Background Ankylosing Spondylitis (AS) is an inflammatory rheumatic disease affecting the axial skeleton (1), with a prevalence of up to 0.5% in Europe (2; 3), and occurs equally in men and women [4]. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) and biological Disease-Modifying Antirheumatic Drugs (bDMARDs) such as Tumour Necrosis Factor inhibitors (TNFi) are the first choice in pharmacological treatments for AS patients (5; 6). Data on employment situation in correlation with disease activity and disease duration in AS patients treated in real life settings in Europe are limited. Objectives To evaluate the disease activity status and assess participation in professional life in relation to treatment response following at least 12 weeks of treatment with TNFi and/or NSAIDs. Methods INVISIBLE was a multinational, cross-sectional, non-interventional study in Germany, Austria and Belgium. Adult patients with confirmed diagnosis of AS for at least 6 months and ongoing treatment with TNFi and/or NSAIDs for at least 12 weeks prior to enrolment were included. Primary data were collected at a single visit using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Ankylosing Spondylitis Disease Activity Score (ASDAS) and collecting current demographic data including employment status using the categories employed, unemployed, parental leave, retired and sick leave (>6 weeks). Medical history data were collected from available patient files. Results 747 adult AS patients (67% male) were included in this analysis. The patients were 47.9±13.1 years of age. Symptom duration was 18.2±13.0 years. 22.9% of patients received NSAIDs, 41.6% TNFi, 29.7% both NSAIDs and TNFi, and 5.4% other treatment for at least 12 weeks prior to enrolment. BASDAI was 3.4±2.3 and ASDAS 2.2±1.0. Patient's disease activity assessment (Visual Analogue Scale 0-100) showed on average 35.8±27.5 and patients with BASDAI ≥4 reported significantly higher disease activity compared to patients with BASDAI <4 (59.7±20.7 vs. 20.7±19.7, p<0.001). 72.5% of patients were employed and 9.9% unemployed or in sick leave for >6 weeks. More patients with a BASDAI ≥4 were unemployed or in sick leave regardless of type of treatment and duration of symptoms compared to patients with a BASDAI <4 (see Table 1). Conclusion Results from the non-interventional study INVISIBLE showed that a higher proportion of AS patients receiving NSAIDs and/or TNFi with high disease activity according to BASDAI are not participating in professional life. These results suggest that in some patients, disease control and treatment response are suboptimal under current treatment strategy. References [1]Braun, J & J, Sieper. Lancet. 2007, Bd. 369, S. 1379–90.[2]Braun, J, et al. Arthritis Rheum. 1998, Bd. 41, 1, S. 58-67.[3]Dean, LE, et al. Rheumatology. 2014, Bd. 53, S. 650-657.[4]Rudwaleit, M, et al. Ann Rheum Dis. Jun 2009, Bd. 68, 6, S. 777-83.[5]Dougados, M, et al. Ann Rheum Dis. 2002, Bd. 61, suppl 3, S. 40–50.[6]Braun, J, et al. Ann Rheum Dis. 2006, Bd. 65, S. 316–20.[7]Feldtkeller, E, et al. Rheumatol Int. 2003, Bd. 23, S. 61–66. Acknowledgements: NIL. Disclosure of Interests Jan Brandt-Juergens Consultant of: Abbvie, Affibody, BMS, Gilead, Janssen, Lilly, Medac, MSD, Novartis, Pfizer, Roche, Sanofi-Aventis, UCB, Judith Haschka Speakers bureau: Eli Lilly, Amgen, Consultant of: Eli Lilly, Richard Finsterwalder Employee of: Novartis Pharma GmbH, Aurélie Casier Employee of: N.V. Novartis Pharma S.A., Angela Kill Employee of: Novartis Pharma GmbH, Stéphanie Dierckx: None declared.Table 1Participation in professional life in relation to disease activityBASDAI <4 (N = 445)BASDAI ≥4 (N = 292)NEmployed 333 (74.8%)Unemployed* 29 (6.5%)NEmployed 192 (65.7%)Unemployed* 41 (14.0%)n%n%n%n%Treatment- NSAID857183.522.4826376.867.3- TNFi20014472.0199.5996363.61414.1- NSAID & TNFi1239879.764.9915863.71920.9- Other262076.927.713646.2215.4Duration of Symptoms- <5 years575189.547.0453782.2613.3- ≥5 years37528174.9256.724215564.03514.5* unemployed and sick leave >6 weeks

Background Ankylosing Spondylitis (AS) is an inflammatory rheumatic disease affecting the axial skeleton (1), with a prevalence of up to 0.5% in Europe (2; 3), and occurs in men twice as often than in women (4). 80% develop AS symptoms before the age of 30 (4). Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) and biological Disease-Modifying Antirheumatic Drugs (DMARDs) such as Tumour Necrosis Factor inhibitors (TNFi) are the first choice in pharmacological treatments for AS patients (5; 6). Data on disease activity and disease control in AS patients treated in real life settings are limited. Objectives To evaluate the average disease activity status in the study population and assess the percentage of AS patients with a suboptimal treatment response following at least 12 weeks of treatment with TNFi and/or NSAIDs. Methods INVISIBLE was a real-world evidence study in Germany, Austria and Belgium. Adult patients with confirmed diagnosis of AS for at least 6 months and ongoing treatment with TNFi and/or NSAIDs for at least 12 weeks prior to enrolment were included. Primary data were collected at a single visit and medical history data from available patient files. Results 747 adult AS patients were included. 67% of patients were male, mean symptom duration 18.2 years. 77% of 696 patients were HLA-B27 positive. Appr. 23% of patients received NSAIDs, 42% received TNFi, 30% received both NSAIDs and TNFi, 5% received other treatment for at least 12 weeks prior to enrolment. Mean Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score (ASDAS) scores were 3.4 and 2.2, respectively. Appr. 40% of patients achieved a BASDAI score ≥4 and appr. 50% an ASDAS ≥2.1. Patients with a BASDAI score ≥4 achieved higher scores in most physician and patient reported outcomes. Overall appr. 35% of patients were not satisfied with their current symptom state according to Patient Acceptable Symptom State (PASS). Of patients with a BASDAI score ≥4 appr. 61% were not satisfied according to PASS. Conclusion Results from the non-interventional study INVISIBLE showed that a high proportion of AS patients receiving NSAIDs and/or TNFi have high disease activity according to BASDAI and ASDAS in real life settings. These results suggest that in some patients, disease control and treatment response are suboptimal under current treatment strategy. Table 1. Demographics and Disease Characteristics Total (N=747) BASDAI <4 (N=445) BASDAI ≥4 (N=292) N Result N Result N Result Age in years, mean (±SD) 746 47.9 (±13.1) 445 46.7 (±13.4) 292 49.6 (±12.3)* Male, n (%) 746 500 (67.0) 445 318 (71.5) 292 176 (60.3) Age at AS diagnosis in years, mean (±SD) 745 35.2 (±12.3) 445 34.1 (±12.0) 292 36.8 (±12.5)* Treatment with NSAIDs, n (%) 744 171 (23.0) 445 85 (19.1) 290 85 (29.3) Treatment with TNFi, n (%) 744 311 (41.8) 445 208 (46.7) 290 100 (34.5) Treatment with NSAIDs and TNFi, n (%) 744 222 (29.8) 445 126 (28.3) 290 92 (31.7) Treatment with other, n (%) 744 40 (5.4) 445 26 (5.8) 290 13 (4.5) Patient disease activity assessment (VAS 0-100), mean (±SD) 735 35.8 (±27.5) 444 20.7 (±19.7) 286 59.1 (±20.7)*** BASDAI Score, mean (±SD) 737 3.4 (±2.3) 445 1.8 (±1.1) 292 5.8 (±1.3)*** ASDAS Score, mean (±SD) 599 2.2 (±1.0) 363 1.7 (±0.6) 236 3.1 (±0.7)*** ASDAS ≥2.1, n (%) 599 297 (49.6) 363 73 (20.1) 236 224 (94.9)*** PASS: current state not satisfactory, n (%) 679 240 (35.3) 403 72 (17.9) 271 166 (61.3)*** SD – Standard Deviation; Level of significance: *< 0.05; ** < 0.01 *** < 0.001 (analyses refer to differences between BASDAI <4 and ≥4 and to respective available patient number). References [ [1] Braun, J and J, Sieper. Lancet. 2007, Vol. 369, pp. 1379–90. [2] Braun, J, et al. Arthritis Rheum. 1998, Vol. 41, 1, pp. 58-67. [3] Dean, LE, et al. Rheumatology. 2014, Vol. 53, pp. 650-657. [4] Feldtkeller, E, et al. Rheumatol Int. 2003, Vol. 23, pp. 61–66. [5] Dougados, M, et al. Ann Rheum Dis. 2002, Vol. 61, suppl 3, pp. 40–50. [6] Braun, J, et al. Ann Rheum Dis. 2006, Vol. 65, pp. 316–20. Disclosure of Interests Jan Brandt-Juergens Consultant of: Abbvie, Affibody, BMS, Gilead, Janssen, Lilly, Medac, MSD, Novartis, Pfizer, Roche, Sanofi-Aventis, UCB, Judith Haschka Speakers bureau: Eli Lilly, Amgen, Consultant of: Eli Lilly, Richard Finsterwalder Employee of: Novartis Pharma GmbH, Aurélie Casier Employee of: N.V. Novartis Pharma S.A., Angela Kill Employee of: Novartis Pharma GmbH, Stéphanie Dierckx: None declared

BackgroundAnkylosing Spondylitis (AS) is an inflammatory rheumatic disease affecting the axial skeleton (1), with a prevalence of up to 0.5% in Europe (2; 3), and occurs in men twice as often than in women (4). 80% develop AS symptoms before the age of 30 (4). Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) and biological Disease-Modifying Antirheumatic Drugs (DMARDs) such as Tumour Necrosis Factor inhibitors (TNFi) are the first choice in pharmacological treatments for AS patients (5; 6). Data on disease activity and disease control in AS patients treated in real life settings are limited.ObjectivesTo evaluate the average disease activity status in the study population and assess the percentage of AS patients with a suboptimal treatment response following at least 12 weeks of treatment with TNFi and/or NSAIDs.MethodsINVISIBLE was a real-world evidence study in Germany, Austria and Belgium. Adult patients with confirmed diagnosis of AS for at least 6 months and ongoing treatment with TNFi and/or NSAIDs for at least 12 weeks prior to enrolment were included. Primary data were collected at a single visit and medical history data from available patient files.Results747 adult AS patients were included. 67% of patients were male, mean symptom duration 18.2 years. 77% of 696 patients were HLA-B27 positive. Appr. 23% of patients received NSAIDs, 42% received TNFi, 30% received both NSAIDs and TNFi, 5% received other treatment for at least 12 weeks prior to enrolment. Mean Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score (ASDAS) scores were 3.4 and 2.2, respectively. Appr. 40% of patients achieved a BASDAI score ≥4 and appr. 50% an ASDAS ≥2.1. Patients with a BASDAI score ≥4 achieved higher scores in most physician and patient reported outcomes. Overall appr. 35% of patients were not satisfied with their current symptom state according to Patient Acceptable Symptom State (PASS). Of patients with a BASDAI score ≥4 appr. 61% were not satisfied according to PASS.ConclusionResults from the non-interventional study INVISIBLE showed that a high proportion of AS patients receiving NSAIDs and/or TNFi have high disease activity according to BASDAI and ASDAS in real life settings. These results suggest that in some patients, disease control and treatment response are suboptimal under current treatment strategy.Table 1.Demographics and Disease CharacteristicsTotal (N=747)BASDAI <4 (N=445)BASDAI ≥4 (N=292)NResultNResultNResultAge in years, mean (±SD)74647.9 (±13.1)44546.7 (±13.4)29249.6 (±12.3)*Male, n (%)746500 (67.0)445318 (71.5)292176 (60.3)Age at AS diagnosis in years, mean (±SD)74535.2 (±12.3)44534.1 (±12.0)29236.8 (±12.5)*Treatment with NSAIDs, n (%)744171 (23.0)44585 (19.1)29085 (29.3)Treatment with TNFi, n (%)744311 (41.8)445208 (46.7)290100 (34.5)Treatment with NSAIDs and TNFi, n (%)744222 (29.8)445126 (28.3)29092 (31.7)Treatment with other, n (%)74440 (5.4)44526 (5.8)29013 (4.5)Patient disease activity assessment (VAS 0-100), mean (±SD)73535.8 (±27.5)44420.7 (±19.7)28659.1 (±20.7)***BASDAI Score, mean (±SD)7373.4 (±2.3)4451.8 (±1.1)2925.8 (±1.3)***ASDAS Score, mean (±SD)5992.2 (±1.0)3631.7 (±0.6)2363.1 (±0.7)***ASDAS ≥2.1, n (%)599297 (49.6)36373 (20.1)236224 (94.9)***PASS: current state not satisfactory, n (%)679240 (35.3)40372 (17.9)271166 (61.3)***SD – Standard Deviation; Level of significance: *< 0.05; ** < 0.01 *** < 0.001 (analyses refer to differences between BASDAI <4 and ≥4 and to respective available patient number).References[[1] Braun, J and J, Sieper.Lancet. 2007, Vol. 369, pp. 1379–90.[2]Braun, J, et al.Arthritis Rheum. 1998, Vol. 41, 1, pp. 58-67.[3]Dean, LE, et al.Rheumatology. 2014, Vol. 53, pp. 650-657.[4]Feldtkeller, E, et al.Rheumatol Int. 2003, Vol. 23, pp. 61–66.[5]Dougados, M, et al.Ann Rheum Dis. 2002, Vol. 61, suppl 3, pp. 40–50.[6]Braun, J, et al.Ann Rheum Dis. 2006, Vol. 65, pp. 316–20.Disclosure of InterestsJan Brandt-Juergens Consultant of: Abbvie, Affibody, BMS, Gilead, Janssen, Lilly, Medac, MSD, Novartis, Pfizer, Roche, Sanofi-Aventis, UCB, Judith Haschka Speakers bureau: Eli Lilly, Amgen, Consultant of: Eli Lilly, Richard Finsterwalder Employee of: Novartis Pharma GmbH, Aurélie Casier Employee of: N.V. Novartis Pharma S.A., Angela Kill Employee of: Novartis Pharma GmbH, Stéphanie Dierckx: None declared

Background and Objectives IgG4-related diseases are often characterised by a generalised inflammatory fibrosis which is also present in patients suffering from systemic sclerosis (SSc). Recent findings indicate the importance of autoantibodies (Aabs) against the angiotensin II type-1 receptor (AT1R) and the endothelin type-A receptor (ETAR) in the pathogenesis of SSc. Therefore, we analysed the levels of anti-AT1R/ETAR IgG sublasses in patients with SSc to determine a possible role of IgG subclasses as markers for disease manifestations in SSc. Material and Methods Sera from 91 SSc patients, 59 patients suffering from systemic lupus erythematosus (SLE), and from 199 healthy donors were analysed for the levels of anti-AT1R and anti-ETAR Aabs as well as for the different anti-AT1R and anti-ETAR IgG subclasses by ELISA. The results were associated with clinical manifestations using Mann-Whitney test and correlated with the time since onset of disease manifestations by Spearman correlation test. Results IgG3 followed by IgG1 was found to show highest anti-AT1R/ETAR Aab levels in all analysed groups, in which SSc patients as well as SLE patients had higher IgG1 and IgG3 anti-AT1R/ETAR Aab levels as compared to healthy donors. Comparing SLE and SSc patients IgG1 and IgG3 showed slightly higher anti-AT1R/ETAR Aab levels in SLE. Within the SSc group patients with diffuse SSc had higher anti-AT1R/ETAR IgG3 levels as compared to those with limited disease or overlap forms. Correlation analysis with SSc-related clinical manifestations revealed that levels of anti- AT1R/ETAR IgG3 negatively correlated with time since onset of Raynaud’s phenomenon, and with time since first detection of PAH. Of note, there were negative correlations between levels of anti-AT1R/ETAR IgG3 levels and diffusion capacity for carbon monoxide (DLCO) as well as between anti-AT1R/ETAR-IgG3 levels and forced vital capacity (FVC) values (p = 0.02/0.07 and p = 0.01/0.03, respectively). Conclusion Interestingly, not IgG4 but IgG3 showed highest anti-AT1R/ETAR Aab levels when compared to other IgG subclasses. However, in SSc patients, anti-AT1R/ETAR IgG3 levels are strongly correlated to certain disease manifestations, whereby high anti-AT1R/ETAR IgG3 levels are associated with low DLCO and FVC indicating deterioration of lung function. According to these findings high anti-AT1R/ETAR IgG3 levels could predict for lung function deterioration and represent a new marker for SSc complications.

Background Functional autoantibodies against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients suffering from systemic sclerosis (SSc) [1]. Objectives Aim of the study was to validateAT1R and ETAR expression in peripheral blood mononuclear cells (PBMCs) and to examine pathological effects mediated through these receptors by the corresponding autoantibodies (Aabs). Methods Protein expression of AT1R and ETAR on PBMCs from healthy individuals and SSc patients was analyzed using flow cytometry, mRNA expression was examined by real-time PCR in PBMCs from healthy donors. In addition, PBMCs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin G (IgG) from SSc patients positive for AT1R- and ETAR-Aabs, and with IgG from healthy donors serving as control. Alterations in chemotactic motility and cytokine secretion were analyzed using chemotaxis assays and ELISA, respectively. Results were correlated with characteristics/clinical findings of the IgG donors. Results Both AT1R and ETAR were expressed on human peripheral lymphocytes and monocytes. Protein expression of both receptors was decreased in the tested cohort of SSc patients when compared to healthy donors and correlated negatively with disease duration. In addition, purified IgG from SSc patients induced T cell migration in an anti-AT1R and anti-ETAR Aab level-dependent manner. Moreover, IgG from SSc patients was capable of stimulating PBMCs to produce more IL-8 and CCL18 than IgG from healthy donors. All effects could be significantly abrogated by the application of selective AT1R and ETAR antagonists. Statistical analysis revealed a negative correlation between SSc IgG-induced IL-8 concentrations and disease duration, between SSc IgG-induced CCL18 concentrations and time since onset of lung fibrosis as well as an association of CCL18 concentrations with vascular complications of the corresponding SSc IgG donors. Conclusions We demonstrated the expression of both, AT1R and ETAR, on human PBMCs, and found a decreased receptor expression on cells from SSc patients suggesting downregulation due to chronic activation. The inflammatory and profibrotic effects upon Aab stimulation in vitro, as shown by T cell migration as well as by the induction of IL-8 and CCL18 secretion, and their associations with clinical findings indicate a role for autoantibody-mediated activation of immune cells mediated through the AT1R and ETAR in the pathogenesis or even the onset of the disease. References Riemekasten et al. Involvement of functional autoantibodies against vascular receptors in systemic sclerosis. Ann Rheum Dis. 2011 Mar;70(3):530-6. doi: 10.1136/ard.2010.135772 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3649

Background Stimulating autoantibodies against angiotensin (II) type-1 receptor (AT1R) and endothelin type-A receptor (ETAR) are present in the majority of systemic sclerosis (SSc) patients. High antibody (ab) levels are associated with severe clinical disease manifestations and with mortality. Current data indicates their contribution in disease pathogenesis. Male patients often exhibit SSc symptoms early in the disease and often have cardiac involvement and very progressive fibrotic lung disease associated with high levels of the profibrotic chemokine CCL18. Since anti-AT1R/ETAR ab levels are similar in female and male SSc patients we asked whether differences in the receptor expression exist that could explain sex differences presented especially in the early phase of SSc. Objectives To identify whether expression of angiotensin-II and endothelin receptors on peripheral blood mononuclear cells (PBMC) is influenced by sex, by disease duration or by organ manifestations in SSc patients and to analyze the functional relevance of receptor expression by measuring ab-mediated CCL18 production in PBMC with known receptor expression of AT1R and ETAR as well as their respective functional counterparts AT2R and ETBR. Methods Protein and mRNA receptor expression (for AT1R, AT2R, ETAR and ETBR) was measured on PBMC of 30 SSc patients by flow cytometry and by real-time PCR, respectively and correlated with sex, disease duration, and clinical findings of these patients. In addition, PBMCs of 11 healthy donors were similarly analyzed for receptor expression and were in vitro stimulated with affinity purified immunglobulin G (IgG) of either SSc patients or healthy donors. Unstimulated PBMC were used as further control. Afterwards the concentration of CCL18 was measured in the supernatants by ELISA and was correlated with receptor expression by Spearman rank correlation. Results SSc patients with lung fibrosis and left ventricular dysfunction have higher expression of ETAR and ETBR compared to those without these symptoms (p<0,05 for both receptors). In addition, male patients present significantly higher levels of receptor density as well as more receptor positive cells compared to female SSc patients for all 4 examined receptors. AT1R, AT2R, ETAR and ETBR expression correlated significantly with the modified Rodnan Skin Score. AT1R density was highest in early disease and the expression of AT1R, AT2R, ETAR and ETBR correlated significantly negative with the time since onset of Raynaud9s phenomenon with various correlation coefficients for the different PBMC subsets. After stimulation of PBMC with SSc-IgG we observed a significant positive correlation between the concentration of CCL18 in the supernatant and the ratio of AT1R/AT2R protein expression on CD14+ (r=0,73; p=0,03). Conclusions Angiotensin and endothelin receptor expressions on PBMC could explain sex differences in SSc patients and different effects of the antibodies among individual SSc patients. References Riemekasten G et al. Involvement of functional autoantibodies against vascular receptors in systemic sclerosis. Ann Rheum Dis 2011;70(3):530-6 Tiev KP et al. Serum CC chemokine ligand-18 predicts lung disease worsening in systemic sclerosis. Eur Respir J. 2011;38(6):1355-60 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5024

Background IgG4-related diseases are often characterized by a generalized inflammatory fibrosis which is also present in patients suffering from systemic sclerosis (SSc). Recent findings indicate the importance of autoantibodies (Aabs) against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) in the pathogenesis of SSc. Objectives Aim of the study was to analyse associations between the serum levels of anti-AT1R/ETAR IgG sublasses of SSc patients and their clinical features in order to determine a possible role of certain subclasses as markers for disease manifestations. Methods Sera from 91 SSc patients, 59 patients suffering from systemic lupus erythematosus (SLE), and 199 healthy donors were analysed for the levels of anti-AT1R and anti-ETAR Aabs as well as for the different anti-AT1R and anti-ETAR IgG subclasses by ELISA. The results were associated with clinical manifestations using Mann-Whitney test and correlated with the time since onset of disease manifestations by Spearman correlation test. Results IgG3 followed by IgG1 was found to show highest anti-AT1R/ETAR Aab levels in all analyzed groups, in which SSc patients as well as SLE patients had higher IgG1 and IgG3 anti-AT1R/ETAR Aab levels as compared to healthy donors. Comparing SLE and SSc patients IgG1 and IgG3 showed a bit higher anti-AT1R/ETAR Aab level in SLE. Within the SSc group patients with diffuse SSc had higher anti-AT1R/ETAR IgG3 levels as compared to those with limited disease or overlap forms. Correlation analysis with SSc-related clinical manifestations revealed that levels of anti-AT1R/ETAR IgG3 negatively correlated with time since onset of Raynaud9s phenomenon, and with time since first detection of pulmonary arterial hypertension. Of note, there were negative correlations between levels of anti-AT1R/ETAR IgG3 levels and diffusion capacity of the lung for carbon monoxide (DLCO) as well as between anti-AT1R/ETAR IgG3 levels and forced vital capacity (FVC) values (p=0.02/0.07 and p=0.01/0.03, respectively). Conclusions Interestingly, not IgG4 but IgG3 showed highest anti-AT1R/ETAR Aab levels when compared to other IgG subclasses. However, in SSc patients, anti-AT1R/ETAR IgG3 levels are strongly correlated to certain disease manifestations, whereby high anti-AT1R/ETAR IgG3 levels are associated with low DLCO and FVC indicating deterioration of lung function. According to these findings high anti-AT1R/ETAR IgG3 levels could predict for lung function deterioration and represent a new marker for SSc complications. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3699

The present study was designed to evaluate the clinical outcome of cell-based therapy with cultured adipose derived stromal cells (ASCs) for the treatment of cutaneous manifestations in patients affected by systemic sclerosis (SSc). ASCs have an extraordinary developmental plasticity, including the ability to undergo multilineage differentiation and self-renewal. Moreover, ASCs can be easily harvested from small volumes of liposuction aspirate, showing great in vitro viability and proliferation rate. Here we isolated, characterized, and expanded ASCs, assessing both their mesenchymal origin and their capability to differentiate towards the adipogenic, osteogenic, and chondrogenic lineage. We developed an effective method for ASCs transplantation into sclerodermic patients by means of a hyaluronic acid (HA) solution, which allowed us to achieve precise structural modifications. ASCs were isolated from subcutaneous adipose tissue of six sclerodermic patients and cultured in a chemical-defined medium before autologous transplantation to restore skin sequelae. The results indicated that transplantation of a combination of ASCs in HA solution determined a significant improvement in tightening of the skin without complications such as anechoic areas, fat necrosis, or infections, thus suggesting that ASCs are a potentially valuable source of cells for to improve dermal repair in rare diseases such as SSc and generally in skin disorders. Poster Tours – Basic

Background Autopsy studies showed cardiac fibrosis in the majority of patients with systemic sclerosis (SSc). Male SSc patients are at higher risk for left ventricular failure than their female counterparts suggesting sex-related differences in activation and migration of inflammatory cells into the heart leading to tissue fibrosis. Angiotensin II and endothelin-1 as well as activation of their specific receptors by autoantibodies (AB) may contribute to these abnormalities. This may depend on expression of angiotensin-II type-1 (AT1R) and endothelin-1 type-II (ETAR). Methods In our single-center SSc data base cardiac involvement was evaluated by echocardiography and electrocardiogram (ECG) and defined by one abnormality either in ejection fraction (EF), in diastolic dysfunction or ECG changes. AB levels against AT1R and ETAR were measured by a commercially available ELISA in arbitrary units (AU, One Lambda, Inc., USA). Peripheral blood mononuclear cells (PBMC) from 8 healthy female and 6 healthy male as well as from 10 female and 8 male SSc patients were analyzed for AT1R and ETAR expression by flow cytometry. Statistics were performed using Mann-Whitney-test. Results Clinical data for cardiac involvement were available for 623/733 SSc patients. Cardiac involvement was significantly more often present in male than in female patients (52/103, 50.5% males versus 154/520, 29.6% females, p< 0.0001). Overall 206/623 patients showed cardiac involvement with a male/female ratio for EF: 13%/7% (p=0.0105), diastolic dysfunction: 36%/29% (p=0.0514) and ECG: 27%/16% (p=0.0044). In cardiac disease mean AT1R AB were higher in male patients whereas ETAR AB were higher in female versus male SSc patients (not significant, n.s.). In patients without cardiac involvement mean levels of AT1R AB were higher in males than in females whereas ETAR AB did not differ between males and females (n.s.). AT1R and ETAR were expressed on all PBMC. In healthy individuals, males showed higher percentages of cells expressing AT1R and ETAR compared to females. Similar results were obtained for receptor density. In SSc patients, no sex differences in receptor expression were observed. Comparing healthy males to male SSc patients, AT1R density and the percentage of cells expressing AT1R were significantly reduced in nearly all PBMC subsets of male SSc patients. The percentage and density of T cells expressing ETAR were diminished, too. Conclusions Sex-specific differences in clinical manifestations of cardiac involvement in SSc patients, in autoantibody levels against AT1R and ETAR, different expression of the receptors in healthy males versus females, as well as sex-specific differences in their activation status as reflected by more pronounced downregulation of the receptors in male patients suggest a possible contribution of AT1R/ETAR-mediated processes. Disclosure of Interest None Declared

Background Systemic sclerosis (SSc) is characterised by autoimmunity, vasculopathy and fibrosis. The functional link between these three pathophysiological components is still missing. Recent research suggests an involvement of the vasoconstrictive soluble peptides endothelin-1 and angiotensin II, and of the activation of their receptors by the natural ligands and agonistic autoantibodies against these receptors in SSc–associated vasculopathy. Objectives We could identify auto-antibodies against the angiotensin receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) in SSc patients. The pathophysiological effects of these antibodies on immune cells and their association with clinical data have not been studied so far. Materials and methods Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by gradient centrifugation and stimulated in vitro by affinity-purified IgG from SSc patients containing anti-AT1R and anti-ETAR antibodies as well as by IgG from healthy donors. After stimulation the expression of several markers and cytokines were measured by flow cytometry or ELISA. Results Stimulation of PBMC from healthy donors by SSc patient IgG resulted in a significantly increased expression of IL-8 compared to the stimulation by IgG of healthy donors. This effect can be blocked by commercial AT1R and ETAR blockers. In contrast, expression of soluble CD14 as well as of the surface marker CD14 was significantly decreased. Correlation analysis of the IL-8 expression with clinical data of the SSc patients whose IgGs were used revealed an association of the high IL-8 expression with the early stage of disease, in fact in vitro stimulation of PBMC with IgG of patients who were within the first 8 years after onset of the disease caused a significantly higher IL-8 secretion in comparison to the stimulation with IgG of patients at a later stage. Conclusion SSc patient IgGs containing anti-AT1R and anti-ETAR antibodies seem to have effects on inflammation and immune regulation. Especially the release of IL-8, which is a strong inflammatory cytokine mainly secreted by monocytes among the immune cells, may play an important role in the early stage of disease contributing to vascular inflammation and injury, which are regarded to be the first events leading to tissue damage and fibrosis.

Background Functional autoantibodies to angiotensin II type 1 receptor (AT1R) and endothelin 1 type A receptor (ETAR) are found in elevated levels in systemic sclerosis (SSc) and they show an association to increased risk of SSc-related manifestations and reduced cumulative survival. Here, functional effects of these autoantibodies (anti-AT1R and anti-ETAR autoantibodies) were studied in vitro and in vivo. Objectives To analyze anti-AT1R and anti-ETAR functions in vitro and in vivo. Methods In vitro, autoantibody-mediated effects were studied on human microdermal endothelial cells-1 (HMEC-1), on healthy donor dermal fibroblasts and healthy donor neutrophils. In vivo, autoantibody-mediated effects were studied by passive autoantibody-transfer treatment into healthy C57Bl/6J mice. For each setting anti-AT1R and anti-ETAR autoantibody-positive IgG of SSc patients was used and IgG of healthy donors as a control. Angiotensin and endothelin receptor inhibitors were used in vitro to analyze receptor-mediated effects. In vitro effects were measured by endothelial cell activation (mRNA and protein levels), endothelial wound repair and trans-endothelial neutrophil migration. Fibroblast activation was measured by type I collagen protein expression. Systemic autoantibody-mediated effects were measured in mice by cellular BALF composition, lung histology and plasma chemokine levels after single and repeated autoantibody-transfer. Results Treatment with anti-AT1R and anti-ETAR autoantibody-positive IgG of SSc patients resulted in several effects, both in vitro and in vivo. In vitro, endothelial cells showed an activation mediated by anti-AT1R and anti-ETAR autoantibodies reflected by enhanced expression of adhesion molecule VCAM-1, of IL-8 and by increased neutrophil trans-endothelial migration. Furthermore, endothelial cells displayed a reduced wound repair capacity, while fibroblasts showed increased type I collagen expression. Neutrophil migration, wound repair and collagen expression were induced in an anti-AT1R and anti-ETAR autoantibody-level dependent manner. Neutrophil counts were elevated in BALF of treated mice, accompanied by increased chemokine KC (functional homologue to human interleukin-8) plasma levels with a correlation between KC levels and neutrophil counts after a single transfer. Repeated transfer led to marked structural alterations in the lung architecture. Conclusions Our findings demonstrate the potential to induce features of SSc pathogenesis on cellular and systemic level. The in vitro findings indicate direct receptor activation by anti-AT1R and anti-ETAR autoantibody-positive SSc-IgG, and activation of the angiotensin and endothelin receptors by these autoantibodies could account in part for effects seen here in animal experiments. These findings stress the autoimmunity aspect in the disease pathogenesis and provide further evidence for the significant involvement of the angiotensin and endothelin receptor activation in SSc. Therefore, receptor inactivation studies will be performed in vivo to assess a deeper knowledge of anti-AT1R and anti-ETAR autoantibody-mediated effects and to improve our current understanding of SSc pathogenesis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5196

Background and Objectives Agonistic autoantibodies against the angiotensin receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in the majority of systemic sclerosis (SSc) patients. We proved the expression of the AT1R and the ETAR on human peripheral immune cells. Here, we investigated the effects of these autoantibodies on human immune cells in vitro, the association with clinical data, and their possible role in the pathogenesis of systemic sclerosis. Methods Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by gradient centrifugation and in vitro stimulated by affinity-purified IgG from SSc patients containing anti-AT1R and anti-ETAR antibodies as well as by affinity-purified IgG from healthy donors. After stimulation the protein expression of cytokines was measured by ELISA. T cells were isolated from PBMCs by magnetically activated cell sort (MACS) and cultivated in antiCD3/CD28 coated plates. Afterwards, migration assays were performed towards IgGs of SSc patients or healthy donors. Results IgG of SSc patients induced a significantly increased expression and secretion of IL-8 and an abundant but due to a broad range not significant secretion of CCL18. T cell migration towards IgG of SSc patients was increased. All these effects were significantly reduced by commercial AT1R and ETAR antagonist, confirming the specifity of the biological activity of the autoantibodies. Correlation analysis with clinical data of the SSc IgG donors revealed a negative correlation of IL-8 expression with the time since disease onset, and an association of CCL18 expression with the incidence of vascular complications. The chemotactic motility of T cells correlated with the autoantibody titers of the used SSc IgG. Conclusions SSc patient IgGs containing anti-AT1R and anti-ETAR antibodies seem to have effects on inflammation and fibrosis. IL-8 is a major inflammatory cytokine, inducing migration and inflammatory effects. CCL18 is a fibrotic cytokine, also contributing to leucocyte infiltration, and to lung fibrosis in SSc and other diseases. Thus, anti-AT1R and anti-ETAR antibodies may play an important role particularly in the early stage of SSc, contributing to leucocyte infiltration, vascular inflammation and injury, which are regarded to be the first events leading to tissue damage and fibrosis.