G Nagy

CD3ζ-Chain Expression of Human T Lymphocytes Is Regulated by TNF via Src-like Adaptor Protein-Dependent Proteasomal Degradation

Decreased expression of the TCR ζ-chain has been reported in several autoimmune, inflammatory, and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. Although ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is less clear. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data have reported that SLAP regulates immunoreceptor signaling. We have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation. TNF treatment of human T lymphocytes (15–40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose-dependent manner (p < 0.05) and decreases activation-induced IL-2 expression (p < 0.01). Although blocking of the lysosomal compartment fails to restore TNF-induced CD3 ζ-chain downregulation, inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalization of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p < 0.05 and p < 0.01, respectively), whereas TNF-induced ζ-chain downregulation was inhibited by gene silencing of SLAP with small interfering RNA. SLAP levels of the CD4+ T lymphocytes isolated from patients with rheumatoid arthritis were more than 2-fold higher than that of the healthy donors’ (p < 0.05); moreover, TNF treatment did not alter the SLAP expression of the CD4+ cells of anti-TNF therapy-treated patients. Our present data suggest that TNF modulates T cell activation during inflammatory processes by regulating the amount of CD3 ζ-chain expression via a SLAP-dependent mechanism. These data provide evidence for SLAP-dependent regulation of CD3 ζ-chain in the fine control of TCR signaling.

Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition.

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.

Identification of a new citrullinated epitope on filaggrin for the early diagnosis of rheumatoid arthritis

Anti-citrullinated protein antibodies (ACPA) are sensitive and specific markers for diagnosis and prognosis in rheumatoid arthritis (RA). Citrullination is a post-translational modification of arginine by deimination, induced by peptidylarginine deiminase. It is a physiologically occurring phenomena during apoptosis, inflammation or keratinisation. Citrullination has been observed in different synovial proteins, including fibrinogen, vimentin and collagen. Antibodies specific for cyclic citrullinated filaggrin peptides (CCP) were detected in RA sera and anti-CCP positivity is widely used for diagnostic purposes. However, to determine …

A2.18 Induction and characterisation of the dominant IL-10 producing B cell subset in healthy blood donors and rheumatoid arthritis patients

Background and objectives The most important feature of B-cells is the production antibodies upon activation; additionally, B-cells produce both pro-inflammatory and anti-inflammatory cytokines in response to certain stimuli. IL-10 producing B10 cells represent a major subset of regulatory B-cells (Bregs). Bregs suppress autoimmune and inflammatory responses by multiple mechanisms. B-cells play crucial role in the development and maintenance of the chronic inflammatory autoimmune disease, Rheumatoid arthritis (RA); however, controversial data are available on B10 population in RA. Our aim was to identify the optimal conditions inducing B10 cells in samples from healthy controls and RA patients; furthermore, to shed light on signalling pathways resulting in the expansion of the B10 subset. Materials and methods Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors and RA patients. B cells were purified by magnetic separation, using negative selection. We assessed IL-10 and TNF expressing B-cells after intracellular staining by flow cytometry, and measured the secreted cytokines with multiplex bead array. Phosphorylation of key signalling molecules was monitored by phospho-flow method. Results The results show that dual stimulation by CpG and CD40L for 48h was optimal for IL-10 induction, which was synergistically boosted by IL-21. We identified CD19+ CD27+ memory B-cells as the major source of B10 cells. IL-21 increased the ratio of BLIMP1 and IL-10 double positive plasmablasts. In RA patients we detected significantly less CD27+ B10 cells as compared to the controls, while addition of IL-21 to the dual stimuli significantly elevated the number of B10 cells both in the CD19+CD27+ and in the CD19+ CD27- population. Different combinations of stimuli induced preferentially the secretion of pro-inflammatory cytokines (TNF, IFNγ, IL-17) and the suppressor cytokine, IL-10. We assumed that activation of ERK, p38 and CREB are indispensable to induce IL-10, while STAT3 appears to be a co-activator for IL-10 transcription in human Bregs. Conclusions CD19+CD27+ memory B-cells are the major source of human B10 cells, which may expand and differentiate to IL-10 producing plasmablasts in the presence of IL-21. CREB and the co-activator STAT3 are the key transcription factors responsible for the expansion of the B10 population. Support: National Research, Development and Innovation Office (OTKA104846), and ELTE TÁMOP 4.2.1./B-09/1/KMR-2010–0003

A2.39 Cytokine-induced regulation of human TH17 differentiation

Background and objectives Th17 cells have a central role in the inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, and tumour necrosis factor-α. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Materials and methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation method. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days the IL-17 and IL-22 production were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although the anti-CD3/CD28 activation increased the IL-17 and IL-22 production; it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc and T-bet without stimulation or cytokine treatment and also higher amount of IL-17 compared to the CD45RO- cells. By contrast, there was no difference between the RORc expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. The production of IL-17 and IL-22 of the differentiated CD45RO- cells from RA patients were both increased by IL-23 treatment. Conclusions The IL-17 and IL-22 production are differently regulated during the human Th17 differentiation. Furthermore, our present data suggest that the increased baseline RORc expression of the CD45RO- cells may contribute to the accelerated Th17 differentiation in RA.

AB0023 The Regulation of Human In Vitro Th17 Cell Differentiation by Cytokines

Background Th17 cells have a central role in inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, tumor necrosis factor-α and the expression of master regulator transcription factor RORc. The Th17 cells have essential role in many autoimmune diseases via induction of the development of inflammation. Objectives We studied the in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with an anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days IL-17 and IL-22 productions were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR methods. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although anti-CD3/CD28 activation increased the IL-17 and IL-22 productions, it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc, T-bet and IL17 without stimulation or cytokine treatment compared to the CD45RO- cells. However, there was no difference between the RORc and Tbet expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. IL-23 treatment increased both of IL-17 and IL-22 cytokine production of the differentiated CD45RO- cells. Conclusions IL-17 and IL-22 production are differently regulated during human Th17 differentiation. Our data suggest that the increased baseline RORc expression of CD45RO- cells may contribute to the accelerated Th17 differentiation in RA. Disclosure of Interest None declared

AB0074 The Effect of Extracellular Vesicles on Human in Vitro Osteoclastogenesis in Health and in Arthritis

Background Extracellular vesicles (EVs) like microvesicles (MVs) and exosomes (EXOs) are released in an evolutionary conserved manner by cells. EVs mediate intercellular communication with the transmission of molecules from their parent cell to their targets. Objectives Our objective was to investigate the effect of EVs on human in vitro osteoclastogenesis in healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected. EVs were isolated by filtration and differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection, and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs. Then the samples were treated with 50 ng/ml recombinant human RANKL and blood-derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartarate resistant acid phosphatase (TRAP). TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results Healthy and RA-derived exosomes significantly (p<0.01) inhibited osteoclast differentiation of the CD14+ cells from the same donors, while PsA-derived exosomes had a stimulatory effect (p<0.05) on osteoclastogenesis. In cross-induction experiments where EXOs and cells were interchanged between the 3 groups healthy (n=5) and RA (n=5) derived EXOs inhibited (p<0.01) the generation of osteoclasts in both groups but PsA (n=7) derived EXOs did not have an inhibitory effect. Microvesicles did not alter the number of mature osteoclasts in any groups. Conclusions Our data suggest that EXOs profoundly regulate human osteoclastogenesis. PsA-derived EXOs stimulate, while healthy and RA-derived EXOs inhibit the osteoclast differentiation. Disclosure of Interest None declared

SP0241 Extracellular Vesicles in Rheumatic Diseases

Extracellular vesicles (EVs) including microvesicles, apoptotic bodies and exosomes are regarded as a crucial form of intercellular communication. EVs are present in body fluids, may carry proteins, lipids, RNA, microRNA and DNA between cells. In addition to their widely studied physiological roles, EVs represent a promising and rapidly growing research field in inflammatory diseases, including rheumatologic conditions. Several lines of evidence support the role of synovial and blood EVs in the pathogenesis of rheumatoid arthritis (RA). Platelet derived microvesicles may carry proinflammatory cytokines in the inflamed synovium in RA. Recent data also indicate the possible role of EVs in the pathogenesis of systemic lupus erythematosus, systemic sclerosis and juvenile idiopathic arthritis. EVs may serve as biomarkers and may represent therapeutic targets in inflammatory conditions. Disclosure of Interest None declared

A7.4 Association of Galectin Single Nucleotide Polymorphisms with Autoimmune Diseases

Background and Objectives Galectins are potent immune regulators. Surprisingly, genetic association of galectin genes with autoimmune diseases have not yet been studied. A polymorphism in the coding region of the galectin-8 gene (Rs2737713; F19Y) and a novel galectin-1 and interleukin 2 receptor β haplotype were investigated for association with rheumatoid arthritis and myasthenia gravis. Materials and Methods A case-control analysis and a related quantitative trait-association study were performed to investigate the association of the galectin 8 gene polymorphism in patients (myasthenia gravis 149, rheumatoid arthritis 214 and 134 as primary and repetitive cohorts, respectively) and 365 ethnically matched (Caucasian) healthy controls. Distribution was also investigated in patients grouped according to their antibody status and age at disease onset. Comparative testing for lectin activity was carried out in ELISA/ELLA-based binding tests with both wild-type and F19Y mutant galectin-8 from peripheral blood mononuclear cell lysates of healthy individuals with different genotypes as well as with recombinant wild-type and F19Y mutant galectin-8 proteins. Furthermore, we evaluated the association of regulatory region polymorphisms of the LGALS1 (Rs4820293, Rs4820294) and IL2Rβ (Rs743777, Rs228941) genes in 146 Caucasian myasthenia gravis patients compared to 291 ethnically matched controls. Results We found a strong association of the F19Y galectin 8 gene polymorphism with rheumatoid arthritis, and a mild one with myasthenia gravis. Moreover, the polymorphism also correlated with age at disease onset in the case of rheumatoid arthritis. The F19Y substitution did not appear to affect carbohydrate binding in solid-phase assays markedly. Also, a significant difference was found in the distribution of the Rs4820293/Rs743777 polymorphism haplotypes (p < 0.01) in patients with myasthenia gravis and controls but not in rheumatoid arthritis. The Rs4820293 polymorphism of LGALS1, previously not described to be associated with any disease, did not affect LGALS1 expression in peripheral mononuclear cells and skeletal muscle. Conclusions This is the first study of an association between a galectin-based polymorphisms leading to a mutant protein and autoimmune diseases, with evidence for antagonistic pleiotropy.

CD3ζ-chain expression is regulated by tumor necrosis factor via Src-like adaptor protein dependent proteosomal degradation in human T lymphocytes

Background and objectives decreased expression of the T cell receptor (TCR) ζ-chain has been reported in several autoimmune, inflammatory and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. While ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is not known. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data indicated that SLAP regulates immunoreceptor signaling. The authors have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation. Materials and methods CD3ζ and SLAP protein levels of Jurkat cells and primary human T lymphocytes were measured by Western blot. Jurkat cells were transiently transfected with siRNAs to silence SLAP, knockdown efficiency of the siRNAs was measured by real-time RT-PCR and by Western blot. For confocal microscopy experiments cells were transfected with eGFP-SLAP expression-ready, full-length cDNA vector or control eGFP vector. The colocalisation between CD3ζ and SLAP were determined by laser confocal microscopy. CD3ζ mRNA was measured by quantitative real-time RT-PCR, IL-2 level was measured by ELISA method. Results TNF treatment of human T lymphocytes (15–40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose dependent manner (p<0.001), and decreases the activation induced IL-2 synthesis (p<0.01). Although blocking of the lysosomal compartment fails to restore the TNF-induced CD3 ζ-chain downregulation, the inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalisation of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p<0.05; p<0.009 respectively), while TNF induced ζ-chain downregulation was inhibited by silencing SLAP with siRNA (p<0.01). Conclusions Our present data suggest that TNF modulates T cell activation during inflammatory processes, by regulating the amount of CD3 ζ-chain expression via SLAP dependent mechanism. These data indicate that SLAP dependent regulation of CD3 ζ-chain provides fine control of TCR signaling.