E-mail: gerlir{at}unipg.it

FRI0180 Correlation Between Clinimetric Approach and German US7 Score in Rheumatoid Arthritis Patients Treated with Tocilizumab: A Pilot Study

Background Tocilizumab (TCZ) is a humanized monoclonal antibody against interleukin (IL)-6 receptor employed in rheumatoid arthritis (RA). The inhibition of acute phase reactant liver production is associated with rapid normalization of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), often predating synovitis improvement. Thus, ESR and CRP are not suitable to assess drug efficacy. Joint ultrasonographic (US) examination is commonly employed to detect RA subclinical synovitis. However, US assessment of the whole articular sites may be time consuming. To overcome such limitation, a novel US score, the German US7 score, has been recently proposed as rapid method to assess synovitis, tenosynovitis, power Doppler (PD) signal and erosions by grey scale (GS) in seven joints. Objectives Since the strong effect of TCZ on acute phase reactants may underestimate disease activity, we investigated German US7 usefulness in detecting subclinical synovitis in TCZ-treated patients. Methods Sixteen RA patients diagnosed according the EULAR/ACR 2010 classification criteria and treated with TCZ since at least 3 months were enrolled. Disease activity was evaluated by DAS28 and HAQ. German US7 score assessment was performed by two blinded investigators (Esaote myLab 70; linear probe 10-18 Mhz). The following sites of the clinically dominant hand and foot were evaluated: wrist, 2nd and 3rd metacarpophalangeal and proximal interphalangeal, and 2nd and 5th metatarsophalangeal joints. Details of the score are provided in Table 1. Inter-observer agreement was very high (Cohens κ=0.9). Spearman correlation coefficient (rho, ρ) was assessed to evaluate correlation between clinic parameters, laboratory data and domains of German US7 score. Results As shown in Table, GSUS synovitis significantly correlated with DAS28-ESR (ρ=0.557; p<0.05), DAS28-CRP (ρ=0.721; p<0.019 and HAQ (ρ=0.634; p<0.01). GSUS tenosynovitis correlated with DAS28-ESR and DAS28-CRP (ρ=0.726; p<0.01 and ρ=0.668; p<0.01, respectively). GSUS erosions correlated with DAS28-ESR (ρ=0.535; p<0.05). When each DAS28 item was considered separately, tender and swollen joint number correlated with GSUS synovitis (ρ=0.744; p<0.001 and ρ=0.605; p<0.01, respectively), GSUS tenosynovitis (ρ=0.626; p<0,01 and ρ=0.527 p<0.05, respectively) and PDUS synovitis (ρ=0.488; p<0.05 and ρ=0.577; p<0.05, respectively). No association between ESR and CRP and German US 7 score domains was observed. Conclusions The present study suggests that German US7 score is a rapid tool to assess subclinical disease activity in RA patients receiving TCZ where ESR and CRP are not suitable to assess drug efficacy. US evaluation of TCZ-treated patients correlates with changing of clinical parameters, is able to detect subclinical activity and represents a prognostic parameter for new erosion development. In addition, our data, showing a strong correlation between number of tender and swollen joints and presence of PD signal, further support PD usefulness as independent diagnostic and prognostic tool in the evaluation of TCZ-treated patients. Despite limitations due to low number of enrolled patients and cross-sectional design, this study supports the role of German US7 score as a simple, reproducible and time-effective tool to monitor clinical and functional response in RA patients receiving TCZ. Disclosure of Interest None declared

A3.1 Pathogenic role of IL-17 producing double negative (DN) T cells in primary sjögren’s syndrome

Background and Objectives We recently demonstrated that a CD3+ T cell population, that lacks both CD4 and CD8 molecules, defined as double negative (DN), is expanded in the peripheral blood of patients with long-standing primary Sjögren’s syndrome (pSS), produces IL-17, accumulates in minor salivary glands (MSGs) and is resistant to corticosteroids (CS) in vitro. Since IL-17 represents a key cytokine in the pathogenesis of pSS, we aimed to investigate glandular and peripheral DN T cells in early phases of the disease in order to verify a possible correlation with clinical parameters, MSGs histological patterns and possibly ectopic lymphoneogenesis. Materials and Methods Paired samples of peripheral blood mononuclear cells and MSGs from early pSS patients were evaluated by flow cytometry and immunofluorescence staining respectively to quantify DN T cells. Histological analysis to assess histological scores, B/T cell segregation and the presence of germinal center-like structures (GCs) was also performed. Ten patients with long-standing disease and 15 normal controls (NC) were also enrolled. Disease activity was assessed by the EULAR Sjögren’s syndrome disease activity index (ESSDAI) and patient reported dryness, fatigue, pain and global disease activity were recorded on a 100 millimeters (mm) visual analogic scale (VAS). Unstimulated salivary flow was also collected. Results In early stages of pSS, circulating DN T cells appeared not to be expanded as it occurs in patients with long-standing disease and were inversely correlated to circulating CD4+Th17 cells. The number of infiltrating DN T cells was associated with the extent of glandular involvement, defined with Tarpley biopsy score, and with the presence of GCs. Furthermore, dryness symptoms were directly correlated with glandular DN T cells and inversely correlated with circulating DN T cells. Circulating DN T cells displayed an in vivo activated phenotype, confirmed by the expression of CD25, CD69 and HLA-DR, which was not affected by the addition of CS to the culture, as it occurred for IL-17 expression. Conclusions Our findings suggest that DN T cells are in vivo activated Th17 cells involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS. Furthermore, DN T cells may play a role in ectopic lymphoneogenesis development occurring during the disease.

SAT0030 Circulating Endothelial Microparticles and Endothelial Progenitors Cells in Primary SjÖGren's Syndrome: New Markers of Chronic Endothelial Damage?

Background Subclinical atherosclerosis can be considered a form of systemic involvement in patients with chronic inflammatory joint and connective tissue diseases. Multiple factors, including inflammatory and immune-mediated mechanisms, have been identified to promote atherosclerotic endothelial injury [1]. Recent studies focused on role played by the altered balance between endothelial microparticles (EMPs) release and endothelial progenitor cells (EPCs) generation in endothelial wall integrity disruption and subsequent endothelial dysfunction. EPCs represent a new marker of endothelial dysfunction cardiovascular (CV) disease patients and increased EMP levels have been demonstrated in diseases characterized by high inflammatory response, such as polymyalgia rheumatica [2]. There is evidence that primary Sjögrens syndrome (pSS) patients, free of previous CV manifestations, display signs of subclinical atherosclerosis, suggesting a greater CV risk [3,4]. However, exact pathogenic mechanisms involved in vascular damage in SS are still unclear. Increased levels of leukocyte and platelet MPs, reflecting systemic cell activation, have been demonstrated in SS. On the other hand, the role of EMPs and EPCs in SS has been never investigated. Objectives To evaluate endothelial injury degree in SS by EMP quantification and their repair potential by EPC and mature EPC measurement and to analyze possible correlation with disease-specific clinical and immunologic features. Methods 34 female pSS patients and 18 age- and sex-matched normal controls (NC) were enrolled. Number of circulating EMPs (CD31+/CD42–), EPCs (CD34+/KDR+/CD133+) and mature EPCs (CD34+/KDR+/CD133–) was quantified by FACS analysis. Parameters of disease activity and damage were measured by ESSDAI and SSDDI, respectively. Disease-related clinical features, laboratory markers of immunologic dysfunction and traditional CV risk factors were recorded. Results SS patients displayed higher EMP number with respect to NC (450±155 vs 231±110 n/μl mean ± SD; p<0.0001). Mean concentrations of EPCs as well as mature EPCs resulted significantly higher in patients in comparison to NC (226±181 vs 69±53 n/mL mean ± SD; p<0.001 and 166±161 vs 36±32 n/mL mean ± SD; p<0.0001, respectively). SS patients exhibited a significantly lower EPC/mature EPC ratio in comparison to NC (p<0.01). EMP levels directly correlated with disease duration from symptoms and diagnosis (rho=0.5; p<0.01). EPCs inversely correlated with disease duration from symptoms (rho= -0.5; p<0.01) and diagnosis (rho= -0.4; p<0.05). Conclusions Present data highlight for the first time a chronic persistent endothelial fragmentation characterizing pSS patients. Endothelial layer reparative potentiality appears to be preserved in the earliest stages of disease. Following disease course, a progressive exhaustion of the precursor endothelial pool may be hypothesized, leading to defective vascular layer restoration and endothelial dysfunction. Further investigations are needed to ascertain the role of EMPs and EPCs in promoting subclinical atherosclerosis in pSS. References Bartoloni E et al. Arthritis Care Res 2011;63:178 Pirro M et al. J Intern Med 2012;272:177 Vaudo G et al. Arthritis Rheum 2005;52:3890 Gerli R et al. Arthritis Care Res 2010;62:712 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2887

THU0194 Role of Platelets in the Pathogenesis of Osteoarthritis and Biological Effects of Hyaluronic Acid: in Vivo and in Vitro Study

Background Although osteoarthritis (OA) is a degenerative joint disorder, it has been demonstrated that inflammation takes part in the development and progression of this condition. Matrix metalloproteinases (MMPs), including MMP-2, are involved in the cartilage breakdown observed in OA. Platelets (PLTs) are involved in different inflammatory conditions, including rheumatoid arthritis, contain and release several MMPs, including MMP-2, and enhance MMPs expression by other cell types. However, their role in OA has not been investigated yet. Hyaluronic acid (HA) is a normal component of extracellular matrix and synovial fluid (SF). Intra-articular (IA) administration of HA in OA is widely employed in clinical practice with good clinical efficacy. Objectives Aims of our study were to investigate the role of platelets and MMP-2 in the pathogenesis of OA, to test the biologic activity of IA-HA in OA patients and to analyze the effects of HA on the interaction between platelets and synoviocytes in vitro. Methods Thirty-eight patients with knee OA were screened and twenty-four patients were included in the study. Systemic therapy with non-steroidal anti-inflammatory drugs or corticosteroids and previous IA corticosteroid treatment represented exclusion criteria. Five injections of IA sodium hyaluronate (500-730 KDa) were administrated weekly. SF samples were collected prior to the first (T0) and at the last (T1) injection. Total cell and PLT count, platelet-leukocyte aggregates (PLT activation marker) and MMP-2 levels were measured in SF. For clinical assessment at T0, T1 and 3 months after the last injection (T3) the Western Ontario and Mc Masters University (WOMAC) scale and VAS for knee pain were employed. Fibroblast-like synoviocytes (FLS) were isolated from untreated OA-SF and cultured with PLTs in presence or absence of HA at different concentrations for 24 hours. Platelet P-selectin was measured by flow-cytometry and MMP-2 in culture supernatants was quantified by zymography. Results Total cell count in SF was significantly reduced after IA-HA (p<0.05). PLT count, platelet-leukocyte aggregates and MMP-2 concentration were significantly decreased at T1 (all p<0.05). WOMAC scale and pain VAS were reduced at T1 (p<0.0001) and this decrease was maintained at T3. MMP-2 production by FLS was significantly higher in the presence of PLTs and this increase was significantly reduced by co-incubation with HA. P-selectin expression, marker of PLT activation, was significantly higher in PLTs cultured with FLS and P-selectin blockade reduced MMP-2 release in culture supernatants. Conclusions Our results show that PLTs, in addition to other inflammatory cells, participate in the pathogenesis of OA by the induction of MMP-2 release by FLS possibly via P-selectin, thus contributing to cartilage breakdown. Treatment with HA decreases count and activation of PLT and levels of MMP-2 in the SF. PLT and synoviocytes interaction leads to platelet activation and MMP-2 release enhancement. Hence, PLTs may represent a new therapeutic target in OA. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4585

A3.4 CD3+CD4–CD8– Double Negative Th17 Cells: New Insights in the Pathogenesis of Primary Sjögren’s Syndrome

Background and Objectives IL-17 axis is widely recognised to be involved in the pathogenesis of autoimmune disorders. Besides conventional CD4+ Th17 cells, a small IL-17 producing T-cell population, that lacks of both CD4 and CD8 molecules, defined as double negative (DN) was recently found to be expanded in the peripheral blood (PB) and to accumulate in the kidney in patients with lupus nephritis. Since IL-17 production is enhanced in minor salivary gland (MSG) infiltrates of patients with primary Sjögren’s syndrome (pSS), we sought to investigate whether DN T cells may be involved in pSS pathogenesis. Materials and Methods Thirty patients with pSS and 16 normal controls (NC) were studied. PBMCs were separated by density gradient and phenotypic characterisation was performed by flow cytometry on both freshly isolated cells and after culture (24, 48, 72 and 96 hours). Total PBMCs were cultured in anti-CD3 coated plates in presence or absence of dexamethasone (Dex) at different concentrations. In selected experiments, real time PCR at the same time-points was performed. The study of pSS-MSGs was performed by immunofluorescence. Results Total circulating DN T cells were increased in pSS compared to NC. NC and pSS freshly isolated DN T cells expressed RORγ t, activation markers (CD25, CD69, HLA-DR) and produced consistent amounts of IL-17. Despite IL-6/TGFβ ratio became abnormal in pSS patients after 72 hour-culture, Dex was able to down-regulate IL-17 in vitro production in NC and pSS CD4+Th17 cells and in NC DN T cells from this time-point on. Surprisingly, IL-17 production by pSS-DN T cells was not affected at all by Dex at any time-point. Dex could also reduce the expression of activation markers on CD4+ cells, but not in pSS and NC-DN T cells. Among DN T cells, those expressing αβTCR were expanded in patients with active pSS compared to those with inactive pSS. DN T cells were present in pSS-MSG infiltrate. Conclusions To our knowledge, this is the first study identifying and characterising DN T cells in pSS. It shows that DN T cells are expanded in the PB of pSS, display an in vivo activated Th17 phenotype, infiltrate MSG and are resistant to corticosteroids. Taken together, these data suggest a key role of this T-cell subset in the perpetuation of chronic sialoadenitis and eventually in SS prognosis and provide the clue to target DN T cells for therapeutic purposes in pSS.

THU0174 Endothelial microparticles and circulating endothelial progenitor cells in primary sjægren’s syndrome patients: A potential marker of endothelial damage?

Background Patients with systemic inflammatory and autoimmune diseases display an increased risk of cardiovascular (CV) mortality. Inflammatory and immune-mediated mechanisms have been identified to promote induction and progression of atherosclerotic endothelial injury (1). Sjögren’s syndrome (SS) represents an interesting model to investigate pathogenetic mechanisms involved in atherosclerotic damage. Young SS patients are characterized by an accelerated subclinical atherosclerotic damage and disease-related immune mechanisms may be involved in this process (2,3). However, the exact pathogenetic mechanisms involved in the precocious vascular damage in SS are still unknown. Recent evidence suggests that endothelial integrity loss may involve defects in the vascular regenerative capacity provided by circulating endothelial progenitor cells (EPC) and generation of circulating microparticles (MPs). Endothelial (E) MPs represent an emerging marker of endothelial dysfunction in patients with CV diseases and an increased number of EMPs have been demonstrated in diseases characterized by high inflammatory response, as polymyalgia rheumatica (4). Increased levels of leukocyte and platelet MPs have been recently demonstrated in SS and have been identified as biomarkers reflecting systemic cell activation. On the other hand, the role of EMPs in SS has never been investigated. Objectives Evaluate the degree of endothelial injury in SS patients by measuring number of circulating EMPs and their repair potential by EPC measurement. Methods 31 SS patients (30 female, 1 male, mean age 52±12 SD) and 31 age- and sex-matched normal controls (NC) were enrolled. Number of circulating EMPs (CD31+/CD42-) and EPC (CD34+/KDR+) was quantified by FACS analysis. Parameters of disease activity and damage were measured by ESSDAI (inactive ≤2, active >2) and SSDDI, respectively. Disease-related clinical features, laboratory markers of immunologic dysfunction and traditional CV risk factors were recorded. Results SS patients displayed higher levels of EMPs and lower count of EPC than NC (634±21548 vs 399±28 n/microL: p<0.001 and 203±16 vs 563±21 n/mL: p<0.001, respectively). No correlation was depicted between EMPs, EPCs and EMP/EPC ratio and parameters of disease activity/damage or disease-related clinical and immunologic features. Of interest, a correlation was demonstrated between number of cigarettes smoked and active disease (p=0.001). Conclusions This is the first demonstration of a significant imbalance between endothelial injury and repair in SS, as demonstrated by increased number of EMPs associated with EPC count reduction. In this setting, increased endothelial fragmentation in association with a reduced endothelial repair may advocated as adjunctive potential pathogenic mechanism promoting subclinical atherosclerotic damage in SS. Among traditional CV risk factors, smoke may be associated with an higher risk of active disease. References Bartoloni E et al. Arthritis Care Res 2011;63:178 Vaudo G et al. Arthritis Rheum 2005;52:3890 Gerli R et al. Arthritis Care Res 2010;62:712 Pirro M et al. J Intern Med 2011 [Epub ahead of print] Disclosure of Interest None Declared

THU0312 Endothelial Microparticles and Circulating Endothelial Progenitor Cells in Primary SjöGren’s Syndrome Patients: Correlation with Clinical and Immunological Features

Background Patients with systemic autoimmune diseases display an increased risk of cardiovascular (CV) mortality. Inflammatory and immune-mediated mechanisms have been identified to promote atherosclerotic endothelial injury (1). Sjögren’s syndrome (SS) represents an interesting model to investigate pathogenetic mechanisms involved in atherosclerotic damage. Young SS patients are characterized by an accelerated subclinical atherosclerotic damage (2,3). However, exact pathogenic mechanisms involved in the precocious vascular damage in SS are still unclear. Recent evidence suggests that loss of endothelial integrity may involve defects in the vascular regenerative capacity provided by circulating endothelial progenitor cells (EPC) and generation of circulating microparticles (MPs). Endothelial (E) MPs represent an emerging marker of endothelial dysfunction in patients with CV diseases and increased numbers of EMPs have been demonstrated in diseases characterized by high inflammatory response, such as polymyalgia rheumatica (4). Increased levels of leukocyte and platelet MPs have been demonstrated in SS and have been identified as biomarkers of systemic cell activation. On the other hand, the role of EMPs in SS has been never investigated. Objectives To evaluate endothelial injury degreein SS by quantification of circulating EMPs and their repair potential by EPCmeasurement and to analyze possible correlation with disease-specific clinical and immunologic features. Methods 49 SS patients (48 female, 1 male) and 30 age- and sex-matched normal controls (NC) were enrolled. Number of circulating EMPs (CD31+/CD42-) and EPC (CD34+/CD133+/KDR+ and mature CD34+/KDR+) was quantified by FACS analysis. Parameters of disease activity and damage were measured by ESSDAI (inactive ≤ 2, active > 2) and SSDDI, respectively. Disease-related clinical features, laboratory markers of immunologic dysfunction and traditional CV risk factors were recorded. Results SS patients displayed higher levels of EMPs and triple positive EPCs than NC (mean±SD: 565±223 vs 399±28 and 144±117 vs 74±54 n/mL, respectively, p<0.05) and lower count of mature EPCs than NC (234±188 vs 563±21 n/mL, p<0.05). The number of both triple positive and mature CD34+/KDR+ EPCs were inversely correlated with disease duration. Intriguingly, it was of interest the observation that, among patients, smokers displayed higher ESSDAI in comparison to non-smokers (p=0.004). Conclusions This is the first evidence that SS may be associated with endothelium integrity loss toward generation of EMPs. Progenitors seem to be adequately released, particularly in early stages of the disease, so suggesting an attempt at revascularization. However, their ability to mature differs from healthy subjects and this may contribute to endothelial dysfunction. Of interest, among traditional CV risk factors, smoking may be associated with an higher risk of active disease. Further investigations, however, are needed to ascertain the role of EMPs and EPCs in promoting subclinical atherosclerotic damage in SS. References Bartoloni E et al. Arthritis Care Res 2011;63:178 Vaudo G et al. Arthritis Rheum 2005;52:3890 Gerli R et al. Arthritis Care Res 2010;62:712 Pirro M et al. J Intern Med 2012;272:177 Disclosure of Interest None Declared