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Background Aberrant regulation of proliferation and survival of immune and stromal cells contributes to the pathogenesis of rheumatoid arthritis (RA). Forkhead box O (FoxO) transcription factors integrate extracellular signals to modulate expression of genes regulating cell cycle and apoptosis, and alterations in activity and expression of FoxO proteins have been reported in several inflammatory diseases, including RA. Objectives The aim of this study was to examine the relationships between inflammation and FoxO expression in RA, and analyze the mechanisms and biological consequences of cytokine-mediated regulation of FoxO1 expression in RA fibroblast-like synoviocytes (FLS). Methods RNA was isolated from synovial biopsies obtained by arthroscopy from 20 RA patients and expression of FoxO1, FoxO3a, FoxO4 and IL-6 was measured by quantitative PCR (qPCR). FoxO1 DNA binding, FoxO1 expression and mRNA stability were measured by ELISA-based assays and qPCR in RA FLS stimulated with IL-1β, TNFα, or LPS in the absence or presence of mitogen-activated protein kinase (MAPK) or protein kinase B (PKB) inhibitors. RA FLS were transduced with adenovirus encoding control GFP or a constitutively active FoxO1 mutant (FoxO1ADA) to examine the effects on cell survival and gene expression. FLS viability was analyzed by MTT and ELISA-based apoptosis detection assay. Changes in expression of genes regulated by FoxO1 were analyzed by low density qPCR arrays and confirmed by immunoblotting. Results In RA synovial tissue expression of FoxO1 negatively correlated with clinical parameters of disease activity: serum C-reactive protein ( R = -0.771, P = 0.0008), erythrocyte sedimentation rate ( R = -0.739, P = 0.0003), and DAS28 ( R = -0.575, P = 0.01), as well as synovial IL-6 mRNA levels ( R = -0.628, P = 0.004). In vitro, RA FLS stimulation with IL-1β or TNFα caused rapid reduction of mRNA levels of FoxO1, but not other FoxO family members. Downregulation of FoxO1 transcript was followed by reduction of FoxO1 protein expression and DNA binding ( P Conclusions Collectively, our findings suggest that suppressed synovial FoxO1 expression is strongly associated with RA pathology and demonstrate that reduction of FoxO1 expression might contribute to perpetuation of inflammation and joint destruction in RA by promoting FLS survival and proliferation. Our data also identify JNK-mediated modulation of FoxO1 mRNA stability as an important mechanism underlying regulation of FoxO1 by inflammatory cytokines. Disclosure of Interest None Declared

The angiogenic factors angiopoietin (Ang)-1 and Ang-2, and their shared receptor Tie2, are expressed in rheumatoid arthritis (RA) synovial tissue. Clinical and translational studies have suggested important roles for Ang-1, Ang-2 and Tie2 in the pathology of RA, but the cellular targets of Ang signalling and the specific contributions of Ang-1 and Ang-2 to disease are poorly understood. Here, I discuss recent findings that synovial macrophages are prominent targets of Ang signalling, highlighting the distinct contributions that Ang-1 and Ang-2 make to macrophage activation, the role of macrophage polarization in regulating Tie2 expression and signalling, and evidence that engagement of synovial Tie2 by Ang-1 drives the development of persistent erosive RA in early arthritis patients. Together, these studies suggest a previously unappreciated role for angiogenic macrophage Tie2 signalling in promoting the initiation and persistence of pathology in RA. Disclosure of Interest None Declared

Background Rheumatoid arthritis (RA) is a chronic and progressive autoinflammatory disorder characterized by the infiltration of inflammatory cells, including B-cells, T-cells and macrophages, in the synovial membrane ultimately leading to cartilage destruction and bone erosion. Clinical disease activity in RA correlates strongly with macrophage numbers in synovial tissue and expression of macrophage-derived cytokines. Bruton’s tyrosine kinase (Btk) is important not only in B cell activation, but also mediates immune complex-dependent activation of monocytes. Pharmacological inhibition of Btk has been shown to be effective in suppressing pathology in murine models of arthritis Objectives The objective of this study was to determine the potential role of Btk in the pathology of RA. Methods Btk expression was detected by immunohistochemistry and digital image analysis in synovial tissue from 16 RA and 12 PsA patients, naive to treatment with biologicals. Immunofluorescent double labelling confocal microscopy was performed to identify which cell types express Btk in RA synovial tissue. Validating qRT-PCR and immunoblotting experiments were performed on isolated relevant cell populations. Effects of a specific Btk inhibitor, RN486, on activation-dependent human macrophage IL-6 production (n=8) and RA synovial tissue explant cultures (n=6) were assessed by ELISA. RN486 influence on macrophage expression of genes related to angiogenesis, cellular adhesion, tissue remodelling and innate immune responses was determined using low density qPCR arrays. Results Btk was expressed at equivalent levels in patients with RA and PsA. No relationship was observed between expression levels and patient clinical characteristics (CRP, ESR, DAS28, RF-positivity). In RA, but not PsA, Btk expression was significantly related to numbers of synovial macrophages (R= 0.63, p Conclusions Btk is expressed in RA synovial tissue and macrophages would be prominent synovial targets of strategies aimed at inhibiting Btk in RA. Btk activity is needed to drive macrophage activation in response to multiple stimuli relevant to RA, and drives IL-6 production in RA synovial tissue. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA. Disclosure of Interest L. Hartkamp: None Declared, I. van Es: None Declared, J. Fine Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, M. Smith Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. Woods Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, S. Narula Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. DeMartino Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, P. Tak Consultant for: GlaxoSmithKline, Employee of: GlaxoSmithKline, K. Reedquist Grant/research support from: Hoffmann-La Roche

Background Rheumatoid arthritis (RA) is a rheumatic disease mainly affecting women. It is known that prolactin (PRL) is a sex hormone, which apart from inducing lactation, also has immunomodulatory properties. PRL levels are elevated in the serum of both female and male RA patients compared to healthy individuals1. Furthermore, hyperprolactinemia is observed in 6% of patients with RA compared to 3% in the normal population2. It has also been observed that serum PRL levels are positively correlated with different disease parameters (eg. IgM rheumatoid factor)3. Limited open label clinical trials, using bromocriptine, leading to decreased PRL levels, show improvement in disease activity of RA patients4. Recently the prolactin receptor (PRLR), belonging to the family of cytokine receptors, has been described in synovial tissue of RA and PsA patients, mainly on macrophages. Objectives The objectives of this study were to unravel the mechanisms underlying the effects of PRL and its receptor in RA. Methods PRL levels in paired serum and synovial fluid (SF) were measured using immunofluorescent metric assay in 18 patients with active RA and 12 with PsA. PRLR expression was determined by RT-PCR in monocyte-derived macrophages from healthy donors polarized with granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-y), interleukin 10 (IL-10), macrophage colony stimulating factor (M-CSF), and RA SF. Polarized monocyte-derived macrophages were stimulated with TNF (10 ng/ml) or LPS (1 ug/ml) plus PRL (25 ng/ml) for 24 hours. Proinflammatory cytokines were measured by ELISA. Results PRL levels were comparable in serum and SF of patients with RA, respectively 10.3 (IQR 6.0-17.6) and 10.5 (IQR 7.5-17.5) ug/L. Similar PRL levels were found in PsA patients, respectively 14.5 (IQR 8.6-18.9) and 12.5 (7.5-17) ug/L. There was a correlation between the serum PRL levels and the levels in SF (r=0.6 p=0.000), suggesting systemic production of PRL. In IFN-y and IL-10 polarized monocyte-derived macrophages, PRLR mRNA expression was 100-200 times higher (p=0.042) compared to macrophages polarized in GM-CSF, M-CSF and RA SF. IFN-y differentiated macrophages displayed a dose-dependent increase in IL-6 production afer stimulation with TNFα plus PRL and LPS plus PRL (respectively p=0.042 and p=0.000, compared to TNFα or LPS alone). Similar results were found in IL-10 differentiated macrophages. Conclusions Our observations in SF of inflammatory joint diseases suggest systemic rather than local production of PRL. In vitro, PRLR was expressed mainly by IFN-y and IL-10 polarized monocyte-derived macrophages. Upon stimulation of these macrophages with TNFα or LPS plus PRL, a dose-dependent increase of IL-6 production was observed. These results suggests that PRL may promote inflammation in RA and PsA via enhancing macrophage activation. References Orbach et al. Ann N Y Acad Sci 2007 Ram et al. Rheumatology (Oxford) 2004 Seriolo et al. Ann N Y Acad Sci 2002 Figueroa et al. Rev Med Chil 1998 Disclosure of Interest None Declared

Background The production of inflammatory cytokines is tightly regulated by epigenetic and non-epigenetic mechanisms, among which acetylation and deacetylation of histones and non-histone proteins plays a prominent role. Histone deacetylase (HDAC) inhibitors (HDACi) suppress cytokine production by rheumatoid arthritis (RA) patient immune and stromal cells, and are potent therapeutics in animal arthritis models. Recently, the HDACi ITF2357 has demonstrated initial clinical efficacy in the treatment of systemic onset juvenile idiopathic arthritis. In our previous work we identified acceleration of mRNA degradation as a mechanism contributing to HDACi suppression of IL-6 production. Objectives This study was undertaken to characterize the role of reduced mRNA stability in HDACi-mediated regulation of RA stromal synovial cell inflammatory activation. Methods RA fibroblast-like synoviocytes (FLS) were treated with IL-1β in the presence or absence of 100-250 nM ITF2357 and total RNA was extracted. mRNA expression and stability of IL-1β-responsive genes was analyzed using a low density quantitative PCR array system. The frequencies of AU-rich elements (AREs) in the 3’-untranslated regions (UTRs) of the analyzed genes were determined using R Scripts (Bioconductor). Production of IL-8, MMP-1, MMP-3 and TIMP-1 was measured by ELISA. Results FLS exposure to ITF2357 significantly reduced the mRNA levels of 85% of genes induced by IL-1β, including cytokines (IL-6, TNFα, IL-1α, IL-1β), chemokines (CCL2, IL-8, CXCL2, CXCL3, CXCL6, CXCL9-11), matrix-degrading enzymes (MMP-1, MMP-3, MMP-13), and intracellular molecules regulating cellular inflammatory responses (COX-2, IRAK2, NFKB1, IRF1). At the same time ITF2357 failed to modulate expression of genes non-responsive to IL-1β. Consistent with mRNA expression data, ITF2357 dose-dependently suppressed protein secretion of IL-8, MMP-1 and MMP-3, without affecting TIMP-1 production. Analyses of mRNA stability confirmed accelerated decay of IL-6 mRNA after FLS treatment with HDACi, and identified a number of other transcripts, including IL-8, COX-2, CXCL2, Bcl-XL and ADAMTS1, which are regulated by HDACi in a similar fashion. 3’ UTRs of these transcripts were characterized by significantly higher frequencies of AREs than other HDACi-sensitive genes in RA FLS. In contrast, mRNA kinetics experiments revealed other sets of genes, including MMPs, CXCL9-11, IL-1β, and E-selectin, which were suppressed by HDACi independently of effects on mRNA degradation. Conclusions We demonstrate that HDACi prevent induction of the vast majority of IL-1β -inducible genes in RA FLS. HDACi suppress a subset of these genes via the acceleration of mRNA degradation, identifying this as an important general mechanism by which HDACi mediate their anti-inflammatory effects. However, our analyses also reveal distinct mechanisms by which HDACi can also regulate gene expression in RA FLS, awaiting further molecular characterization. Our findings provide additional evidence that therapies targeting HDAC activity may be useful in suppressing inflammation in RA. Disclosure of Interest None Declared

Background and Objectives Aberrant regulation of proliferation and survival of immune and stromal cells contributes to the pathogenesis of rheumatoid arthritis (RA). Forkhead box O (FoxO) transcription factors integrate extracellular signals to modulate expression of genes regulating cell cycle and apoptosis, and alterations in activity and expression of FoxOs have been reported in several inflammatory diseases, including RA. In this study, we examined the relationships between inflammation and FoxO expression in RA, and analysed the mechanisms and biological consequences of cytokine-mediated regulation of FoxO1 expression in RA fibroblast-like synoviocytes (FLS). Materials and Methods RNA was isolated from synovial biopsies obtained by arthroscopy from 20 RA patients and expression of FoxO1, FoxO3a, FoxO4 and IL-6 was measured by quantitative PCR (qPCR). FoxO1 DNA binding, FoxO1 expression and mRNA stability were measured by ELISA-based assays and qPCR in RA FLS stimulated with IL-1β, TNFα, or LPS in the absence or presence of mitogen-activated protein kinase (MAPK) or protein kinase B (PKB) inhibitors. RA FLS were transduced with adenovirus encoding control GFP or constitutively active FoxO1ADA to examine the effects on cell viability and gene expression. Results In RA synovial tissue expression of FoxO1 negatively correlated with clinical parameters of disease activity: serum C-reactive protein ( R = –0.771, P = 0.0008), erythrocyte sedimentation rate ( R = –0.739, P = 0.0003), and DAS28 ( R = –0.575, P = 0.01), as well as synovial IL-6 mRNA levels ( R = –0.628, P = 0.004). In vitro, RA FLS stimulation with IL-1β or TNFα caused rapid downregulation of FoxO1 mRNA levels, followed by reduction of FoxO1 protein expression and DNA binding. This effect was independent of PKB signalling, and was associated with acceleration of FoxO1 mRNA degradation in the presence of IL-1β. Inhibition of c-Jun N-terminal kinase (JNK), but not other MAPKs, prevented downregulation of FoxO1 expression and binding by IL-1β, and blocked IL-1β-induced reduction of FoxO1 mRNA stability. Overexpression of constitutively active FoxO1 in RA FLS induced apoptosis associated with altered expression of genes regulating cell cycle and apoptosis: BIM and p27 Kip1 were induced while expression of Bcl-XL was suppressed in cells expressing active FoxO1. Conclusions Collectively, our findings suggest that suppressed synovial FoxO1 expression is strongly associated with RA pathology and demonstrate that reduction of FoxO1 expression might contribute to perpetuation of inflammation in RA by promoting FLS survival and proliferation. Our data also identify JNK-mediated modulation of FoxO1 mRNA stability as an important mechanism underlying regulation of FoxO1 by inflammatory cytokines.

The protooncogene product Cbl has emerged as a novel signal transduction protein downstream of a number of cell surface receptors coupled to tyrosine kinases. Recently, we and others have reported the activation-dependent association of Cbl with the Syk and ZAP-70 tyrosine kinases through presently undefined mechanisms. Potential Src homology 2 and 3 domain binding sites within the C-terminal half of Cbl mediate in vivo interactions with several signaling proteins; however, the N-terminal transforming region (Cbl-N) lacks recognizable catalytic or protein interaction motifs. Here, we show that in vitro Cbl-N (amino acids 1-357) but not Cbl-C (amino acids 358-906) binds to ZAP-70 in a T cell-activation-dependent manner. A point mutation in Cbl-N, G306E, corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue, SLI-1, severely compromised Cbl-N/ZAP-70 binding. Cbl-N/ZAP-70 binding was direct and phosphotyrosine-dependent, thus identifying a phosphotyrosine-binding domain within the transforming region of Cbl. In vivo, Cbl-N expressed in T cells selectively associated with the ZAP-70/zeta complex. These results identify a novel mechanism for the direct participation of the N-terminal region of Cbl in ZAP-70 signal transduction, and suggest a biochemical mechanism for the leukemogenicity of the oncogene v-cbl through potential interaction with proliferation-related phosphotyrosyl proteins.

We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we show that Cbl associates with all three forms of the human Crk protein, predominantly CrkL, following T cell receptor activation of Jurkat T cells. Association between Cbl and Crk proteins was confirmed in normal human peripheral blood-derived T cells. In vitro, Cbl was able to interact with the Crk SH2 domain but not the SH3 domain. A phosphopeptide corresponding to a potential Crk SH2 domain-binding motif in Cbl (pYDVP) specifically inhibited binding between Cbl and Crk SH2 domain. Anti-Cbl antibody completely immunodepleted the CrkL-associated 120kDa phosphotyrosyl polypeptide, suggesting that the recently described p130cas-related Crk-associated p116 of T cells may be Cbl. Consistent with this possibility, the 4F4 antibody used to characterize the p116 polypeptide cross-reacted with Cbl protein when it was resolved on one- or two-dimensional gels. CrkL was constitutively associated with a substantial amount of the guanine nucleotide exchange protein C3G, and a fraction of the C3G protein was coimmunoprecipitated with Cbl in activated Jurkat T cells. These results suggest the possibility that Cbl may participate in a signaling pathway that regulates guanine nucleotide exchange on small G-proteins in T cells.

We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3-kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120cbl in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors.

Backgroundand objectives Forkhead box O (FoxO) transcription factors, regulated by phosphatidlyinositol 3-kinase and reversible acetylation, integrate environmental signals to orchestrate inflammatory responses, cell cycle and apoptosis. Here, the authors examined the relationship between inflammation and FoxO expression and activity in rheumatoid arthritis (RA) synovial tissue, determined the effects of histone deacetylase inhibitors (HDACi) on FoxO expression and activity in RA fibroblast-like synoviocytes (FLS), and identified genes regulated by FoxO1 in RA FLS. Materials and methods Total RNA was isolated from synovial biopsies obtained by arthroscopy from 20 RA patients. FoxO1, FoxO3a, FoxO4, TNFα, MMP-1 and IL-6 expression were measured by quantitative PCR (qPCR) in synovial tissue and RA and osteoarthritis (OA) FLS. RA FLS were stimulated with IL-1β or TNFα, in the absence or presence of the HDACi trichostatin A, and FoxO family member expression and FoxO1 DNA binding activity were measured by qPCR and ELISA-based assays, respectively. RA FLS were transduced with adenovirus encoding control GFP or constitutively active FoxO1ADA to examine the effects on RA FLS gene expression using low density qPCR arrays. Results Negative correlations were observed between RA synovial tissue expression of FoxO1 and the levels of serum C reactive protein (CRP) (R=−0.771, p=0.0008), erythrocyte sedimentation rate (ESR) (R=−0.739, p=0.0003), and disease activity score 28 (DAS28) (R=−0.575, p=0.01). A strong negative correlation was also observed between synovial FoxO1 and IL-6 mRNA levels (R=−0.628, p=0.004), but not TNFα or MMP-1. FoxO1, FoxO3a and FoxO4 mRNA were each detected in RA and OA FLS, and FoxO1 mRNA levels were significantly reduced in RA FLS compared to OA FLS (p Kip1 . FoxO1ADA also suppressed IL-1β-mediated induction of inflammatory mediators, including CCL2, CXCL6, PDGF and ICAM1. Conclusions The authors demonstrate that inflammatory stimuli decrease FoxO1 expression and DNA binding activity, effects reversed by exposure of RA FLS to HDACi. Restoration of FoxO1 activity suppresses a subset of genes regulated by HDACi during FLS activation, identifying the enhancement of FoxO1 function as a novel mechanism by which HDACi might mediate their anti-inflammatory effects in RA synovial cells.

Background and objectives Vascular remodeling promotes immune cell infiltration and provides nutrients to hyperplastic tissue in the synovial tissue of patients with rheumatoid arthritis (RA). In solid cancers, angiopoietin (Ang-1 and Ang-2) signaling to Tie2-expressing immunosuppressive monocytes (TEMs), is critical in allowing tumor establishment. A similar role for TEMs in chronic inflammatory disease has not been assessed, but the authors have recently found that macrophages are the primary cell types in which Tie2 is activated in RA synovial tissue. Here, the authors examined how the differentiation stimuli and the RA synovial microenvironment might regulate expression of Tie2 on macrophages, as well as their effects on expression of macrophage angiogenic factors and gene expression responses to Ang stimulation. Material and methods Human healthy donor peripheral blood monocytes were differentiated in the presence of pro-inflammatory/classically activating (GM-CSF, IFN-γ), anti-inflammatory/alternatively activating (M-CSF, IL-10) cytokines and RA synovial fluid (SF). Tie-2 expression was analysed by flow cytometry and quantitative PCR. Macrophage mRNA expression of 84 angiogenic factors in the absence or presence of TNF stimulation, alone or in combination with Ang-1 was analysed using low density quantitative PCR Array. Results Tie2 protein and mRNA expression was observed under all conditions, but expression levels failed to correspond to pro- or anti-inflammatory phenotypes – expression levels were equivalent and significantly highest in macrophages differentiated in IFN-γ and IL-10. Tie2 expression was also maintained in macrophages differentiated in the presence of RA SF.Gene expression analysis of angiogenic factors demonstrated distinct expression profiles under each polarisation condition, although macrophages differentiated in RA SF (RASF macrophages) showed significantly enhanced expression of pro-angiogenic chemokines (CXCL3, 5 and 6, IL-8 and CCL2) and decreased expression of antiangiogenic chemokines (CXCL2, 9, 10 and 11) (p values ranging from Conclusion Our results suggest that the RA synovial environment promotes pro-angiogenic macrophage differentiation, and potentiates macrophage production of chemokines which perpetuate synovial monocyte influx in response to TNF and Ang-1.

Background and objectives The angiogenic factors angiopoietin-1 (Ang-1) and Ang-2 are differentially expressed in the serum and synovial fluid of patients with different forms of arthritis. Ang-1 and Ang-2 contribute to inflammation and joint destruction in rheumatoid arthritis (RA), but the underlying mechanisms for their differential expression and their consequences for synovial Tie2 activation are unknown. Here the authors examined relationships between synovial expression of Ang-1 and Ang-2, and Tie2 activation in patients with RA and psoriatic arthritis (PsA), and the expression of transcription factors (TF) regulating Ang expression. Materials and methods Ang-1, Ang-2, Tie2 and phosphorylated (p) active Tie2 expression was examined by immunohistochemistry combined with digital image analysis in synovial biopsies from 20 RA and 19 PsA patients. Relationships between Ang expression and Tie2 activation were compared. In silico analysis was conducted to identify possible TF candidates in the Ang-1 promoter, using four different TF binding site prediction programmes. RA synovial expression of TF and Ang-1 was examined in publicly available gene expression data sets. Candidate TF were characterised by immunohistochemistry. Results Synovial Ang-1 expression was elevated approximately fourfold in patients with RA (p Conclusions In RA, synovial Tie2 engagement is driven primarily by enhanced local Ang-1 production. Expression of Ang-1 is differentially regulated in RA and PsA, correlating with enhanced expression of putative Ang-1 TF in RA.

Rap1 is signalling molecule that modulates T lymphocyte trafficking and activation upon antigen stimulation. We demonstrated that transgenic mice with T cells expressing constitutively active Rap1 (RapV12) are protected from experimental arthritis. Here, we aimed to identify the mechanisms of protection by using a TCR transgenic model of MOG-induced EAE (2D2 mice).

Angiogenic growth factors Ang-1 and Ang-2 and their receptor Tie2 are expressed in inflamed synovial tissue and thought to contribute to disease initiation and joint destruction in rheumatoid arthritis (RA). The goal of this study was to examine the predictive value of synovial Ang-1 and Ang-2 expression and Tie2 activation with regard to disease outcome and erosive disease progression in disease-modifying anitrheumatic drug (DMARD)-naive patients with early …

The authors have previously found that macrophages are predominant targets of the angiogenic factors angiopoietin (Ang)-1 and -2 in synovial tissue of patients with rheumatoid arthritis (RA). How macrophage Tie2 ligation by Ang might contribute to disease in RA is currently …

Little is known about the contribution of mitogen-activated protein kinase (MAPK) to disease in rheumatoid arthritis (RA), although inhibitors targeting one of these enzymes (p38) have already entered the clinic. We have investigated synovial MAPK activation status in disease-modifying antirheumatic drug (DMARD)-naive patients with early arthritis. 50 patients with DMARD-naive early arthritis (disease duration

Introduction: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA).Methods: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests.Results: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor α, interferon γ and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help.Conclusions: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.

The purpose of this study was to investigate the profile of histone deacetylase (HDAC) expression in the synovial tissue of rheumatoid arthritis (RA) compared with that of normal control and osteoarthritis (OA), and to examine whether there is a link between HDAC activity and synovial inflammation.HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of total synovial tissue surgically obtained from normal, OA and RA joints. The level of cytoplasmic tumor necrosis factor a (TNFα) fraction was measured by ELISA. Total RNA of synovial tissue was used for RT-PCR of HDAC1-8. In synovial fibroblasts from RA (RASFs), the effects of TNFα on nuclear HDAC activity and class I HDACs (1, 2, 3, 8) mRNA expressions were examined by quantitative real-time PCR. The protein expression and distribution of class I HDACs were examined by Western blotting.Nuclear HDAC activity was significantly higher in RA than in OA and normal controls and correlated with the amount of cytoplasmic TNFα. The mRNA expression of HDAC1 in RA synovial tissue was higher than in OA and normal controls, and showed positive correlation with TNFα mRNA expression. The protein level of nuclear HDAC1 was higher in RA synovial tissue compared with OA synovial tissue. Stimulation with TNFα significantly increased the nuclear HDAC activity and HDAC1 mRNA expression at 24 hours and HDAC1 protein expression at 48 hours in RASFs.Our results showed nuclear HDAC activity and expression of HDAC1 were significantly higher in RA than in OA synovial tissues, and they were upregulated by TNFα stimulation in RASFs. These data might provide important clues for the development of specific small molecule HDAC inhibitors.

Cellular activation, proliferation and survival in chronic inflammatory diseases is regulated not only by engagement of signal trans-duction pathways that modulate transcription factors required for these processes, but also by epigenetic regulation of transcription factor access to gene promoter regions. Histone acetyl trans-ferases coordinate the recruitment and activation of transcription factors with conformational changes in histones that allow gene promoter exposure. Histone deacetylases (HDACs) counteract histone acetyl transferase activity through the targeting of both histones as well as nonhistone signal transduction proteins important in inflammation. Numerous studies have indicated that depressed HDAC activity in patients with inflammatory airway diseases may contribute to local proinflammatory cytokine production and diminish patient responses to corticosteroid treatment. Recent observations that HDAC activity is depressed in rheumatoid arthritis patient synovial tissue have predicted that strategies restoring HDAC function may be therapeutic in this disease as well. Pharmacological inhibitors of HDAC activity, however, have demonstrated potent therapeutic effects in animal models of arthritis and other chronic inflammatory diseases. In the present review we assess and reconcile these outwardly paradoxical study results to provide a working model for how alterations in HDAC activity may contribute to pathology in rheumatoid arthritis, and highlight key questions to be answered in the preclinical evaluation of compounds modulating these enzymes.

We investigated the therapeutic potential and mechanism of action of IFN-β protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-β or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-κB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-β. We also examined the effect of IFN-β on NF-κB activity. IFN-β, at 0.25 μg/injection and higher, significantly reduced disease severity in two experiments, each using 8–10 mice per treatment group. IFN-β-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-κB ligand and c-Fos. Tumor necrosis factor α and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-β treatment. IFN-β reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-κB activity. The data support the view that IFN-β is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.

IFN-β is emerging as a pivotal molecule involved in synovial inflammation and bone homeostasis. We conducted in vitro studies as well as studies using the collagen-induced arthritis model to investigate the effects of IFN-β. DBA/1 male mice were immunized intradermally with bovine type II collagen in complete Freund's adjuvant. On the first clinical sign of disease, mice were treated for 7 days by daily intraperitoneal injections of recombinant mouse IFN-β or saline. Disease progression was monitored by validated visual clinical scoring and by measurement of paw swelling using calipers. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis on decalcified wax-embedded paw sections and quantified. Safranin O staining was performed to determine proteoglycan depletion. In addition, cytokine profiles in the synovium were evaluated by immunohistochemistry. Cytokine expression was also measured in supernatants of rheumatoid arthritis (RA) and osteoarthritis fibroblast-like synoviocytes (FLS) in culture after incubation with IFN-β. We incubated RA FLS, which were transfected with a NF-κB/luciferase construct, with IFN-β and measured luciferase activity to determine the effects on NF-κB activity. In two independent experiments with eight to 10 mice per treatment group in each experiment, IFN-β at doses from 0.25 μg/injection and higher significantly reduced disease severity. Moreover, IFN-β-treated animals had significantly less cartilage and bone destruction than control animals, demonstrating a protective effect of the treatment. There was a significant reduction in c-Fos expression and osteoclast numbers. The proinflammatory cytokines tumor necrosis factor alpha and IL-6 were significantly reduced, and IL-10 production was increased after IFN-β treatment. In vitro studies revealed reduced NF-κB activity and expression of proinflammatory cytokines in RA FLS after IFN-α treatment. Taken together, our data show that frequent exogenous administration of IFN-β protein may reduce synovial inflammation and protects against cartilage and bone destruction by inhibition of NF-κB activity, immunomodulation, and impairment of osteoclastogenesis through inhibition of c-Fos. Continuous IFN-β expression at the site of inflammation may be required to reach these therapeutic effects in RA patients. The exciting biological effects could translate into clinically meaningful improvement if the cytokine is administered frequently, when pegylated IFN-β is used, or when IFN-β gene therapy is used. Therefore, IFN-β gene therapy studies are now underway.

Synovial fluid (SF) T lymphocytes isolated from rheumatoid arthritis (RA) patients display constitutive activation of the integrin LFA-1. LFA-1-dependent T cell interactions with ICAM-bearing fibroblast-like synoviocytes and macrophages contribute to the inflammatory state of the joint via retention of T lymphocytes in the synovium and induction of inflammatory cytokines. SF components, notably TGF-β, are sufficient and required to sustain activation of LFA-1 in SF T lymphocytes, but little is known about the intracellular signalling pathways regulating LFA-1-dependent adhesion in SF T lymphocytes. We have recently found that deregulated signalling via the small GTPases Ras and Rap1 is responsible for chronic oxidative stress in SF T lymphocytes. Although activated Rap1 is a critical mediator of T cell receptor and CD31 -stimulated LFA-1 adhesion, SF T lymphocytes adhere via LFA-1 in the complete absence of Rap1 signalling. We find that both N-Ras and K-Ras isoforms of Ras are constitutively activated in SF T cells, and that transient expression of activated N-Ras, but not K-Ras or H-Ras, stimulates LFA-1-dependent adhesion in Jurkat T lymphocytes. Adhesion induced by N-Ras is insensitive to overexpression of the Rap1 negative regulatory protein RapGAP, consistent with the observation that SF T cells are highly adherant despite undetectable levels of activated Rap1. We provide pharmacological evidence indicating that N-Ras and Rap1 induce adhesion by distinct signalling mechanisms. Identification of N-Ras as a key intracellular signalling protein mediating lymphocyte retention in RA joints provides potential targets for new therapeutic strategies for RA.

Objective To examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA. Methods Clinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex. Results Tie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients. Conclusion Tie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases.