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Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses.

Career situation of first and presenting author Student for a master or a PhD. Introduction Increasing evidence points to a mucosal origin of the autoimmune process in the development of rheumatoid arthritis. A B cell subset identified by surface expression of Fc receptor-like 4 (FcRL4) associates with inflammation in the mucosa associated lymphoid tissue.1 Our group identified these cells in the synovium of RA patients.2 They express high levels of RANKL and participate in the autoimmune response to citrullinated proteins.3 4 Recent in vitro work suggested that FcRL4 may be a low-affinity receptor for aggregated IgA.5 FcRL4 +B cells in RA SF express low levels of CD21 and high levels of CD11c, a phenotype they share with Age-Associated B cells (ABC). Very low numbers of ABC are found in blood of healthy individuals but their frequency increases with age however their exact role in RA is not yet understood.6 Objectives 1) Investigate the binding activity of IgA immune complexes to FcRL4+ B cells in tonsil, RA synovial fluid, and the peripheral blood of healthy individuals and RA patients. 2) Determine if FcRL4 can specifically capture IgA from the RA SF. 3) Examine the specificity of FcRL4 for secreted and serum IgA and for different IgA isotypes. Methods SF, peripheral blood and tonsil mononuclear cells were labelled for FcRL4, IgA, IgG, and CD19 (PBMCs were also labelled for CD21 and CD11c) and analysed by flow cytometry. Cells pre-treated with a pH3 buffer to remove receptor-bound immunoglobulins were compared with untreated cells to distinguish IgA B cell receptor expression from receptor- bound IgA Immune complexes. An FcRL4 expressing cell line was incubated with RA synovial fluid and purified human IgA to assess FcRL4s specificity. Results Receptor-bound IgA Immune complexes were observed on ex vivo RA SF and tonsil FcRL4+ B cells but not on FcRL4- B cells (p=0.001). Intriguingly, this IgA capture was not observed in peripheral blood FcRL4 +ABC from HC or RA patients. Furthermore, the proportion of B cells with IgA BCRs was increased in tonsil and SF FcRL4 +B cells but not in peripheral FcRL4+ B cells, compared to FcRL4- B cells. Using a cell line, we show that FcRL4 specifically binds IgA immune complexes from SF and shows a preference for IgA1 over IgA2 (p=0.02). Conclusions FcRL4 is a receptor for IgA immune complexes in RA joints. RA SF and tonsil FcRL4 +B cells RA SF differ from peripheral blood FcRL4 +ABC in their in vivo capture of IgA immune complexes and in their frequency of IgA BCRs, suggesting distinct function and origin of these cells. References Ehrhardt GR, et al. J Exp Med 2005. Yeo L, et al. Ann Rheum Dis 2011. Yeo L, et al. Ann Rheum Dis 2015. Amara K, et al. J Autoimmun 2017. Wilson TJ, et al. J Immunol 2012; 188. Marrack P, et al. J Immunol 2015. Disclosure of Interest None declared.

Career situation of first and presenting author Student for a master or a PhD. Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterised by joint inflammation and bone destruction. The presence of autoantibodies, years before the clinical onset of disease, and the efficacy of Rituximab, a B-cell depleting therapy, highlight a pathogenic role for B cells. Different groups have recently identified a novel subset of B cells named age-associated B cells (ABCs). Studies in mice autoimmune models and patients suffering from autoimmune diseases described these cells as CD19high CD21- CD11c+. Moreover, a subset of synovial fluid B cells with low levels of CD21, expresses FcRL4 and produces the cytokine RANKL, which stimulates the differentiation and activation of osteoclasts. The ABCs found in peripheral blood could therefore be the precursors of this FcRL4 positive subset found in synovia. Objectives We aimed to investigate the proportion and phenotype of peripheral blood ABCs in patients suffering from early drug naive RA. Methods Newly presenting patients, naive to immunomodulatory treatment, were recruited from the Newcastle Early Arthritis Clinic, and followed until diagnoses were confirmed. B-cell subsets in peripheral blood were detected and phenotyped using flow cytometry. NanoString nCounter Immunlogy v2 Panel (NanoString Technologies) was used to detect mRNA from cell lysates of sorted ABCs, naive, memory and CD5+ B cells from RA patients, age-matched healthy controls and disease controls (Psoriatic Arthritis patients). Results Our work showed that there are no significant differences in the frequency of ABCs between RA patients, disease controls and age-matched healthy controls. There is a possible trend for increased frequencies of ABCs with age as well as those who have a high disease activity. Our results also show that ABCs resemble a memory B cells phenotype with regard to class-switch immunoglobulins expression, with a significant percentage of them being positive for IgG and IgA. Interestingly, the FcRL4+, the proliferating Ki67+ and the T-bet expressing B cells were enriched in the ABC population compared to the other B cell subsets. Furthermore, ABCs expressed high levels of MHC class II and co-stimulatory molecules, as well as the activation marker, CD69. In addition, gene expression analysis using NanoString showed differentially expressed genes between ABCs and memory B cells. Conclusions These data supports an activated phenotype of the ABCs, which supports the idea that ABCs have a pathogenic role in RA, potentially via autoantibody and T cell stimulatory ability. However, further characterisation of this subset and functional studies are needed. Disclosure of Interest None declared.

Introduction Increasing evidence points to the autoimmune process of rheumatoid arthritis (RA) originating at mucosal surfaces. Previous work from our group described a subset of B cells in the synovium and synovial fluid (SF) of RA patients distinguishable by their expression of Fc-like receptor 4 (FcRL4) and elevated expression of RANKL, indicating a unique pathogenic function.1,2 B cells expressing FcRL4 were originally described as a distinct memory B cell subset in human tonsils where they are localised in the epithelium.3 We have recently shown in RA that they are enriched in clones recognising citrullinated autoantigens.3 Recent in vitro work suggested that FcRL4 is a low affinity receptor for aggregated IgA.4 Objectives Investigate the interactions between IgA and FcRL4 and FcRL4+ B cells using tonsil and SF B cells, and transfected cell lines. Examine the distribution of Ig classes by flow cytometry and PCR. Methods Mononuclear cells were isolated from SF (n=10) and tonsil, labelled for CD19, FcRL4, IgA, and IgG and analysed by flow cytometry. In experiments identifying BCRs and relative loads of surface bound IgA; SFMCs were washed in an acidic buffer to remove receptor-bound proteins before staining. Heat-aggregated purified human IgA was added to assess IgA binding to FcRL4 +B cells. Single B cells were sorted from SF of RA patients and their constant region genes probed for identification of their Ig subclass by PCR. Results We show that ex vivo SF FcRL4+ B cells have a significantly higher load of IgA bound to their surface (p=0.0001) than FcRL4- B cells. Following in vitro removal of surface bound IgA, FcRL4 +B cells bind heat-aggregated IgA (p=0.03 vs control). We also demonstrate that a significantly higher proportion of FcRL4+ B cells use IgA BCRs (p=0.0061) by flow cytometry, and by probing constant region genes by PCR an enrichment for Ig genes for coding the IgA1 isotype (p=0.009) was found. Furthermore, three out of eight of the antibodies recognising citrullinated peptides had been cloned from FcRL4+ IgA+ B cells. Conclusions In conclusion, their specificity for citrullinated antigens, ability to capture IgA immune complexes through via FcRL4 and enrichment in IgA1 subclass expression, suggest a role for FcRL4 +B cells in the mucosal origin of joint inflammation. References . Yeo L, et al. Ann Rheum Dis2011;70:2022–2028. doi:10.1136/ard.2011.153312 . Yeo L, et al. Ann Rheum Dis2015;74:928–935. doi:10.1136/annrheumdis-2013-204116 . Amara K, et al. J. Autoimmun2017. doi:10.1016/j.jaut.2017.03.004 . Wilson TJ, et al. J Immunol2012;188;4741–4745. doi:10.4049/jimmunol.1102651 Acknowledgements This work was supported by the Medical Research Council UK, Rheumatoid Arthritis Pathogenesis Centre of Excellence, and Karolinska Institutet Foundations for Rheumatology Research. Disclosure of interest None declared

Background Increasing evidence points to the autoimmune process of rheumatoid arthritis (RA) originating at mucosal surfaces. Previous work from our group described a subset of B cells in the synovium and synovial fluid (SF) of RA patients which can be distinguished by their expression of the Fc-like receptor 4 (FcRL4) and elevated expression of RANKL, indicating a unique pathogenic function1,2. B cells expressing FcRL4 were originally described as a distinct memory B cell subset in human tonsils where they accumulate in the epithelium3. We have recently shown that they are enriched in cells recognizing citrullinated autoantigens (Amara, K. et al. under revision). Recent in vitro work suggested that FcRL4 is a low affinity receptor for aggregated IgA4. Objectives 1) To investigate the interaction of RA synovial fluid FCRL4+ B cells with IgA 2) To examine the distribution of Ig classes by flow cytometry and PCR Methods SF mononuclear cells were isolated, labelled for FcRL4, IgA and CD19 and analysed by flow cytometry. In experiments identifying IgA B cell receptors, SF mononuclear cells were briefly incubated in an acidic buffer to remove surface receptor bound antibodies before staining. Heat-aggregated purified human IgA was added to assess IgA binding to FcRL4+ B cells. Single B cells were sorted from SF of RA patients and their constant region genes probed for identification of their Ig subclass by PCR. Results Ex vivo, FcRL4+ B cells in SF of RA patients have a higher load of IgA bound to their surface compared to their FcRL4- counterparts. After in vitro removal of surface bound IgA, they can bind heat-aggregated IgA (p=0.0313). We also demonstrate that a significantly higher proportion of FcRL4+ B cells use IgA BCRs (p=0.0061) by flow cytometry and further more by probing constant region genes by PCR an enrichment for Ig genes for coding the IgA1 isotype (p=0.009) was found. Conclusions Both their ability to capture IgA immune complexes through binding to FcRL4 and their enrichment in IgA Ig gene expression suggest a potential role for synovial fluid FcRL4+ B cells in the mucosal origin of joint inflammation. References Yeo, L. et al. Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis. Ann Rheum Dis 70, 2022–2028, doi:10.1136/ard.2011.153312 (2011). Yeo, L. et al. Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis. Ann Rheum Dis 74, 928–935, doi:10.1136/annrheumdis-2013–204116 (2015). Falini, B. et al. Expression of the IRTA1 receptor identifies intraepithelial and subepithelial marginal zone B cells of the mucosa-associated lymphoid tissue (MALT). Blood 102, 3684–3692, doi:10.1182/blood-2003–03–0750 (2003). Wilson, T. J., Fuchs, A. & Colonna, M. Cutting edge: human FcRL4 and FcRL5 are receptors for IgA and IgG. J Immunol 188, 4741–4745, doi:10.4049/jimmunol.1102651 (2012). Disclosure of Interest None declared

ObjectivesTo examine how rituximab may result in the inhibition of joint destruction in rheumatoid arthritis (RA) patients.MethodsTwenty-eight patients with active RA were treated with rituximab. Radiographs of hands and feet before and 1 year after therapy were assessed using the Sharp–van der Heijde score (SHS). Expression of bone destruction markers was evaluated by immunohistochemistry and immunofluorescence of synovial biopsies obtained before and 16 weeks after the initiation of treatment. Serum levels of osteoprotegerin, receptor activator of nuclear factor κB ligand (RANKL), osteocalcin and cross-linked N-telopeptides of type I collagen (NTx) were measured by ELISA before and 16 weeks post-treatment.ResultsAfter 1 year, the mean (SD) change in total SHS was 1.4 (10.0). Sixteen weeks after treatment there was a decrease of 99% in receptor activator of nuclear factor κB-positive osteoclast precursors (p=0.02) and a decrease of 37% (p=0.016) in RANKL expression in the synovium and a trend towards reduced synovial osteoprotegerin expression (25%, p=0.07). In serum, both osteoprotegerin (20%, p=0.001) and RANKL (40%, pConclusionsRituximab treatment is associated with a decrease in synovial osteoclast precursors and RANKL expression and an increase in the osteoprotegerin/RANKL ratio in serum. These observations may partly explain the protective effect of rituximab on the progression of joint destruction in RA.

Objectives In rheumatoid arthritis (RA), a complex cytokine network drives chronic inflammation and joint destruction. So far, few attempts have been made to identify the cellular sources of individual cytokines systematically. Therefore, the primary objective of this study was systematically to assess the cytokine messenger RNA expression profiles in the five largest cell populations in the synovial fluid and peripheral blood of RA patients. To reflect the in vivo situation as closely as possible, the cells were neither cultured nor stimulated ex vivo. Methods Inflammatory cells from 12 RA patients were sorted into CD4 and CD8 T cells, B cells, macrophages and neutrophils. mRNA expression for 41 cytokines was determined by real-time PCR using microfluidic cards. Receptor activator nuclear factor kappa B ligand (RANKL) (TNFSF11) expression by B cells was further confirmed by flow cytometry and by immunofluorescence staining of frozen sections of synovial tissue from patients with RA. Results The detection of cytokines characteristic for T cells and myeloid cells in the expected populations validated this methodology. Beyond the expected cytokine patterns, novel observations were made. Striking among these was the high expression of mRNA for RANKL in B cells from synovial fluid. This observation was validated at the protein level in synovial tissue and fluid. Conclusions RANKL, the key cytokine driving bone destruction by osteoclast activation, is produced by synovial B cells in RA. This observation is of importance for our understanding of the role of B cells in RA and their therapeutic targeting.

Rheumatoid arthritis (RA) is a persistent systemic inflammatory disease characterized by inflammation involving multiple cell types, with the progressive destruction of involved joints (1). An essential component of the switch to persistence that underlies joint destruction is the production of chemokines, which recruit mononuclear cells, such as lymphocytes and monocytes, to the inflamed joint (2). Galectins, an evolutionarily conserved family of animal lectins, have diverse roles in cellular homeostasis and have been shown to modulate inflammatory responses, functioning as either proinflammatory or antiinflammatory regulators, in part through their ability to cluster and modulate signaling through glycan receptors associated with multiple ligands (3). This ability to influence immune responses has been demonstrated in animal models of a number of diseases, including arthritis (4). Galectin 3, a chimera-type member of the galectin family, has a C-terminal carbohydrate recognition domain responsible for carbohydrate binding but exhibits an N-terminal domain that is responsible for interactions between subunits facilitating its oligomerization (5,6). The biologic functions attributed to this lectin are likely to depend on both ligand crosslinking and oligomerization (6,7). Galectin 3 has been associated with a proinflammatory role in models of fibrotic disease affecting the lung and liver (8,9) and has been shown to promote monocyte chemotaxis and macrophage activation (10-12) in addition to neutrophil activation, degranulation, and superoxide production (13-15), suggesting a critical role in the development of innate immune responses. Furthermore, a key role of galectin 3 has also been shown in the survival and progression of tumor metastases by modulating different processes, including homotypic and heterotypic cell adhesion, migration, angiogenesis, and tumor-immune escape (16). In the context of synovitis, Ohshima and colleagues (17) have demonstrated increased levels of galectin 3 and its binding protein in RA synovial tissue compared with osteoarthritis (OA) synovial tissue. Furthermore, galectin 3 levels are increased in RA in both synovial fluid and peripheral blood compartments, where levels correlate with C-reactive protein (17). Interestingly, up-regulated expression of galectin 3 correlates with abnormal cell apoptosis in synovial tissue from patients with juvenile RA (18). In contrast, galectin 1, a prototype member of the galectin family composed of 1 conserved carbohydrate recognition domain that can dimerize, has a predominantly antiinflammatory role, suppressing experimental models of inflammatory diseases, such as hepatitis, experimental autoimmune encephalomyelitis, uveitis, colitis, and arthritis (19-22). Furthermore, this glycan-binding protein appears to play an important role in the mechanisms involved in Treg cell–mediated suppression of immune responses (23), inhibition of T cell receptor–mediated signal transduction (24), and differential regulation of T helper cell viability (25). Intriguingly, synovial fibroblasts engineered to overexpress galectin 1 ameliorated collagen-induced arthritis and induced a bias toward a Th2-mediated cytokine profile in vivo (22). Synovial fibroblasts have an established role as sentinel cells for immune cell activation in the joint (2), and in RA, these cells are responsible for secreting significant quantities of inflammatory cytokines (26). RA synovial fibroblasts actively contribute to destruction of cartilage and bone via secretion of matrix metalloproteinases (MMPs) and cathepsins, and via expression of RANKL, resulting in promotion of monocyte-to-osteoclast differentiation (27). The expanded population of synovial fibroblasts in RA is also a prolific source of chemokines responsible for the recruitment and retention of cells within the joint (2). It is clear that synovial fibroblasts are an important source of galectin 3 within the joint, as shown by messenger RNA (mRNA) and proteomic analyses (17,28). Following stimulation by the products of cartilage degradation, synovial fibroblasts also produce galectin 3 (29). However, there are likely to be many other sources of galectin 3 within the joint, including macrophages, which synthesize this glycan-binding protein in significant amounts (30). Although considerable information is available on the cellular sources of galectins in the synovium, the downstream effects of galectin 3 on different cell types in rheumatoid synovium remain largely unexplored. We therefore examined the effects of exogenous galectin 3 on RA synovial fibroblasts, comparing them with genetically matched control skin fibroblasts. We show that inflammatory cytokines, such as interleukin-6 (IL-6), and neutrophil-attracting chemokines, such as IL-8, are produced equally by galectin 3– and tumor necrosis factor α (TNFα)–stimulated synovial and skin fibroblasts. However, in response to galectin 3, synovial fibroblasts, but not skin fibroblasts, secrete mononuclear cell–attracting chemokines such as CCL2, CCL3, and CCL5. The molecular basis for this selectivity is due to the differential activation of MAPK and phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways in response to galectin 3. The increased expression of galectin 3 found in fibroblast-rich areas of the synovium may therefore have significant functional consequences in terms of recruitment of monocyte and lymphocyte infiltrates.

According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1-mediated survival was as efficient as that induced by the cytokine IFN-β and provided an additive, increased delay in apoptosis when given in combination with IFN-β. VCAM-1 delivered its antiapoptotic effect through binding the integrin α9β1. The α9β1 signaling pathway shares significant features with the IFN-β survival signaling pathway, requiring PI3 kinase, NF-κB activation, as well as de novo protein synthesis, but the kinetics of NF-κB activation by VCAM-1 were slower and more sustained compared with IFN-β. This study demonstrates a novel functional role for α9β1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues.

Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic. From five fusions clones were identified with predominant reactivity with: 1) fibroblasts and endothelial cells; or 2) broad stromal elements (fibroblast, endothelium, epithelium, follicular dendritic cells). A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.

A fundamental mechanism of immune privilege in the eye is the induction of T lymphocyte apoptosis. Intraocular inflammation in uveitis implies compromise of immune privilege. This study sought to determine whether apoptosis of T cells is actively inhibited in patients with uveitis and by what pathways this may occur. Apoptotic lymphocytes were found to be absent from aqueous humor (AqH) of virtually all patients with recent-onset uveitis. However, T cells removed from the eye were highly susceptible to both spontaneous and Fas ligand-induced apoptosis in vitro. AqH from patients with uveitis had no modulatory effect on Fas ligand-induced apoptosis, but strongly suppressed survival factor deprivation-induced apoptosis. In contrast, noninflammatory AqH from patients undergoing cataract surgery had no modulatory effects on apoptosis at all. These data suggest that triggering of the Fas pathway is diminished in uveitis, and also that homeostatic resolution through survival factor deprivation-induced apoptosis is inhibited by factors present in AqH. The most widely recognized pathways, common γ-chain cytokines and type I IFNs, did not contribute to AqH-mediated T cell survival. High levels of both IL-6 and soluble IL-6R were found in AqH. IL-6 alone did not induce T cell survival, because IL-6R expression on T cells in AqH was too low to facilitate signaling. However, combinations of IL-6 and soluble IL-6R were highly effective inhibitors of T cell apoptosis, suggesting that the trans-signaling pathway is likely to be a key mediator of T cell apoptosis inhibition mediated by uveitis AqH.

Neutrophils are very abundant, short-lived leucocytes and their death by apoptosis is central to homoeostasis and the resolution of inflammation, yet the trigger for apoptosis is still a topic of debate. Depolarization of the mitochondrial membrane has been supposed to initiate neutrophil spontaneous apoptosis, as neutrophils gradually lose the anti-apoptotic protein Mcl-1 and Bax translocates and inserts into the mitochondrial membrane. However, other reports show that caspase 8 is required for neutrophil apoptosis, suggesting the involvement of DR (death receptor) signalling. As DR ligation is not required for neutrophil apoptosis, this raises the intriguing possibility that activation of caspase 8 during neutrophil apoptosis occurs via a novel mechanism. In the present paper, we discuss the current evidence for mechanisms occurring in neutrophil apoptosis, which could trigger DR signalling in the absence of DR ligation.

OBJECTIVE The aim of this study was to investigate the cellular populations and regulatory factors responsible for B-lymphocyte stimulator (BLyS) overexpression in SLE patients. METHODS Surface and intracellular BLyS levels were quantified by flow cytometry in healthy and SLE monocytes cultured in the presence of TNF-α, IFN-α, IFN-γ, GM-CSF and SLE immune complexes (SLE-ICs), while soluble BLyS was measured by ELISA. Also, both surface and intracellular BLyS expression by different cell subsets was determined in 23 SLE patients and 16 healthy controls. Disease activity was assessed using classic BILAG index. RESULTS In vitro experiments using healthy monocytes showed that IFN-α and SLE-ICs induced a progressive increase in surface-bound BLyS with respect to the intracellular stores. IFN-α-treated SLE monocytes, especially from patients with high anti-dsDNA levels or disease activity, exhibited higher intracellular levels of BLyS that was mobilized to the membrane more rapidly and subsequently released. Furthermore, ex vivo analysis of SLE patients revealed up-regulated BLyS expression in B cells, myeloid and plasmacytoid dendritic cells (DCs), whereas active patients had an increased surface:intracellular BLyS ratio in monocytes and myeloid DCs. CONCLUSION Monocyte BLyS induction and mobilization from intra- to extracellular compartments seems to be influenced by IFN-α and disease activity or anti-dsDNA levels. Accordingly, monocytes and myeloid DCs from active patients presented the highest membrane-bound:intracellular BLyS ratio. In addition, expression levels in several blood cells support the existence of generalized immune stimulation in SLE patients.

Objective To test the hypothesis that the formation of ectopic germinal center (GC)–like structures in Sjogren's syndrome (SS) is associated with the ectopic expression of the constitutive lymphoid tissue–homing chemokines B cell–attracting chemokine 1 (BCA-1; or, CXCL13) and stromal cell–derived factor 1 (SDF-1; or, CXCL12). Methods Immunohistochemical and immunofluorescence analysis was used to determine the expression of the constitutive chemokines BCA-1 (CXCL13) and SDF-1 (CXCL12) in salivary glands from 5 SS patients and 3 non-SS patients. In addition, the expression of their respective receptors (CXCR5 and CXCR4) was examined on infiltrating lymphocytes. Human tonsil was used as a positive control for secondary lymphoid tissue. Results BCA-1 (CXCL13) was expressed within lymphoid aggregates in SS, which shared many structural features with GCs in tonsil. BCA-1 (CXCL13) was completely absent in control biopsy samples from patients who did not have SS. High levels of BCA-1 (CXCL13) were also found on endothelial cells in salivary glands from SS patients. Diseased SS tissue was infiltrated by CXCR5-expressing B cells which organized into GC-like clusters. In complete contrast, SDF-1 (CXCL12), a constitutive chemokine involved in leukocyte retention within lymphoid tissue, was expressed by epithelial cells in both diseased and control samples. The chemokine receptor for SDF-1, CXCR4, was expressed on T cells that accumulated in a periductal distribution in diseased tissue. Conclusion The ectopic expression of BCA-1 (CXCL13) on endothelial cells and within GC-like structures, together with the strong expression of SDF-1 (CXCL12) on ductal epithelial cells, is a unique feature of inflamed glands in SS. By creating a local microenvironment supportive of focal B cell aggregation and differentiation, with structural features that are remarkably similar to GCs, BCA-1 (CXCL13) and SDF-1 (CXCL12) may contribute to the excessive production of high-affinity, class-switched autoantibodies and to the high incidence of B cell lymphomas classically associated with SS.

Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl.

Neutrophils play a central role in host defense and are recruited in vast numbers to sites of infection where they phagocytose and kill invading bacterial pathogens. Neutrophils have a short half-life that is extended at the inflamed site by pro-inflammatory cytokines and contact with bacterial cell walls. Normal resolution of inflammation involves the removal of neutrophils and other inflammatory cells by the induction of apoptosis. Spontaneous neutrophil apoptosis does not require Fas ligation, but is mediated by caspases 3, 8 and possibly caspase 9 and also involves activation of protein kinase C-delta. With chronic inflammatory disease, neutrophil apoptosis is delayed by pro-inflammatory cytokines, leading to persistence of neutrophils at the inflamed site and non-specific tissue damage. Here we discuss the evidence for inhibition of neutrophil apoptosis via signaling though PI-3-kinase and downstream pathways, including PDK-1 and PKB. Therapeutic strategies to resolve chronic inflammation could therefore usefully target neutrophil apoptosis and the PI-3-kinase or PKC-delta signaling pathways.

Type I interferons rescue activated human T cells from cytokine deprivation-induced apoptosis. Our data now show that IFN-beta also rapidly inhibits apoptotic signals induced through the Fas receptor (CD95) in human T cells. To identify upstream signaling elements that could be targets of IFN-beta, we have studied protein kinase C (PKC). PKC-delta is actively involved in the regulation of apoptosis and immunofluorescence staining revealed that early in apoptosis PKC-delta accumulated in the nucleus. Addition of IFN-beta to T cells already deprived of survival factors or treated with anti-Fas antibody caused a rapid retranslocation of PKC-delta away from the nucleus. Furthermore, the generation of a constitutively active catalytic fragment by cleavage of PKC-delta by caspase 3 occurred only after translocation of full-length PKC-delta to the nucleus. IFN-beta also inhibited caspase 3 and the proteolytic activation of PKC-delta. We conclude from these studies that nuclear translocation of PKC-delta is an early event in T cell apoptosis and that IFN-beta rapidly reverses this process.

In a previous study using the monoclonal anti-CD26 antibody MIB-DS2/7 in leprosy and other granulomatous diseases, it was shown that CD26 may be a candidate for use as an operational marker of a human Th1-like reaction. In this follow-up study, we compared seven different monoclonal anti-CD26 antibodies with respect to their staining pattern in lepromatous and tuberculoid leprosy tissues. Three distinct staining patterns became apparent in this anti-CD26 antibody panel: staining of T-lymphocytes and of connective tissue; staining of T-lymphocytes, connective tissue and macrophages; and almost no staining of T-lymphocytes but staining of connective tissue and macrophages. The two antibodies assigned to the first staining pattern, including MIB-DS2/7, were found to be most suitable for the operational discrimination between Th1-like and Th2-like reactions in leprosy. The antibodies assigned to staining patterns 2 and 3 did not allow this discrimination. Although all seven monoclonal antibodies investigated were specific for CD26, only two were found to be useful in identifying a Th1-like immune reaction in human tissue.

Objectives. Lactoferrin is an iron-binding protein that is released from activated neutrophils at sites of inflammation and has anti-microbial as well as anti-inflammatory properties. This study set out to determine whether lactoferrin can delay neutrophil apoptosis and could act as a survival factor for neutrophils in SF.\ud \ud Methods. Human peripheral blood and SF neutrophils were incubated with iron-free lactoferrin and apoptosis determined after 9 h. SF from patients with RA was added to isolated neutrophils, with or without immunodepletion of lactoferrin, and effects on neutrophil apoptosis determined. Levels of lactoferrin in SF were assessed and related to disease duration and markers of disease activity.\ud \ud Results. Iron-free lactoferrin significantly delayed apoptosis of peripheral blood neutrophils, in a concentration-dependent manner after 9 h in culture (P < 0.04). Lactoferrin could also delay apoptosis of neutrophils isolated from SF of patients with RA. SF from patients with established RA delayed apoptosis of peripheral blood neutrophils and this effect was significantly reduced by depletion of lactoferrin (P < 0.03). Lactoferrin levels in SF from patients with established RA did not correlate with disease severity, but did correlate with markers of inflammation (CRP) and with the presence of RF. SF from patients with arthritis of

Activated T cells require anti-apoptotic cytokines for their survival. The anti-apoptotic effects of these factors are mediated by their influence on the balance of expression and localisation of pro- and anti-apoptotic members of the Bcl-2 family. Among the anti-apoptotic Bcl-2 family members, the expression level of Bcl-2 itself and its interaction with the pro-apoptotic protein Bim are now regarded as crucial for the regulation of survival in activated T cells. We studied the changes in Bcl-2 levels and its subcellular distribution in relation to mitochondrial depolarisation and caspase activation in survival factor deprived T cells. Intriguingly, the total Bcl-2 level appeared to remain stable, even after caspase 3 activation indicated entry into the execution phase of apoptosis. However, cell fractionation experiments showed that while the dominant nuclear pool of Bcl-2 remained stable during apoptosis, the level of the smaller mitochondrial pool was rapidly downregulated. Signals induced by anti-apoptotic cytokines continuously replenish the mitochondrial pool, but nuclear Bcl-2 is independent of such signals. Mitochondrial Bcl-2 is lost rapidly by a caspase independent mechanism in the absence of survival factors, in contrast only a small proportion of the nuclear pool of Bcl-2 is lost during the execution phase and this loss is a caspase dependent process. We conclude that these two intracellular pools of Bcl-2 are regulated through different mechanisms and only the cytokine-mediated regulation of the mitochondrial pool is relevant to the control of the initiation of apoptosis.

Membrane microdomains known as lipid rafts have been shown recently to be involved in Fas signalling and apoptosis in T and B cell lines. Here, we have investigated further the role of lipid rafts in Fas-induced apoptosis in non-transformed human CD4 T cells. We show that Fas-induced apoptosis in CD4 T cells was inhibited by the lipid raft disrupter methyl-β-cyclodextrin. When lipid rafts were isolated from control and Fas ligand treated cells, we found that a small proportion of Fas was present in the raft fraction in untreated cells and that this was greatly increased upon Fas ligation. The other components of the Death Inducing Signalling Complex (DISC), FADD, and procaspase 8, were also present at higher levels in the raft fraction isolated from Fas ligand treated cells. We conclude that formation of the DISC occurs in lipid rafts and that these membrane microdomains are required for efficient Fas signalling and apoptosis.

Fibroblasts are important sentinel cells in the immune system and, here, it is proposed that these cells play a critical role in the switch from acute inflammation to adaptive immunity and tissue repair. It is suggested that chronic inflammation occurs because of disordered fibroblast behaviour in which failure to switch off their inflammatory programme leads to the inappropriate survival and retention of leukocytes within inflamed tissue.

Background and Objectives The importance of B cells in the pathogenesis of rheumatoid arthritis (RA) is highlighted by the clinical effectiveness of rituximab. Potential mechanisms for B cells in driving disease processes include autoantibody production, antigen presentation and cytokine secretion. Our recent findings suggest a pro-inflammatory and destructive role for B cells via production of RANKL, TNF-α and IL-6. A memory B cell subset in tonsils which expresses Fc receptor-like 4 (FcRL4) has previously been found to produce RANKL. We sought to determine whether the RANKL-expressing B cells present in the RA synovium belong to this subset. Methods: Synovial fluid and peripheral blood cells from RA patients were analysed for B cell differentiation markers by flow cytometry. Synovial tissue samples from RA patients were assessed using immunofluorescence and immunohistochemistry. FcRL4 mRNA expression was determined in synovial samples from RA patients and uninflamed control subjects by real-time PCR. FcRL4+ and FcRL4- CD19 + B cells were sorted from synovial fluid using a MoFlo cell sorter. mRNA expression of 48 cytokines was determined using Taqman microfluidic cards. Results FcRL4 expressing B cells were detected in the synovial fluid (median 14% of B cells) at a significantly higher frequency compared to peripheral blood (median 0.04%; p = 0.004). RANKL was significantly enriched in the FcRL4+ B cell population compared to the FcRL4- population. FcRL4+ B cells were predominantly switched memory B cells, expressing significantly higher levels of CD80, CD86, CD95, CD20 and CD11c than FcRL4- cells. Sorted FcRL4+ B cells expressed significantly higher mRNA levels of RANKL and TNF-α than FcRL4- B cells. FcRL4+ B cells expressing RANKL were present in synovial tissue, and were distributed under the synovial lining layer and in perivascular cuffs. FcRL4 mRNA was detected in synovium from RA patients but not uninflamed controls, and FcRL4 expression in RA synovium correlated positively with the degree of leukocyte infiltration. Conclusions We have identified a novel pro-inflammatory subset of B cells in the RA synovium which expresses RANKL and TNF-α and shows considerable expression of co-stimulatory molecules indicating a potential pathogenic role in RA. As these cells express high levels of CD20 it is conceivable that their removal contributes to the anti-inflammatory clinical effect of rituximab.

Upon activation, in an active process known as netosis, neutrophils can release their chromatin into the extracellular space. The resulting meshwork of DNA is called Neutrophil Extracellular Traps or NETS (1). These NETS are thought to retain bacteria at a site rich in neutrophil derived proteases and thus contribute to the anti-bacterial action of neutrophils. This process, therefore, is regarded as beneficial in the context of bacterial infection. However, recent studies in SLE patients suggest that in other situations, when neutrophils are activated in the context of autoimmunity, NETS can add to disease pathology (2). The release of the normally tightly packed chromatin depends on the citrullination of histone proteins (3), by an enzyme known as peptidylarginine deiminase 4 (PAD4). PAD4 and related other PAD family members have caused great interest in the research of the pathogenesis of rheumatoid arthritis. Proteins citrullinated by this group of enzymes are recognized by acpa, or anti-citrullinated protein specific antibodies. These antibodies are highly specific for rheumatoid arthritis, although their contribution to disease pathology is currently unclear. Here, we have been investigating the role of NETS in the inflamed joints of patients with rheumatoid arthritis. Most sites of chronic inflammation contain few neutrophils. An exception here is the synovial fluid (SF) of patients with chronic arthritides. The majority of the infiltrating immune cells found here are activated neutrophils. We initially quantified extracellular DNA in SF from patients with RA, OA and crystal arthritis, confirming previous reports of the presence of DNA in SF (4). Further, we applied a range of imaging technologies to generate images of NETS and attached proteins such as neutrophil elastase, PAD4 as well as citrullinated proteins. NETS were enriched from in vivo and in vitro activated neutrophils and probed for the presence of citrullinated proteins and PAD4 by western blotting. In functional experiments, we are investigating the consequences of NET generation by synovial fluid neutrophils. Taken together, our data suggest that generation of NETS by locally activated neutrophils contributes to the production of autoantigens in the inflamed joints of patients with rheumatoid arthritis. References Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004 Mar 5;303(5663):1532-5. Garcia-Romo GS, Caielli S, Vega B, Connolly J, Allantaz F, Xu Z, et al. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Science translational medicine. 2011 Mar 9;3(73):73ra20. Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 2009 Jan 26;184(2):205-13. Leon SA, Revach M, Ehrlich GE, Adler R, Petersen V, Shapiro B. DNA in synovial fluid and the circulation of patients with arthritis. Arthritis Rheum. 1981 Sep;24(9):1142-50. Disclosure of Interest None Declared

Background The importance of the B cell lineage in RA pathogenesis is highlighted by the clinical effectiveness of rituximab. Potential mechanisms by which B cells drive disease processes in RA include their ability to produce autoantibodies, act as antigen-presenting cells, or secrete cytokines. Our recent findings suggest a pro-inflammatory and destructive role for this cell type via production of cytokines including RANKL, TNF-α and IL-6 [1]. A memory B cell subset normally resident in epithelial niches which is characterised by expression of the inhibitory receptor FcRL4 has previously been described to express RANKL [2]. Objectives As we previously made the observation that B cells in the RA synovium produce RANKL, we sought to determine whether RANKL-expressing cells belonged to the FcRL4+ B cell subset. Methods Synovial fluid, peripheral blood and synovial tissue samples were obtained from patients fulfilling 1987 ACR criteria for RA. Mononuclear cells were isolated from synovial fluid and peripheral blood by density gradient centrifugation, stained with antibodies against CD19, CD20, CD27, IgD, CD11c, RANKL, FcRL4, CD95 and CD21, and analysed by flow cytometry. Mononuclear synovial fluid cells were stained with antibodies against CD19 and FcRL4 and sorted using a Mo-Flo™ cell sorter. Taqman™ microfluidic cards were used to detect mRNA expression of 48 cytokines by real-time PCR in these samples. Immunofluorescence staining was performed on 5 µm frozen tissue sections with antibodies against CD20, RANKL and FcRL4. Results FcRL4-expressing B cells, comprising 4-22% of the B cell population, were detected in the synovial fluid at a significantly higher frequency compared to matched peripheral blood (n=12, p=0.0004). RANKL was significantly enriched in the FcRL4+ B cell population compared to the FcRL4- population (p=0.006). Both FcRL4+ B cells and RANKL+ B cells were predominantly switched IgD-CD27+/- memory B cells, expressing significantly higher levels of CD11c and CD95, and lower CD21 levels than the FcRL4- or the RANKL- population. FcRL4+ B cells expressing RANKL were also present in synovial tissue (n=4). At the mRNA level, sorted FcRL4+ B cells were found to express considerably higher levels of both RANKL and TNF compared to the FcRL4- fraction (n=5). Conclusions We have identified a novel subset of B cells in the RA synovium which is characterised by expression of FcRL4 and has not previously been described in RA or any other chronic inflammatory joint condition. FcRL4+ B cells produce RANKL, and express high levels of TNF mRNA, indicating a destructive, pro-inflammatory role for this B cell subpopulation in RA pathogenesis. References Yeo L, Toellner KM, Salmon M, Filer A, Buckley CD, Raza K, Scheel-Toellner D: Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis . Ann Rheum Dis 2011, 70 (11):2022-2028. Ehrhardt GR, Hsu JT, Gartland L, Leu CM, Zhang S, Davis RS, Cooper MD: Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells . J Exp Med 2005, 202 (6):783-791. Acknowledgements The University of Birmingham has filed a patent on the subject of this work. Disclosure of Interest None Declared

Objectives Anti-citrullinated protein antibodies (ACPA) are characteristically detected in patients with rheumatoid arthritis (RA) and growing evidence suggests that they are involved in disease pathogenesis. During disease flares large numbers of neutrophils enter the joint space of RA patients. These cells can extrude genomic DNA in an active process termed NETosis. NETosis critically depends on histone citrullination by peptidyl deiminase 4 (PAD4). Here we tested the hypothesis that activation and release of PAD4 during NETosis contributes to the production of autoantigens in the inflamed joint. Materials and Methods Levels of extracellular DNA in the synovial fluid (SF) of patients with RA (n = 23) and osteoarthritis (OA) (n = 15) or crystal arthritis (n = 6) were quantified using the non cell-permeable DNA dye SYTOX Green. PAD4, neutrophil elastase, citrullinated proteins and bound human IgG were labelled in association with DNA on smear preparations of RA SF and detected by immunofluorescence. NETs from SF and in vitro stimulated neutrophils were isolated and western blotting and mass spectrometry were used to identify proteins released during NET formation and proteins co-fractionated with NETs. PAD enzymatic activity was determined after NETosis in vitro and in the SF of patients with RA (n = 9) and OA (n = 9). Results Extracellular DNA was detected in SF from patients with RA and crystal arthritis at significantly higher levels than in OA SF (RA versus OA p Conclusions These findings suggest that neutrophil NETosis, as a source of extracellular activated PAD enzymes, is involved in the production of auto-antigens in the joints of RA patients. In ACPA positive patients the resulting immune complexes can contribute to the perpetuation of the inflammatory response.

Cytokine gene expression was investigated in the germinal centre constituents of follicular dendritic cells and germinal centre T cells and compared to the mRNA expression of nongerminal centre tonsillar cells. Cells were isolated from human tonsils by preenrichment with MACS and subsequent FACS sorting. Cytokine gene expression was investigated by intronspanning RT-PCR for IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha. Frequency of cytokine-producing cells and the cytokine production pattern of single cells were determined by single cell PCR. Furthermore, cytokine protein expression was investigated by immunohistology. Using these methods, we found a strong production of IL-1 beta mRNA and protein in a small percentage of FDC. Germinal centre T cells showed production of IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha mRNA. The mRNAs of these cytokines could also be detected at the single cell level; as they were produced in a high percentage of germinal centre T cells, whereas immunohistological staining was negative, we conclude that a high percentage of germinal centre T lymphocytes produce IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha in low concentrations in contrast to T cells outside the germinal centre, in which strong cytokine production in few cells was shown earlier.

Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined. Here we report that the anti-apoptotic effect of lactoferrin is dependent upon its iron saturation status as iron-saturated lactoferrin did not affect neutrophil apoptosis. We also show that the effect of lactoferrin is mediated at an early stage in apoptosis as it inhibited activation of sphingomyelinase, generation of ceramide, activation of caspase 8 and Bax and cleavage of Bid. Lactoferrin did not inhibit apoptosis induced by exogenous ceramide, supporting the proposal that it acts upstream of ceramide generation. We therefore conclude that raised lactoferrin levels are likely to contribute to chronic inflammation by delaying neutrophil apoptosis and that this is achieved by inhibiting proximal apoptotic signaling events.