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BackgroundPatients with rheumatoid arthritis (RA) have an increased risk for cardiovascular disease, including venous thromboembolic events (VTE)1. The reason behind the increased VTE risk is incompletely understood, but inherent features of RA, such as RA specific autoantibodies, could potentially play a role. For example, studies have linked occurrence and levels of rheumatoid factor (RF) in the general population to increased VTE risk2. We and others have demonstrated an association between ACPA and risk of later ischemic cardiovascular events3. There are also potential mechanistic links; citrullinated fibrinogen (cFib) has been associated to clot stability4.ObjectivesWe aimed to examine the association between anti-modified protein antibodies (AMPAs) and risk of VTE in RA.MethodsWe included 2809 individuals newly diagnosed with RA and included in the Swedish EIRA study 1996-2009. Through linkage to nationwide health care registers we identified past and incident events of VTE based on validated ICD code algorithms. We centrally typed baseline sera for anti-CCP2, 20 different ACPA sub-specificities, RF isotypes, carbamylated antibodies and 10 additional post-translational modifications. We followed all individuals from RA diagnosis up until their first ever VTE event, migration, death or end of study (2020-12-31) whichever occurred first. We used a Cox regression to estimate hazard ratios (HR) with 95% confidence intervals (CI). Individuals with a history of a VTE event (n=27) at RA diagnosis were excluded.ResultsWe included 2782 individuals; 72% were women, median age at RA diagnosis was 54 years (inter quartile range (IQR) 18 years) and median follow-up time was 15.5 (IQR 6.8) years. During follow-up 177 incident VTE events were observed corresponding to an incidence of 5.0 per 1,000 person years.1797 (64.6%) patients were positive for IgG anti-CCP2 and the HR for VTE (vs. being negative for anti-CCP2) was 1.33 (95%CI 1.00-1.78). The risk of VTE increased with the level of anti-CCP2, with an HR of 1.49 (95%CI 0.99-2.22) for the group with extreme levels compared to those negative for anti-CCP2 (p-value for trend 0.048). For IgA anti-CCP2 the HR was 1.35 (95% CI 0.99-1.84) when comparing those expressing IgA anti-CCP2 against those who did not.Of 20 ACPA fine-specificities studied, 18 occurred with a frequency > 10% in our sample. The median number of fine-specificities expressed was 6 (IQR 11). The risk of VTE increased with the number of ACPA fine-specificities expressed (p-value for trend 0.033). At the 0.05 significance level, two fine-specificities were each associated with VTE; cPept Z1 [HR=1.40 (95%CI 1.06-84)] and cPept-1 [HR=1.47 (95%CI 1.12-1.93)]. None of the six antibodies against cFib assessed were statistically significantly associated with VTE risk. No associations were observed for other AMPAs. Among the three RF isotypes, only IgM RF was statistically associated with VTE [HR=1.38 (95%CI 1.04-1.83)].ConclusionRA-related antibodies analysed in clinical practice (anti-CCP2 IgG, RF) are associated not only with risk of myocardial infarction, stroke and cardiovascular death as previously demonstrated but also with VTE. There were no clear specific signals with ACPA fine-specificities, other AMPAs, or IgA RA autoantibodies.References[1]Holmqvist ME,et al. Risk of venous thromboembolism in patients with rheumatoid arthritis and association with disease duration and hospitalization. JAMA. 2012;308(13):1350-6.[2]Meyer-Olesen CL, et al. Increased rheumatoid factor and deep venous thrombosis: 2 cohort studies of 54628 individuals from the general population. Clin Chem. 2015;61(2):349-59.[3]Westerlind H, et al. Anti-citrullinated protein antibody specificities, rheumatoid factor isotypes and incident cardiovascular events in patients with rheumatoid arthritis. Arthritis Rheumatol. 2020.[4]Maners J, et al. A Mendelian randomization of gamma’ and total fibrinogen levels in relation to venous thromboembolism and ischemic stroke. Blood. 2020;136(26):3062-9.Disclosure of InterestsHelga Westerlind: None declared, Alf Kastbom: None declared, Johan Rönnelid: None declared, Monika Hansson: None declared, Lars Alfredsson: None declared, Linda Mathsson-Alm Employee of: LMA an employee of Thermo Fisher Scientific producing the ACPA sub-specificity test, Guy Serre: None declared, Martin Cornillet: None declared, Rikard Holmdahl Consultant of: historically several. Currently paid advisor for Lipum AB and Cyxone AB, Per-Johan Jakobsson Consultant of: UCB – Nov 2021 to Feb 2022., Karl Skriner: None declared, Holger Bang Employee of: HB is an employee of Orgentec Diagnostica, an IVRc company, Lars Klareskog: None declared, Saedis Saevarsdottir Employee of: SS is a part-time employee of deCODE genetics Inc., Karin Lundberg: None declared, Caroline Grönwall: None declared, Johan Askling Grant/research support from: AbbVie, AstraZeneca, Bristol Myers Squibb, Eli Lilly, Janssen, Merck, Pfizer, Roche, Samsung Bioepis, Sanofi, and UCB.

BackgroundPulmonary manifestations in rheumatoid arthritis (RA) are common comorbidities but the underlying mechanisms are largely unknown. We found in a previous study 3 SNPs associated with pulmonary fibrosis (PF); rs35705950 (MUC5B), rs111521887 (TOLLIP), and rs2609255 (FAM13A) besides age, rheumatoid factor positivity and methotrexate treatment.ObjectivesTo evaluate for the added value of a multiplex of anti-citrullinated peptide antibodies (ACPA) for the development of pulmonary fibrosis (PF) in an inception cohort of RA patients.MethodsA total of 1184 patients with early RA were consecutively included and followed prospectively from the date of diagnosis (index date) until death or until 31 December 2016. The diagnosis of PF was based on high resolution tomography. The presence of 21 ACPA fine specificities were analysed in plasma sampled at index date, using a custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden). Data on both ACPA and genetic data was available for 841 RA patients, of whom 50 developed PF. Associations were analysed using logistic regression analysis and presented as the odds ratio (OR) with the 95% confidence interval (CI). Models were adjusted for sex, age, DAS28 and presence of RF at RA diagnosis, smoking ever, and HLA-SE and in a second step for the three SNPs (.rs35705950, rs111521887 and rs2609255), respectively.ResultsIn unadjusted analyses eight ACPA reactivities were found associated with PF development (p< 0.05-0.001). The number of ACPA reactivities was related to PF development, both in crude and adjusted models (pConclusionThe development of PF in an inception cohort of RA patients was associated both with risk genes and, independently of the risk genes, the presence of certain ACPA, and the number of ACPA reactivities.References[1]Jönsson E, et al. Pulmonary fibrosis in relation to genetic loci in an inception cohort of patients with early rheumatoid arthritis from northern Sweden. Rheumatology (Oxford). 2021 May 16:keab441. doi: 10.1093/rheumatology/keab441.AcknowledgementsI have no acknowledgements to declare. The staff and patients at the departments of rheumatology in northern Sweden.Disclosure of InterestsNone declared

Background:Seropositive rheumatoid arthritis (RA) is characterized by the presence of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) with different fine-specificities. Yet, other serum anti-modified protein autoantibodies (AMPA), e.g. anti-carbamylated (Carb), anti-acetylated (KAc), and anti-malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. By using RA patient single-cell derived monoclonal antibodies we have previously shown that individual ACPA clones recognize small distinct citrulline-containing epitopes giving them extensive multireactivity when these epitopes are found in many peptides and proteins. Moreover, certain CCP2+ multireactive ACPA clones bind also to cabamylated and acetylated autoantigens [1].Objectives:To provide a comprehensive evaluation of serum IgG and IgA autoreactivity to different post-translational modifications in RA.Methods:We analyzed 30 different IgG and IgA AMPA reactivities to modified antigens by ELISA and autoantigen arrays, in N=1985 newly diagnosed RA patients and population controls. The study utilized both previously established (i.e IgG and IgA CCP2; IgG ACPA fine-specificities; IgG anti-Carb fibrinogen and Carb FCS; IgG and IgA Cit/Carb/KAc/Orn(Ac)-vimentin), and novel assays (e.g. IgG anti-MAA and IgG anti-acetylated histones). Association with patient characteristics such as smoking and disease activity were explored. The newly developed assays were also evaluated in SLE disease controls and CCP2+ RA-risk individuals without arthritis.Results:Carb and KAc reactivities by different assays were primarily seen in patients also positive for citrulline-reactivity. Modified vimentin (mod-Vim) peptides were used for direct comparison of different AMPA reactivities, revealing that IgA AMPA recognizing mod-Vim was mainly detected in subsets of patients with high IgG anti-Cit-Vim levels and a history of smoking. IgG acetylation reactivity was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone 2B reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking. This reactivity was also found to be present in CCP2+ RA-risk individuals without arthritis. Our data further demonstrate that IgG autoreactivity to MAA was increased in RA compared to controls with highest levels in CCP2+ RA, but was not RA-specific, and showed low correlation with other AMPA. Anti-MAA was instead associated with disease activity and was not significantly increased in CCP2+ individuals at risk of RA. Notably, RA patients could be subdivided into four different subsets based on their AMPA IgG and IgA reactivity profiles.Conclusion:We conclude that autoantibodies exhibiting different patterns of ACPA fine-specificities as well as Carb and KAc reactivity are present in RA and may be derived from multireactive B-cell clones. Anti-Carb and anti-KAc could be considered reactivities within the “Cit-umbrella” similar to ACPA fine-specificities, while MAA is distinctly different.References:[1]Sahlström P, Hansson M, Steen J, Amara K, Titcombe PJ, Forsström B, Stålesen R, Israelsson L, Piccoli L, Lundberg K, Klareskog L, Mueller DL, Catrina AI, Skriner K, Malmström V, Grönwall C. Different Hierarchies of Anti-Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis. Arthritis Rheumatol. 2020 Oct;72(10):1643-1657. PMID: 32501655Caroline Grönwall: None declared, Lisa Liljefors: None declared, Holger Bang Employee of: Employee at ORGENTEC Diagnostika GmbH, Aase Hensvold: None declared, Monika Hansson: None declared, Linda Mathsson-Alm Employee of: Employee at Thermo Fisher Scientific, Lena Israelsson: None declared, Anna Svärd: None declared, Cyril CLAVEL: None declared, Elisabet Svenungsson: None declared, Iva Gunnarsson: None declared, Guy Serre: None declared, Saedis Saevarsdottir: None declared, Alf Kastbom: None declared, Lars Alfredsson: None declared, Vivianne Malmström: None declared, Johan Rönnelid: None declared, Anca Catrina: None declared, Karin Lundberg: None declared, Lars Klareskog: None declared

Background:Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding the shared epitope (SE), a 5 amino acid motive. RA is usually preceded by the emergence of anti-citrullinated protein antibodies (ACPAs) detected by anti-CCP2 tests. Citrullin is a neutral amino acid resulting from post translational modification of arginin by Peptidyl Arginyl Deiminases (PADs). ACPAs recognize epitopes from citrullinated human fibrinogen (Fib-cit) and can be specifically detected by the AhFibA assay. Five peptides derived from Fib-cit together represent almost all of the epitopes recognized by patients with ACPA-positive RA: β60–74cit, α36–50cit, α621–635cit, α501–515cit and α171–185cit. As RA is a pleiomorphic disease, whose evolution is difficult to predict, the use of antibody fine specificity as a marker of clinical phenotypes has become a major challenge.Objectives:Our objective was to study whether clinical characteristics and HLA-DRB1 genetic background were associated with a specific reactivity against these epitopes.Methods:184 ACPA positive RA patients fulfilling the 2010 ACR/EULAR criteria were studied. Patients characteristics, including HLA-DRB1 genotype, were collected from their medical files. Anti-CCP2, AhFibA, Rheumatoid Factors (RF), and antibodies against the five major Fib-cit peptides were analyzed using ELISA assays.Results:Anti-CCP2 and AhFibA titres were strongly correlated (rs: 0.7037; p = 5.69x10-29, Pearson’s). Anti-α505-515cit antibodies were associated with HLA-DRB1*04:01 (OR = 5.52 [2.00 – 13.64]; p = 0.0003). High level anti-α505-515cit antibodies were significantly associated with rheumatoid nodules (OR = 2.71 [1.00 – 7.16], p= 0.044). Anti α501–515cit antibodies were associated with RF (OR=2.31 [1.10 – 4.78], p= 0.026).Conclusion:Immune complexes containing anti-α501–515cit antibodies and rheumatoid factors might be involved in the development of rheumatoid nodules on the HLA-DRB1*04:01 background. These findings highlight the role played by the HLA-DRB1*04:01 molecule and its rapid intracellular route into the lysosomes, enabling original antigen processing. Finally, purifying these epitope specific antibodies might be a new therapeutic opportunity for rheumatoid nodules.Disclosure of Interests:None declared

OBJECTIVE—Antiperinuclear factor (APF), "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin (AFA), are highly specific serological markers of rheumatoid arthritis (RA), which recognise epitopes on various isoforms of (pro)filaggrin. It was proposed that these antibodies are globally named antifilaggrin autoantibodies. Here the diagnostic value of the detection of each one is compared and the overlap between the three tests evaluated. METHODS—492 serum samples were tested, including 279 RA serum samples, taken from patients in France and Belgium. APF and "AKA" titres were estimated by indirect immunofluorescence, and AFA titres by immunoblotting on filaggrin enriched human epidermis extracts. RESULTS—By a convenient choice of the positivity thresholds, the diagnostic sensitivity and specificity of the tests were shown to be similar (0.52 and 0.97, respectively). Although the antibody titres were strongly correlated, the associations APF-AFA or AFA-"AKA" permitted more than 52% or 55% of RA to be diagnosed, with a specificity of 0.99. CONCLUSION—APF, "AKA", and AFA detection have a similar diagnostic value. However, because the three tests do not totally overlap, associating APF with "AKA" or AFA with "AKA" can improve diagnostic sensitivity. None of the three antigens used bear all the epitopes recognised by anti-filaggrin autoantibodies. Keywords: rheumatoid arthritis; antifilaggrin autoantibodies; antiperinuclear factor; antikeratin antibodies

To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated proteins autoantibodies (ACPA) reactive to α36-50Cit₃₈,₄₂ and/or β60-74Cit₆₀,₇₂,₇₄, two peptides identified as bearing the immunodominant epitopes of their major target, citrullinated fibrin. To analyse the relationships of anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies with autoantibodies reactive to the complete citrullinated human fibrinogen molecule (AhFibA) and with anti-CCP2 antibodies.617 sera from 181 patients with established RA and 436 with non-RA rheumatic diseases were tested by ELISA for AhFibA, anti-CCP2, anti-α36-50Cit₃₈,₄₂, anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies, and by nephelometry for rheumatoid factor (RF). Diagnostic indexes, correlations and concordances between tests were analysed. Crossreactivity of anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies was assessed in competition experiments.At a diagnostic specificity of 95%, the diagnostic sensitivity of AhFibA (83%) was significantly higher than that of all other tests. The diagnostic sensitivity of anti-β60-74Cit₆₀,₇₂,₇₄ (71%) was significantly higher than that of anti-α36-50Cit₃₈,₄₂ autoantibodies (51%) but similar to that of anti-CCP2 (74%). Titres of RF, anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies were weakly correlated with each other, whereas titres of anti-β60-74Cit₆₀,₇₂,₇₄ were strongly correlated with those of AhFibA (r=0.633) and anti-CCP2 (r=0.634). Anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ mainly corresponded to two non-crossreactive subfamilies of ACPA. More than 90% of AhFibA-positive or anti-CCP2-positive sera recognised the α36-50Cit₃₈,₄₂ and/or the β60-74Cit₆₀,₇₂,₇₄ peptide.Autoantibodies reactive to α36-50Cit₃₈,₄₂ and β60-74Cit₆₀,₇₂,₇₄ form two distinct, non-overlapping subfamilies of ACPA that, together, cover practically all the ACPA reactivity to citrullinated fibrinogen and to CCP2 antigens. In established RA, anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies show diagnostic indexes similar to those of anti-CCP2.

Background Antibodies (Ab) towards citrullinated (cit) peptides (ACPA) are specific serological markers for a subset of rheumatoid arthritis (RA) patients with distinct genetic and environmental associations [1,2]. On the autoantigen-derived peptide level, the ACPA responses in RA patients are heterogeneous. Better understanding of the relationships between immunity to defined peptides, and structural variants of HLA molecules may provide novel insights concerning HLA-driven pathology in RA. Objectives To investigate whether RA specific Ab towards cit peptides can detect subsets of patients with distinct associations to different HLA structures. Methods We used a multiplex peptide array [3] to detect reactivities in sera of patients (n=6400) and selected controls (n=12000) across cohorts from Sweden (EIRA), United Kingdom (WTCCC), and the United States (NARAC). Genetic data from the immunochip platform was used to investigate variation in the HLA region. First, we tested the association between AA-alleles of HLA molecules and Ab defined strata of RA patients. In order to quantify the similarity between different peptide reactivities we used permutation, scaled Jaccard distance metrics and hierarchical clustering. Next, we assessed genetic associations with antibody titers in RA cases, and estimated interactive effects on susceptibility to Ab-positive RA. Results Comparing 20 different ACPAs reactive with different cit peptides, we observed that reactivities to specific peptides are highly correlated across patients (>87% pairs co-occurs more than expected). The majority of Abs are highly co-expressed in patients who have high frequencies of HLA-alleles known to be associated to ACPA-positive RA. AA at positions 11/13 of HLA-DRB1 display the strongest susceptibility to 18 fine-specific ACPAs (omnibus p≤9.3x10 –64 ), the exceptions being reactivities towards cit Fibrinogen (Fib) β 62–81 with citrullination at position 72 or 74. The reactivities towards two Fib peptides are not highly co-occurring with other Ab; The expression of Ab against cit Fib α peptide 36–50 is independent of HLA variants and for Ab towards cit Fib β 62–81 (72) there is even a negative association to HLA-variants previously shown to associate with ACPA-positive RA. Finally, we observed several allelic combinations showing interactive effects on fine specific ACPAs, and not or weakly with CCP2. Conclusions RA patients often co-express many different RA specific Ab, and the expressions of most of these are associated to HLA variants linked with ACPA-positive RA. However we also identified ACPAs with distinct expression profiles and HLA associations. References Klareskog L et al. PMID: 18173373 Raychaudhuri S et al. PMID: 22286218 Hansson M et al. PMID: 23025688 Disclosure of Interest None declared

Background Anti-CCP2 antibodies are associated with parenchymal lung changes in early RA. Objectives To examine the association between ACPA fine specificities and parenchymal lung changes in early RA. Methods Patients with newly diagnosed RA, glucocorticoids and DMARDs-naive, were included. High-resolution computed tomography (HRCT) was performed to assess parenchymal lung changes (ground glass changes, nodules, infiltrates or fibrosis). ImmunoCAP was used to detect RF IgA, RF IgM, anti-CCP2 IgA, anti-CCP2 IgG and ISAC microarray system was used to detect antibodies against 10 citrullinated (Cit) peptidic antigens: CCP-1 (Filaggrin), CEP-1 (α-enolase), Vim 2–17, Vim 60–75 (Vimentin), Fib β 36–52, Fib α 573, Fib α 591, Fib α 36–50, Fib β 60–74, Fib α 621–635 (Fibrinogen). Logistic regression was performed to examine associations between lung changes and autoantibodies, adjusted for age and sex. Due to risk for effect modification of smoking and co-linearity between smoking and Cit peptides, we stratified the cohort according to ever vs. never smokers. Results A total of 106 patients were included. The mean (SD) age was 57 (14) years. 69% were females; 73% were ever smokers; 70% were RF positive and 69% were positive for anti-CCP2. Parenchymal lung changes were found in 58 patients (54.7%). Higher age [≥65 vs Conclusions RF IgA, anti-CCP2 IgG, antibodies to Cit Fib and Cit Vim peptides were strongly associated to parenchymal lung changes in ever smokers with early RA. The more ACPA fine specificities, the higher the risk of parenchymal lung changes. Disclosure of Interest None declared

Background Antibodies against citrullinated peptide antigens (ACPAs) strongly predict the risk of developing RA. ACPA also seem to predict a more severe RA phenotype. Objectives Little is, however, known about the predictive value of specific ACPAs, or other autoantibodies (AAbs), in terms of response to methotrexate treatment. Methods A total of 20 AAbs were measured by a custom-made microarray assay based on the ImmunoCAP ISAC system ( Phadia AB, Uppsala, Sweden ), in baseline samples from patients participating in two randomized controlled trials (RCTs): Swefot, n=340; Improved, n=450). Validation was performed in two large inception cohorts (EIRA linked to the SRQ register, n=1481; Glasgow, n=384). All patients started methotrexate in monotherapy as their first DMARD. Response to methotrexate was defined as EULAR response at first follow-up visit (3–6 months). The likelihood of EULAR good or moderate (vs. no) response was calculated by logistic regression adjusted for potential confounders, and the proportion of patients positive for each AAb is provided (adj. OR, 95%CI; % positive ). Results For the RCTs, 5 out of 20 AAbs were significantly associated with response in Improved, and 1 out of 17 in Swefot (fewer peptides on an earlier version of ISAC). Fib36_52_cit (citrullinated fibrinogen) was significantly associated with good/moderate response in both RCTs (Improved: adj.OR=2.27 (1.19–4.31), 32% positive ; Swefot: adj.OR=1.87 (1.06–3.29), 44% positive ). Fib591_cit (also citrullinated fibrinogen) was associated with good/moderate response in Improved (adj.OR=3.70 (1.27–10.76), 15% positive ) and a non-significant trend was observed in Swefot (adj.OR=1.65 (0.89–3.06), 30% positive ). Vim60_75_cit (citrullinated vimentin) was associated with response in Improved (adj.OR=1.85 (1.06–3.21), 46% positive ) but not in Swefot (adj.OR=1.06, 63% positive ). For CEP_1 , a borderline significant association was observed in Improved (adj.OR=1.70 (0.96–3.00), 40% positive ) but not in Swefot (adj.OR=0.90, 51% positive ). Further, AAbs against citrullinated peptides named pept_Z2 and pept_5 were associated with response in Improved (adj.OR=1.81 (1.02–3.23), 41% positive ; and 2.25 (1.27–4.01), 44% positive , respectively), while no association was observed in Swefot (adj.OR=0.98 and 1.17). Interestingly, none of the above associations were replicated in the inception cohorts (EIRA-SRQ, Glasgow), nor did we observe any trend for a dose-response association with response for the total number of positive AAbs. Conclusions Whilst two AAbs against citrullinated fibrinogen predict response to methotrexate in both early RA RCTs, neither these nor any other of the AAbs tested predicted response in the real-life population-based inception cohort setting. The reasons for the discrepant results are not yet clear, but our results highlight the importance of testing in multiple study populations and settings when identifying predictors to be used for clinical prediction. Disclosure of Interest None declared

ObjectiveTo analyse Fcγ receptor (FcγR) expression on monocytes and macrophages from rheumatoid arthritis (RA) patients versus healthy controls (HC), and to compare their responses to immune complexes containing RA-specific anti-citrullinated proteins auto antibodies (ACPA).MethodsMonocytes and monocyte-derived macrophages were obtained from the peripheral blood of 34 RA patients and 69 HC. FcγR expression was studied by flow cytometry. Cells were stimulated with ACPA-containing immune complexes, and tumour necrosis factor alpha (TNFα) was assayed in culture supernatants.ResultsVariations distinguished RA from HC monocytes, corresponding to a 5% and 6% decrease in the percentages of monocytes expressing FcγRI and FcγRII, respectively, and a 7% increase in the proportion of FcγRIII-positive monocytes. Although in both HC and RA patients macrophage differentiation was accompanied by a dramatic increase in the percentage of FcγRIII-expressing cells (72% vs 74.5%), the parallel decline in the proportion of FcγRI-positive cells was markedly smaller in RA (7% vs 43%). Monocytes and macrophages from patients were responsive to ACPA-containing immune complexes but TNFα production in both cell types neither differed from that observed with the corresponding cells from HC, nor correlated with FcγR expression or clinical or biological data. In RA as in HC, ACPA-containing immune complexes induced secretions of more TNFα in macrophages than in paired monocytes (ninefold). Finally, the proinflammatory potential of ACPA-containing immune complexes was confirmed in CD14-positive monocyte macrophages from the synovial fluid of four RA patients.ConclusionsACPA-containing immune complexes induce TNFα secretion by blood and synovial fluid-derived macrophages from RA patients, fitting with their probable involvement in RA pathophysiology.

Background and objectives Activated neutrophils (PMN) form neutrophil extracellular traps (NET), expelled chromatin fibres. NETosis is dependent on citrullination and may be pathogenic in rheumatoid arthritis (RA), which is characterised by the production of anti-citrullinated protein autoantibodies (ACPA). However, the potential pathogenic mechanisms triggered by NET are not elucidated. Therefore, we have analysed the antigenic and inflammatory properties of NET in RA. Materials and methods Primary blood PMN and monocytes were freshly purified from 115 independent healthy donors (HD) and RA patients. IgG were purified from HD, ACPA-positive RA patients or ACPA-negative patients. Monocytes were differentiated into macrophages. NETosis was induced in vitro by PMA on adherent PMN and analysed by fluorescence microscopy, together with IgG binding. To produce soluble NET (sNET), NET were detached from glass by mild nuclease digestion. They were enriched, quantified by spectrophotometry, and characterised by SDS-PAGE and agarose gel. Macrophages/PMN were cultured with sNET in the presence/absence of LPS, IgG, NH 4 Cl, LL-37, ATP or C1q. Cell purity, IgG binding and cell activation were estimated by flow cytometry. Cytokine secretion was measured by ELISA. Results Low binding of IgG was observed on untreated PMN. Upon activation, a strong recognition by ACPA+ IgG was observed on NET. The NET staining with ACPA from RA patients was stronger than with ACPA-negative IgG. HD NET were as antigenic as RA NET. NETosis was higher with RA PMN. Soluble NET activated steady-state macrophages and PMN, independently of ACPA+ IgG or soluble LL-37. The stimulatory activity of sNET on macrophages was increased in the presence of C1q but was not influenced by inhibition of endosomal acidification/TLR in PMN. Both HD NET and RA NET induced cell activation, although RA NET were more potent. Likewise, both HD and RA PMN and macrophages responded to sNET but IL-1β secretion was not induced. On the contrary, sNET specifically inhibited LPS-induced IL-6 secretion by macrophages. Conclusions ACPA+ IgG from RA patients recognise NET and not untreated PMN. NET are antigenic and possess both pro- and anti-inflammatory properties. Therefore, an excess of NETosis together with higher NET activity may be pathogenic in RA.

Background:Different methods exist to demonstrate anti-citrullinated protein/peptide antibodies (ACPA).Aims:To evaluate discrepancy between four ACPA tests.Patients and methods:Population 1 consisted of patients with a new diagnostic problem, including 86 patients with rheumatoid arthritis (RA) and 450 patients without RA. Population 2 consisted of 155 patients with RA who had long-standing disease. Population 3 consisted of 188 patients with psoriatic arthritis and in population 4 there were 192 patients with systemic lupus erythematosus. Populations 1 and 2 were tested with the anti-human fibrinogen antibody (AhfibA) test, anti-CCP2 from Eurodiagnostica (CCP2-euro), anti-CCP2 from Pharmacia (CCP2-phar) and anti-CCP3 test by Inova (CCP3). Samples were annotated as discrepant if positive in one and negative in at least one other test. Each discrepant sample was re-analysed in a different run. Populations 3 and 4 were analysed in the CCP2-euro and AhFibA test.Results:In population 1, ACPA positivity was found in 17 of 450 (3.8%) patients without RA; 14 (82%) of these 17 samples were discrepant. In contrast, 61 of 86 (70.9%) patients with RA were ACPA positive of whom 18 of 61 (29.5%) were discrepant (70.9% vs. 29.5%, pConclusions:Discrepancy between different ACPA tests was observed attributable to the occurrence of borderline results, inter-assay variability and mainly to inter-test variability. The lowest inter-test discrepancy is observed between tests that use the same substrate.

Background: Autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies can be detected in rheumatoid arthritis (RA) sera. Objective: To determine the diagnostic values of RF, anticitrullinated protein antibodies, and the shared epitope (SE), and their associations with radiological progression rates and extra-articular manifestations. Methods: Population 1 consisted of sera from 315 patients, consecutively sent for detection of anticitrullinated protein antibodies, of which 264 were used to determine the sensitivity and specificity of RF and of antibodies against three synthetic citrullinated peptides: peptide A (pepA), peptide B (pepB), and CCP2. Population 2 consisted of sera from 180 longstanding RA patients and was used to determine associations of RA associated antibodies and the SE with radiological progression rates and extra-articular manifestations. Antibodies to pepA and pepB were detected by line immunoassay, and antibodies to CCP2 by ELISA. HLA Class II typing was performed by LiPA. Results: In population 1, we defined adapted cut offs corresponding to a specificity of ⩾98.5%. This yielded the following sensitivities: RF 12.8%; anti-pepA antibodies 63.6%; anti-pepB antibodies 54.2%; and anti-CCP2 antibodies 73.7%. In population 2, significant differences in radiological progression rates were found between positive and negative patients for different RA antibodies and the SE. RF, but not anticitrullinated protein antibodies or the SE, were more frequent in patients with extra-articular manifestations. Conclusion: A valid comparison of RA associated antibodies shows superior sensitivity of the anticitrullinated protein antibodies compared with RF. The presence of RA associated antibodies and the SE are indicative for poorer radiological outcome, and presence of extra-articular manifestations is associated with RF but not with anticitrullinated protein antibodies.

Background and objectives Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients were they probably contribute to synovitis. We showed that monoclonal IgM paraproteins with RF activity from patients with mixed cryoglobulinemia enhance the FcγR-mediated proinflammatory response of macrophages to ACPA immune complexes (ACPA-IC). Our aim was to investigate the influence of IgM RF from RA patients on the FcR- and complement-dependent effects of ACPA-IC and to examine that of RA-associated IgA RF. Materials and methods M-CSF-differentiated macrophages were stimulated in a serum-free medium by ACPA-IC formed with plate-bound citrullinated fibrinogen in the absence or presence of one of 5 IgM RF or 3 IgA RF fractions purified from RA serum pools by sequential affinity chromatography. Pro-inflammatory and immunoregulatory cytokines were assayed in the culture supernatants. The expression of IgM and/or IgA receptors was analysed by flow cytometry and their contribution to TNF-α secretion prompted by RF-containing ACPA-IC was evaluated. The complement activation capacity of ACPA-IC formed in the absence or presence of IgM or IgA RF was compared by ELISA measuring deposition of the terminal C5b–9 complex. Results When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with an increase of the TNF-α:IL-10 ratio and of the IL-6 and IL-8 secretions, and a decrease of the IL-1Ra:IL 1β ratio. The participation of an IgM receptor in the IgM RF-mediated amplification of the inflammatory response of macrophages was excluded, notably as they did not express any established receptor for IgM. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC. LPS further amplified the TNF-α response of macrophages to IgM or IgA RF-containing ACPA-IC. Finally, the presence of IgM RF or of IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Conclusions Specifically using autoantibodies found in RA patients, the strong complement-dependent and FcR-mediated proinflammatory effect of macro-IC including both ACPA and IgM RF or IgA RF is established, strongly reinforcing the hypothesis of their high pathogenic potential. Simultaneous FcR triggering by these macro-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.

Background and objectives Upon activation, neutrophils (PMN) form neutrophil extracellular traps (NET). Those structures are expelled chromatin fibres composed of DNA and associated proteins. The process, NETosis, is dependent on citrullination, a post-translational modification of proteins. NETosis has been suggested to be pathogenic in certain autoimmune diseases, particularly in rheumatoid arthritis (RA), characterised by the production of anti-citrullinated protein autoantibodies (ACPA) which are specific for the disease. Although RA PMN show enhanced NETosis and RA autoantibodies recognise NET, the pathogenic mechanisms triggered by NET are not elucidated. Therefore, we have analysed the antigenic and immunogenic properties of NET in RA. Materials and methods PMN and monocytes were freshly isolated by dextran sedimentation or purified by magnetic bead sorting, respectively, from the blood of healthy donors and RA patients. IgG were purified from healthy donors, from ACPA-positive RA patients and from ACPA-negative patients with rheumatic diseases. Monocytes were differentiated into macrophages by culturing in the presence of M-CSF. Cell purity and IgG binding were estimated by flow cytometry. NETosis was induced in vitro on poly-L-lysine-adherent PMN and was analysed by fluorescence microscopy, together with IgG binding. NET were detached from plastic by mild nuclease digestion to produce soluble NET which were enriched, quantified by fluorescence (PicoGreen) and by spectrophotometry (NanoDrop) and characterised by SDS-PAGE and agarose gel. Macrophages and PMN were cultured with soluble NET in the presence/absence of LPS or C1q. Cell activation was estimated by measuring cytokine secretion by ELISA. Results Only low binding of IgG was observed by flow cytometry on untreated freshly isolated PMN. Upon NETosis, a strong binding of ACPA+ IgG was observed, specifically on the NET structures, as demonstrated by the co-staining of DNA by fluorescence microscopy. Soluble NET activated both macrophages and PMN, leading to IL-8 secretion, independently of ACPA+ IgG. The stimulatory activity of soluble NET was increased in the presence of C1q. Soluble NET from both healthy donors and RA patients induced cell activation. Likewise, both PMN and macrophages from healthy donors and RA patients responded to soluble NET. On the contrary, soluble NET specifically inhibited LPS-induced IL-6 secretion by macrophages, and to a lower extent TNF secretion, but not IL-8 secretion. Conclusions ACPA+ IgG from RA patients strongly and specifically recognise NET and not untreated PMN. NET are immunogenic and possess both pro- and anti-inflammatory properties depending on the cell type, the activation status and the presence of co-factors like C1q.

OBJECTIVE—To develop a standardisable enzyme linked immunosorbent assay (ELISA), using human filaggrin, for detection of antifilaggrin autoantibodies in rheumatoid arthritis (RA). To compare the diagnostic performance of the ELISA with those of reference tests: "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin detected by immunoblotting (AhFA-IB). METHODS—Two ELISAs were developed using either affinity purified neutral-acidic human epidermis filaggrin (AhFA-ELISA-pur) or a recombinant human filaggrin deiminated in vitro (AhFA-ELISA-rec) as immunosorbent. Antifilaggrin autoantibodies were assayed in 714 serum samples from patients with well characterised rheumatic diseases, including 241 RA and 473 other rheumatic diseases, using the two ELISAs. "AKA" and AhFA-IB tests were carried out in the same series of patients. The diagnostic performance of the four tests was compared and their relationships analysed. RESULTS—The titres of "AKA", AhFA-IB, and the AhFA-ELISAs correlated strongly with each other. The diagnostic sensitivity of the AhFA-ELISA-rec, which was better than that of AhFA-ELISA-pur, was 0.52 for a specificity of 0.95. This performance was similar to those of "AKA" or AhFA-IB. However, combining AhFA-ELISA-rec with AhFA-IB led to a diagnostic sensitivity of 0.55 for a specificity of 0.99. CONCLUSION—A simple and easily standardisable ELISA for detection of antifilaggrin autoantibodies was developed and validated on a large series of patients using a citrullinated recombinant human filaggrin. The diagnostic performance of the test was similar to that of the "AKA" and AhFA-IB. Nevertheless, combining the AhFA-ELISA-rec with one of the other tests clearly enhanced the performance.

Based on their tight specificity for the disease, their association to its most severe forms and their production in the inflamed synovial tissue where citrullinated fibrin, their main antigenic target, is also abundant, the autoantibodies to citrullinated proteins (ACPA) probably play a central role in the pathophysiology of rheumatoid arthritis (RA). To substantiate this hypothesis the authors developed an original and totally human in vitro model …