Giulia Mirabelli

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.

Cardiovascular disease in systemic sclerosis.

Cardiovascular (CV) system involvement is a frequent complication of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). It still remains unclear if a premature atherosclerosis (ATS) occurs even in systemic sclerosis (SSc). Although microvascular disease is a hallmark of SSc, in the last few years a number of studies highlighted a higher prevalence of macrovascular disease in SSc patients in comparison to healthy individuals and these data have been correlated with a poorer prognosis. The mechanisms promoting ATS in SSc are not fully understood, but it is believed to be secondary to multi-system organ inflammation, endothelial wall damage and vasculopathy. Both traditional risk factors and endothelial dysfunction have been proposed to participate to the onset and progression of ATS in such patients. In particular, endothelial cell injury induced by anti-endothelial antibodies, ischemia/reperfusion damage, immune-mediated cytotoxicity represent the main causes of vascular injury together with an impaired vascular repair mechanism that determine a defective vasculogenesis. Aim of this review is to analyse both causes and clinical manifestations of macrovascular involvement and ATS in SSc.

T Regulatory and T Helper 17 Cells in Primary Sjögren’s Syndrome: Facts and Perspectives

Historically, primary Sjögrens syndrome (pSS) was thought to be a T helper (h) 1 driven disease due to the predominance of CD4+T lymphocytes and their products in target organs and peripheral blood of patients. In the last decades, the identification of a number of T cell subsets, including Th17, T regulatory (Treg), and follicular helper T cells, challenged this long-standing paradigm and prompted to identify their role in pSS pathogenesis. In addition the impact of abnormal proinflammatory cytokine production, such as IL-6, IL-17, IL-22, and IL-23, has also attracted considerable attention. However, although several studies have been carried out in experimental models and patients with pSS, many aspects concerning the role of Treg cells and IL-17/Th17 cell system in pSS pathogenesis are not fully elucidated. In particular, the role played by different IL-17-producing T cell subsets as well as the effects of pharmacological therapies on Treg/Th17 cell balance represents an intriguing issue. The aim of this review article is to provide an overview of current knowledge on Treg cells and IL-17-producing T cells in pSS pathogenesis. We believe that these insights into pSS pathogenesis may provide the basis for successful therapeutic intervention in this disease.

Platelets Contribute to the Accumulation of Matrix Metalloproteinase Type 2 in Synovial Fluid in Osteoarthritis

Inflammation plays a role in the initiation and progression of osteoarthritis (OA), a chronic degenerative joint disorder. Platelets are inflammatory cells, contain and release matrix metalloproteinases (MMPs) and favour the release of these enzymes, key effectors of cartilage and subchondral bone degradation, by other cells; however, their role in OA has not been investigated yet. Our aims were (1) to assess the presence of platelets and of MMP-2 in synovial fluid (SF) of OA patients; (2) to evaluate the contribution of platelets to MMP-2 release by fibroblast-like synoviocytes (FLS); and (3) to investigate if hyaluronic acid (HA) interferes with these processes. SF was collected from 27 OA patients before and after treatment with intra-articular HA (20 mg/2 mL). Moreover, FLS were co-cultured with platelets, and the release of MMP-2 in supernatants was measured. Our results show that platelets are present in OA SF and show markers of activation. OA SF also contains relevant amounts of MMP-2. Co-incubation of platelets with FLS favours the release of MMP-2 by the interaction of platelet surface P-selectin with FLS CD44 by a mechanism involving the activation of pAkt and pSrc in FLS. Administration of HA to OA patients decreased the infiltration of platelets in SF and reduced the levels of MMP-2. The addition of HA in vitro inhibited the release of MMP-2 by FLS triggered by the interaction with platelets. In conclusion, our data show that platelets may contribute to joint degeneration in OA by favouring the accumulation of MMP-2 in SF.

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

Unusual Bursal Fluid

A 73-year-old man with seropositive rheumatoid arthritis and extraarticular involvement (rheumatoid nodules on the right elbow) presented with persistent, painless swelling of the left elbow. His medical history was unremarkable except for hypertension.

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