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Leflunomide is a new disease modifying drug that is widely used in the therapy of rheumatoid arthritis (RA). The active metabolite of leflunomide A771726 leads to inhibition of dihydroorotate dehydrogenase, an enzyme necessary for pyrimidine de-novo synthesis. Since activated lymphocytes expand their pyrimidine pool, A771726 leads to a decrease of their proliferation. A771726 also suppresses TNF mediated nuclear factor kappaB activation. We were therefore interested if A771726 also would be capable to influence transendothelial migration (TEM) of peripheral mononuclear cells (PBMC). We investigated the TEM of PBMC, which migrated through endothelial cell monolayers in an in-vitro model. Human umbilical vein endothelial cells (EC) were cultured to confluence on collagen gels and then incubated with human PBMC of healthy blood donors. PBMC were recollected in three groups: 1) cells that did not adhere to the endothelium, 2) cells that bound to the endothelium, 3) cells that had migrated through the endothelium, and then counted by microscope. Experiments in which PBMC as well as EC were treated with A771726 (in the absence or presence of uridine) were compared to simultaneously performed control experiments. No increased toxicity on the PBMC treated with the doses of A771726 used in our experiments, was observed. 24 hour incubation of PBMC and EC with 30 mg/ml A771726 led to a significant decrease in TEM (11 ± 3 % vs. 6 ± 3 % migrated cells, P = 0.016, paired T test). Uridine partly reversed the decrease in TEM when incubating PBMC and EC with both uridine and A771726. Our results demonstrate that leflunomide may have direct anti-inflammatory effects by inhibiting the extravasation of PBMC. These data suggest, that leflunomide, besides its influences on lymphocyte proliferation, also reduces accumulation of cells at inflammatory sites.

Objectives. Both increased and decreased apoptosis may be involved in generating autoimmunity. This study addressed the question of whether apoptosis and apoptosis-regulating proteins are altered in systemic sclerosis (SSc). Patients and methods. Peripheral lymphocytes of 39 SSc patients and 47 healthy control persons were studied for apoptosis, Bcl-2 and Bax levels, expression of Fas (CD95) and activation markers (CD25, HLA-DR) as determined by fluorocytometry. Serum Fas and Fas ligand were measured by ELISA. Results. SSc lymphocytes (mainly CD4 + ) expressed increased amounts of Bcl-2, while Bax was not elevated. Apoptosis rates of SSc lymphocytes were increased in unsupplemented medium, but returned to normal in the presence of autologous plasma. SSc patients had increased percentages of activated and CD95 + lymphocytes and elevated soluble Fas and soluble FasL levels in serum. Activating anti-CD95 antibodies further increased the apoptosis rate. Conclusions. Increased in vitro apoptosis, elevated lymphocytic Bcl-2 content and the increased number of Fas-positive T cells are not specific for peripheral blood from SSc patients, but indicate deregulation of lymphocyte homeostasis in this disease.

Background CD4 + T cells and the Th1 and Th17 subsets in particular play a pivotal role in SLE. Regulatory T cells (Treg) are essential for maintaining peripheral tolerance, but their number and function in SLE is decreased. Objectives We herein characterize CD4+T cell and Treg homeostasis and severity of organ involvement in a murine model of SLE and analyze the effects of in vitro –induced iTreg. Methods Mice were injected i.p. with 0.5ml of pristane or PBS as control and killed after 8 months. Induction of Treg: Naive CD4 + thymocytes were cultured under specific conditions (including TGFβ, IL-2) and tested for CD4 + Foxp3 + expression by FACS (induced Treg, iTreg). Cell suspensions with >80% purity for CD4 + FoxP3 + iTreg were injected intravenously either once at start of experiments (5x10e6, iTreg-boost) or monthly (1x10e6, iTreg-repeated). Animals were monitored for clinical signs of arthritis and in order to analyze and compare disease severity, histological features of arthritis and pneumonitis were quantified by an image analysis system (Osteomeasure®). Lungs were scored for the severity of perivascular inflammation by analyzing the numbers of affected vessels and the area of the inflammatory infiltrate. Lymphocytes were isolated from granulomas (ectopic lymphoid tissue within the peritoneum), lymph nodes (LN) and spleens and were analyzed separately for each mouse by FACS. For analyzes of the Th1, 2, 17 subsets, respectively, cells were restimulated in vivo plate bound with anti-CD3 and anti-CD28abs. Results PIL mice presented with involvement of inner organs, most frequently affected were the lungs (100% pneumonitis); 52% of PIL developed arthritis, both clinically and histologically. Monthly iTreg-injection significantly decreased clinical signs of arthritis and histological lung and joint parameters. Out of 6 mice treated, 4 (66%) did not show any signs of arthritis at all. (Fig. 1A-F) The iTreg-boost did not prevent joint manifestations or pneumonitis, but appeared to have a retarding effect (Fig.1A-F) indicated by a delayed onset of clinical symptoms and by a significantly decreased erosive area at the end of observation (Fig.1B). Intraperitoneal granuloma typical for PIL appeared to be the hotspots of inflammation showing a significantly elevated Teff/Treg ratio of 1.3. Upon re-stimulation, CD4+ cells showed a pronounced Th1 response (27% IFNγ producers) compared to cells from LN and spleens from both PIL and HC (with Th1 percentages ranging from 9-16%). In addition, frequencies of Th2 and Th17 cells were elevated in PIL, again with the highest yield in granuloma. The repeated application of iTreg reduced the Teff/Treg ratio in PIL granuloma to 0.7. Conclusions Repeated injections of exogenously induced iTregs reduce severity of pneumonitis and arthritis as well as the T eff /T reg ratio. A single injection of iTregs (even when applied before the clinical onset) is not effective, but appears to retard onset of symptoms and progression of arthritis and pneumonitis. Thus, iTreg have significant effects on lupus symptoms when applied repeatedly, which may have consequence for future therapeutic considerations. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5386

Objectives Arthritis is frequently seen in human lupus, but rarely in lupus models. Pristane-induced lupus (PIL) can be induced in various mouse strains such as BALB/c and C57Bl/6. We herein characterize clinical and histological features of arthritis in the context of systemic lupus and provide a prudent comparison with models of rheumatoid arthritis (RA). Methods Arthritis is frequently seen in human lupus, but rarely in lupus models. Pristane-induced lupus (PIL) can be induced in various mouse strains such as BALB/c and C57Bl/6. We herein characterize clinical and histological features of arthritis in the context of systemic lupus and provide a prudent comparison with models of rheumatoid arthritis (RA). Results In BALB/c, clinical arthritis started at 3 months, occurred finally in 79% of PIL (but not in controls, p 0.7, p Conclusions PIL in BALB/c mice is characterized by severe organ involvement, typical auto-abs and by a mild-erosive arthritis with similarities, but also with distinct differences to RA. PIL may help to study arthritis along with other key features of SLE after therapeutic interventions or in knockout models based on a BALB/c, but not on a Bl/6 background. Disclosure of Interest None Declared

Background Systemic lupus erythematosus (SLE) is a severe systemic autoimmune disease with involvement of inner organs; autoreactive (CD4+) T cells, B cells and autoantibodies are crucial in its pathophysiology. CD4+FoxP3+ Treg, on the other hand, are important for maintaining peripheral tolerance to self-antigens. In human SLE, however, both the absolute numbers and the functionality of these cells are reduced and correlate inversely with disease activity, but direct evidence for a central role of Treg malfunction in SLE pathophysiology is still missing. Objectives We therefore analyzed, if Treg-deficient scurfy mice share typical features of SLE. Methods Scurfy mice have dysfunctional Treg due to a genetic defect in the transcription factor foxP3 which is crucial for their development and function. 9 scurfy and 9 matched controls (C57Bl/6) were analyzed at 4-5 weeks of age. By immunofluorescence we screened for autoantibodies and also performed hematological workup. Specimen of skin and inner organs were stained with H/E and screened for inflammation by a blinded pathologist; kidneys were also stained with PAS and by direct immunofluorescence. We analyzed joint pathology after staining with H/E (overview), tolloidin blue (cartilage) and TRAP (osteoclasts). Immunhistochemistry allowed for further analysis of the cellular composition of the inflammatory infiltrate, which was finally quantified by image analysis systems (Osteomeasure and HistoQuest, respectively). Results We confirmed previous reports that scurfy mice spontaneously develop severe systemic autoimmune disease which includes pneumonitis and hematological abnormalities (anemia, lymphopenia) similar to those seen in SLE. We here show that scurfy, but not WT control mice, exhibit various additional features typical for SLE: They develop severe interface dermatitis, show elevated serum levels of ANA and dsDNA-abs and develop mesangial glomerulonephritis (resembling lupus nephritis WHO2; seen in 8 out of 9 scurfy [88.9%] vs. 0/6 [0%] of controls, respectively, p=0.0014). In contrast to controls, scurfy mice showed increased cartilage degradation (destained/normal cartilage area 0.050±0.009 vs. 0.018±0.003, p=0.004) and developed inflammatory arthritic infiltrates (mean area 0.38±0.25mm2). There were no osteoclasts within the joint space and, consecutively, no erosions. Besides fibroblasts, the inflammatory infiltrate consisted mainly of CD3+ T lymphocytes (13%), with 7% B cells and Conclusions Our observations support the hypothesis that lupus-like disease develops in the absence of functional Treg. References: Scheinecker C, Bonelli M, Smolen JS. Pathogenetic aspects of systemic lupus erythematosus with an emphasis on regulatory T cells. J Autoimmun. 2010 Nov;35(3):269-75. doi: 10.1016/j.jaut.2010.06.018. Epub 2010 Jul 16. Disclosure of Interest: None Declared

Background Pathogenic autoantibodies and immune complexes are a hallmark of SLE and are effectively removed by immunoadsorption (IAS). We previously reported that IAS performed with columns using sheep IgG as ligand (Ig-Therasorb®) reduces proteinuria and global disease activity in highly active renal SLE (Ref.1). Meanwhile, three different IAS columns using different adsorbing ligands are in use for lupus patients. Objectives We now attempted to answer the question if one column should be preferred in the treatment of active, refractory lupus nephritis. Methods All patients with histologically proven lupus nephritis undergoing IAS (n=26) were included to analyze the effects of IAS during induction therapy (≤3 months) on renal outcome and global disease activity. Patients were grouped according to the IAS column used: IgG-group (sheep IgG, Ig-Therasorb®, n=16), ProtA-group (staphylococcal protein A, Immunosorba®, n=5), and Gam-group (synthetic peptid Gam146, Globaffin®, n=5). Patients underwent IAS therapy when cyclophosphamide or MMF therapy was not effective or contraindicated; characteristics are given in the table. As described, we used response criteria R20 and R50, defined as a reduction by 20% or 50% in at least 2 of the 3 main outcome parameters proteinuria, SLEDAI and pre-treatment anti-dsDNA levels (Ref 1). Results In all 3 groups, IASsignificantly lowered serum levels of IgG, IgM, and anti-dsDNA-abs. Proteinuria significantly decreased in all groups, with a reduction by 48% in IgG versus 67% in ProtA and 64% in Gam groups, respectively (p=n.s.), while serum-creatinine decreased accordingly (table). All groups presented with comparably high disease activity scores (SLEDAI) and achieved a significant reduction within one month. After 3 months, all groups showed good responses with a R20 in >80% and a R50 in >60% of patients. No severe adverse event (allergic reaction, critical hypotension) occurred. Infections were distributed equally among the 3 groups with one infection per patient year necessitating antibiotics. Conclusions IAS reduces proteinuria and global disease activity in refractory lupus nephritis and improves renal function. All three columns performed well in induction therapy with slightly, albeit not significantly better response rates for ProtA and Gam columns. Our findings on efficacy of different columns may facilitate future attempts for randomized controlled trials on IAS in lupus nephritis References Stummvoll et al;IgG immunoadsorption reduces SLE activity and proteinuria: a long term observational study. ARD Juli 2005 Disclosure of Interest None Declared

Background Systemic lupus erythematosus (SLE) is frequently featured by musculoskeletal issues as arthralgia and arthritis, which account for the most important burdens in patient-based questionnaires. In most cases, lupus arthritis appears as non-erosive in projection radiography, but presented as (minimal) erosive disease in recent MRI-based observations. Despite its high prevalence, lupus arthritis is not in the focus of intensive basic research, since it is rarely seen in murine lupus models. The mineral oil pristane can induce a systemic lupus-like disease in mice featured by typical auto-antibodies, the involvement of inner organs, and also by arthritis. Objectives To establish pristane induced lupus (PIL) in BALB/c mice as suitable model for lupus arthritis, provide in-depth analysis of both clinical and histological features by using an established work up procedure originating from RA basic research. Methods For disease induction BALB/c mice were injected i.p. with either 0.5ml of pristane (PIL group n=57) or PBS (healthy controls, HC, n=35) and sacrificed after 8 months. In addition, also Bl/6 mice were treated similarly (10 PIL, 10 HC). Animals were monitored for clinical signs of arthritis (paw swelling, loss of grip strength) and analyzed by histopathology techniques: H/E (overview), TRAP (osteoclasts), toluidine blue (cartilage). In order to analyze and compare disease severity, the histological features were quantified with an image analysis system (osteomeasure) and also combined to define an arthritis severity score (ASS). Specimens were also stained for granulocytes (G1), T- (anti-CD3) and B-Cells (anti-CD45) for immunohistochemistry. Blood samples were tested for anti-chromatin-abs, anti-histone-abs and rheumatoid factor. Results After 3 months, clinical signs of arthritis developed and constantly increased; finally 75% of mice experienced at least one episode of paw swelling, while HC were free of arthritis (p 0.7, p Histological analysis showed arthritis in 58% of PIL-group, but none in HC (p Conclusions In contrast to most murine models of SLE, PIL in BALB/c mice is featured by arthritis. Despite its erosive nature, PIL arthritis does not procede to joint destruction as seen in human RA or murine RA models. PIL may provide a good tool to study lupus arthritis in knock-out or transgenic mouse models based on a BALB/c, but not on a Bl/6 background. Disclosure of Interest None Declared

Background Spondyloarthritis (SpA) is relatively common inflammatory disorder with a frequency of 1-2% in the European population (1). Establishing the diagnosis however may be difficult, since abnormalities in conventional X-ray develop with a latency of several years and so far only HLA-B27 has been established as a laboratory marker of the disorders (2). Besides MRI, there are no sufficient tools for the early diagnosis of SpA (3). Objectives The goal of our study therefore was to identify new autoantibodies as markers of SpA. Methods As a screening procedure, we used protein array technology for detection of possible new autoantigens in SpA. Sera of patients with SpA without peripheral manifestation (n=5) and other diseases as controls (n=45), were studied. In the second step, the results of the protein array were confirmed by ELISA using commercially available recombinant antigens (Abnova, Taiwan and Abcam, UK) and new synthetic derived peptides (Biomatik, USA). Considering the sensitivity and specificity, we developed an ELISA with synthetic peptide Class II-associated invariant chain peptide (CLIP) in cooperation with AESKU.Diagnostics (Wendelsheim, Germany). The sera for the ELISA were obtained from 198 SpA patients visiting the rheumatological outpatients and inpatients clinics, 80 rheumatoid arthritis (RA), 40 systemic lupus erythematosus patients (SLE), 40 human immunodeficiency virus (HIV) infected patients with elevated viral load and 100 blood donors. All donors provided informed consent for the study which was approved by our local ethical committee (project number 4928). Results Using the protein array, we detected IgG antibodies against CD74 in 4/5 SpA sera, but only in 1/45 controls. After we tested different different CD74 proteins as antigens in ELISAs, we chosed a peptide which includes the CLIP within the CD74 protein. Of the SpA patients with disease onset less than 1-6 months ago 24/24 (100%), less than 7-12 months 32/34 (94%) were positive for IgG autoantibodies. After 20yrs, the frequency of IgG autoantibodies against CD74 decreased to 65%. 141/196 (71%) of all SpA patients showed positivity for IgG autoantibodies against CLIP. In the further control groups, the prevalence of IgG autoantibodies against CLIP was 14/80 (17.5%) in RA, 5/40 (12.5%) in SLE, 1/40 (2.5%) in HIV and 0/100 (0%) in blood donors . Conclusions Considering their specifity and sensitivity, antibodies against CLIP will be an useful addition to our diagnostic tools for SpA in the future. References Braun J, Bollow M, Remlinger G. Prevalence of Spondylarthropathies in HLA-B27 positive and negative blood donors. Arthritis Rheum 1998;41:58–67 Dincer U, Cakar E, Kiralp MZ, Dursun H. Diagnosis delay in patients with ankylosing spondylitis: possible reasons and proposals for new diagnostic criteria. Clin Rheumatol. 2008;27:457-62. Bennett AN, Rehman A, Hensor EM, Marzo-Ortega H, Emery P, McGonagle D, The fatty Romanus lesion: a non-inflammatory spinal MRI lesion specific for axial spondyloarthropathy. Ann Rheum Dis. 2010;69:891-4. Disclosure of Interest None Declared