Filomena Panza

Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps

Background Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). Objective The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. Methods Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. Results RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14–34 and 31–50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

Free IL-18 and IL-33 cytokines in chronic spontaneous urticaria

Overproduction of IL-18 has been described in chronic urticaria. To evaluate free IL-18 and IL-33 in chronic spontaneous urticaria (CSU). IL-18, its inhibitor IL-18BP, IL-33 and its soluble receptor ST2 (sST2) were measured (ELISA) in the sera of 73 CSU patients. Free IL-18 was calculated (law of mass action). Autologous serum skin test (ASST) was performed in all patients. Total IL-18, IL-18BP and free IL-18 serum levels were significantly higher in CSU than in controls. IL-18 and IL-18BP increased significantly in both ASST-positive and negative subgroups. Free IL-18 resulted significantly higher in the ASST-negative, but not in the ASST-positive subgroup. No differences in IL-33/sST2 levels were detected between CSU and controls. Increased levels of free IL-18 and IL-18BP, but not IL-33, was detected in CSU. Whether IL-18 up-regulation is a consequence of inflammation or one of the causes of the pathology needs to be addressed.

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides-viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein-Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4-were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10(-6) M>KD>10(-7) M) and interacted with HCP1 with higher apparent affinity (KD=10(-8) M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.

CCL5/RANTES, sVCAM-1, and sICAM-1 in Chronic Spontaneous Urticaria

Background: Chronic urticaria (CU) is a common disease characterized by recurrent itchy wheals and/or angioedema for more than 6 weeks. We aimed to investigate the potential involvement of chemotactic mediators and soluble adhesion molecules as markers of endothelial dysfunction in the pathogenesis of chronic spontaneous urticaria (CSU). The potential relevance of these soluble mediators in the evaluation of disease activity was also investigated. Methods: We measured the levels of CCL5/RANTES, CXCL8/IL-8, sVCAM-1, and sICAM-1 in the sera of 87 patients with CSU and 61 normal healthy subjects (NHS) using ELISA assays. According to the results of autologous serum skin tests (ASST), CSU patients were classified into ASST-positive and ASST-negative subgroups. Furthermore, we investigated in 4 patients whether H1-antihistamine therapy decreases sVCAM-1 and sICAM-1 levels. Results: We detected a significantly higher concentration of CCL5/RANTES (p < 0.0001) but not of CXCL8/IL-8 in CSU patients compared to NHS. The serum levels of sICAM-1 and sVCAM-1 were significantly increased in CSU patients compared to NHS (p = 0.0121 and p = 0.0043, respectively). No difference in chemokine or soluble adhesion molecule levels was detected between the ASST-positive and ASST-negative subgroups. A positive correlation was found between sICAM-1 and sVCAM-1 (p = 0.0022) but not between these and CCL5/RANTES. After H1-antihistamine therapy, sVCAM-1 and sICAM-1 levels did not decrease in the 4 CSU patients tested. Conclusions: Our study suggests that CCL5/RANTES, sICAM-1, and sVCAM-1 play a potential role in the pathogenesis of CSU but they do not parallel disease activity and are not predictive of the response to H1-antihistamine therapy.

Fingerprinting of anti-citrullinated protein antibodies (ACPA): specificity, isotypes and subclasses

Anti-citrullinated protein antibodies (ACPA) are a family of rheumatoid arthritis (RA)-specific autoantibodies that recognize the amino acid citrulline, resulting from the post-translational modification of arginine. Peptidyl arginine deiminase, the enzyme responsible for citrullination, is present in humans in different isoforms with different tissue distribution, enzymatic activity and target specificity; nonetheless, the number of proteins citrullinated in physiological or pathological conditions is wide, but not every citrullinated protein is a target for antibodies. In pre-RA patients the immune response to citrullinated antigens is initially restricted, expands with time and, after the onset of the disease, is relatively stable. ACPA are heterogeneous in terms of not only fine specificity but also isotype and IgG subclasses usage. This heterogeneity may be relevant for the immunopathogenesis of RA, conditioning the interaction of antibodies with complement and Fc receptors.

Immunoglobulin G subclass profile of anticitrullinated peptide antibodies specific for Epstein Barr virus-derived and histone-derived citrullinated peptides.

To the Editor: Studies have shown that the anticitrullinated peptide antibodies (ACPA) response is highly polyclonal, in terms of epitope specificity, V genes, and isotype usage1,2. Longitudinal studies of patients with rheumatoid arthritis (RA) have documented epitope spreading, and ACPA, specific for distinct citrullinated epitopes, have been described. By using different citrullinated antigens, ACPA from immunoglobulin (Ig)G, IgA, and IgM isotype have been detected3. ACPA are polyclonal in the usage of different IgG subclasses, but in this case the pattern is more heterogeneous. So far the studies conducted indicate the dominance of IgG1 and IgG4, while IgG3 have been detected with cyclic citrullinated peptide (CCP) and vimentin, but not with fibrinogen4,5. The production of specific IgG subclasses might help in deciphering the mechanisms eliciting B cell expansion in response to different antigens. Thus, it is of interest to explore the profile of IgG subclasses of antibodies reactive with novel citrullinated substrates, already known to be tools for ACPA detection. Ninety-three patients with RA, 25 with psoriatic arthritis, 15 with ankylosing spondylitis, and … Address correspondence to Professor P. Migliorini, Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 67, 56126 – Pisa, Italy. E-mail: paola.migliorini{at}med.unipi.it

Surface Plasmon Resonance Method to Evaluate Anti-citrullinated Protein/Peptide Antibody Affinity to Citrullinated Peptides.

Surface plasmon resonance (SPR) technique is extremely interesting in immunology because it has the potential to directly visualize biomolecular interactions in real-time monitoring antibody affinity, one of the parameters affecting pathogenicity in autoimmune diseases. Herein we describe the affinity evaluation of anti-citrullinated peptide antibodies (ACPA) to a peptide-based biosensor by SPR. The method describes the purification of ACPA isolated from rheumatoid arthritis (RA) patients using affinity columns, the strategy employed for the immobilization of citrullinated peptides onto a sensor chip, and the evaluation of the specific binding of purified ACPA to immobilized peptides.

AB0500 A Novel DNA-Peptide Complex Detects Anti-DSDNA Antibodies in SLE Sera

Background The detection of anti-dsDNA antibodies is critical for the diagnosis and follow-up of SLE patients. The presently available assays are characterized by a non-optimal specificity (solid phase assays) or sensitivity (CLIF test). Objectives To overcome the limits of CLIFT and solid phase chromatin assays, we explored the diagnostic potential of an assay based on plasmid DNA containing a highly bent fragment of 211 bp from Crithidia Luciliae microcircles, complexed with histone peptides. Methods Electrically neutral complexes of PK201/CAT plasmid (PK) DNA and histone (H) 4 peptides were evaluated by electromobility shift assay. Complexes of H4 peptides and PK were absorbed to the solid phase to detect IgG antibodies in sera. Sera from 109 SLE patients, 100 normals and 69 disease controls (systemic sclerosis, Sjogrens syndrome, unclassified connective tissue disease) were tested. Results H4 (14–34) containing the consensus sequence for DNA binding interacts with PK retarding its migration. H4 (14–34)-PK complexes were used to test sera by ELISA; inhibition assays show that sera react with epitopes present on DNA or on the complex, not on the peptide. Anti-H4-PK antibodies were detected in 56% SLE sera (more frequently in patients with skin or joint involvement) vs 12% disease controls; antibody titer is correlated with ECLAM score and anti-C1q antibodies, negatively with C3 levels. Anti-H4-PK antibodies compared with CLIFT and solid phase dsDNA assays display moderate concordance. Conclusions The H4-PK assay is a simple and reliable test, endowed with high specificity and sensitivity, useful for the differential diagnosis and evaluation of disease activity of SLE patients. This assay increases the variety of anti-DNA antibodies that can be measured, complementing the assays currently used for the detection of anti-DNA antibodies. Disclosure of Interest None declared

A6.10 Anti citrullinated peptides/proteins antibodies induce secretion of CCL5/rantes by fibroblast like synoviocytes

Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder, characterised by a progressive joint damage, caused by a cellular infiltrate localised in the synovia. In RA, fibroblast like synoviocytes (FLS) acquire an aggressive phenotype and contribute to joint destruction also producing a variety of pro-inflammatory cytokines. In the pathogenesis of RA a pivotal role is played by fibroblast like synoviocytes (FLS), that produce cytokines that perpetuate inflammation. One of the serological markers of RA are anti citrullinated protein antibodies (ACPA), a family of autoantibodies recognising a wide variety of antigens all characterised by the deimination of arginine into citrulline. Among the different citrullinated targets, ACPA can bind viral citrullinated peptides (VCPs) and histone citrullinated peptides (HCPs). Despite their strict association with RA, so far little is known about the role of ACPA in the pathogenesis of the disease. Objective The aim of this work was to analyse the effects of anti VCPs and HCPs antibodies on in vitro cultured FLS. Methods FLS from RA and non RA patients were isolated from synovial fluid and used for experiments at 4–5th passage. Anti-VCPs and anti-HCPs antibodies were purified by affinity chromatography from RA patients sera and IgG from normal subjects were used as control antibodies. Anti-VCPs and –HCPs antibodies were incubated on subconfluent FLS; cells and supernatants were recovered at different time points. RANTES protein was quantified in the supernatants by sandwich ELISA and RANTES gene expression was evaluated by RT-PCR. To investigate intracellular signalling after ACPA stimulation, specific MAP kinase, already known to be activated during CCL5/RANTES production, were studied. Results Anti-VCPs and anti-HCPs from 5 RA patients induce RNA expression and secretion of RANTES from FLS as compared with normal IgG (p < 0.001). No differences were observed between RA and non RA FLS. FLS stimulation with ACPA seems to induce phosphorylation of ERK, JNK and p38. Conclusions These results suggest a novel mechanism potentially involved in damage induced by ACPA, mediated by the production of RANTES in FLS. ACPA might play a role in activation and chemotaxis of T cells in synovial tissue with a perpetuation in inflammation and immune response. Studies are in progress to clarify the intracellular pathways implicated in the synthesis of RANTES induced by ACPA in FLS and to define the target of ACPA on FLS surface.

THU0465 Genetic Control of the Immune Response to Histone Derived Citrullinated Peptides in Rheumatoid Arthritis Patients

Background Rheumatoid arthritis (RA) sera contain antibodies specific for deiminated peptide/proteins (ACPA) directed against endogenous (filaggrin, fibrin, vimentin, collagen, enolase) or exogenous antigens. Recently ACPA reacting with citrullinated peptides derived from histone H4 have been detected. It is widely accepted that HLA-DRB1 alleles encoding the shared epitope sequences are associated with the production of ACPAs. However, the genetic background underlying the production of single ACPA specificities has not been thoroughly investigated. Objectives To analyze the reactivity to two different histone H4 derived citrullinated peptides (HCP1 = citrullinated H414–34 and HCP2 = citrullinated H431–50) and to study their association with genetic variants within HLA-DRB1 SE alleles in a RA population. Methods One hundred and seventy-two French RA patients were characterized in terms of HLA-DRB1 genotype, anti VCP-A and anti-VCP-B antibodies. HLA-DRB1 typing was performed using the polymerase chain reaction-sequence specific primer (SSP) method, and SE alleles were classified into four SE+ groups (S1, S2, S3P, S3D) and one SE- group (X) according to the new classification of HLA-DRB1 alleles, reshaping the shared epitope (SE) hypothesis [1]. Anti-HCPs antibodies were assessed by home made ELISA on HCP1 and HCP2 coated plates. Results Setting the threshold value at the 98° percentile of the normal population, anti-HCP1 antibodies are present in 46% and anti-HCP2 antibodies in 62% of RA patients. Anti-HCP1 antibodies are not associated with HLA-SE alleles. On the contrary, anti-HCP2 antibodies are associated with HLA-DRB1 *0102 (p=0.04), *0401 (p=0.03), *0404 (p=0.01). Subgrouping the SE alleles according to the new classification, the presence of anti-HCP2 antibodies is associated with S2 allele (p<0.05). Conclusions Using two histone H4 derived citrullinated peptides as probes, we showed genetic heterogeneity in the control of the immune response to single components of the ACPA family and to different portion of the same molecule. These data underline the need to type RA patients for different ACPA specificities, that may contribute to the identification of different subsets of patients. References du Montcel ST et al. Arthritis Rheum. 2005, 52(4): 1063-8 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5450