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Little is known about the experiences, values, and needs of people without arthritis who undergo predictive biomarker testing for the development of rheumatoid arthritis (RA). Our study aimed to explore the perspectives of these individuals and describe their information needs. A qualitative, multicenter interview study with a thematic analysis was conducted in Austria, Germany and the UK. Individuals were interviewed who underwent predictive biomarker testing for RAand had a positive test result but no diagnosis of any inflammatory joint disease. Participants included patients with arthralgia and asymptomatic individuals. Information and education needs were developed from the qualitative codes and themes using the Arthritis Educational Needs Assessment Tool as a frame of reference. Thematic saturation was reached in 34 individuals (76% female, 24 [71%] with arthralgia, and 10 [29%] asymptomatic individuals). Thirty‐seven codes were summarized into 4 themes: 1) decision‐making around whether to undergo initial predictive testing, 2) willingness to consider further predictive tests, and/or 3) preventive interventions, including medication, and 4) varying reactions after receiving a positive test result. Individuals with arthralgia were more likely to be willing to take preventive action, undergo further testing, and experience psychological distress than asymptomatic individuals. All participants expressed the need for tailored, patient‐understandable information. Individuals at risk of RAare currently the subjects of research aimed at developing better predictive strategies and preventive approaches. Their perceptions and needs should be addressed to inform the future development of interventions combined with education.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα.

Accumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA).

Scurfy mice are deficient in regulatory T cells (Tregs), develop a severe, generalized autoimmune disorder that can affect almost every organ and die at an early age. Some of these manifestations resemble those found in systemic lupus erythematosus (SLE). In addition, active SLE is associated with low Treg numbers and reduced Treg function, but direct evidence for a central role of Treg malfunction in the pathophysiology of lupus-like manifestations is still missing. In the present study, we characterize the multiorgan pathology, autoantibody profile and blood count abnormalities in scurfy mice and show their close resemblances to lupus-like disease. Scurfy mice have dysfunctional Tregs due to a genetic defect in the transcription factor Forkhead box protein 3 (Foxp3). We analyzed skin, joints, lung and kidneys of scurfy mice and wild-type (WT) controls by conventional histology and immunofluorescence (IF) performed hematological workups and tested for autoantibodies by IF, immunoblotting and enzyme-linked immunosorbent assay. We also analyzed the intestines, liver, spleen and heart, but did not analyze all organs known to be affected in scurfy mice (such as the testicle, the accessory reproductive structures, the pancreas or the eyes). We transferred CD4+T cells of scurfy or WT mice into T cell-deficient B6/nude mice. We confirm previous reports that scurfy mice spontaneously develop severe pneumonitis and hematological abnormalities similar to those in SLE. We show that scurfy mice (but not controls) exhibited additional features of SLE: severe interface dermatitis, arthritis, mesangioproliferative glomerulonephritis and high titers of anti-nuclear antibodies, anti-double-stranded DNA antibodies, anti-histone antibodies and anti-Smith antibodies. Transfer of scurfy CD4+T cells (but not of WT cells) induced autoantibodies and inflammation of lung, skin and kidneys in T cell-deficient B6/nude mice. Our observations support the hypothesis that lupus-like autoimmune features develop in the absence of functional Tregs.

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease, which can affect different organs. Increased proportions of CD4+CD25-Foxp3+T cells have been described in SLE patients. The exact role of this cell population in SLE patients still remains unclear. We therefore analyzed this T cell subset in a large cohort of SLE patients with different organ manifestations. Phenotypic analyses, proportions and absolute cell numbers of CD4+CD25-Foxp3+T cells were determined by flow cytometry (FACS) in healthy controls (HC) (n = 36) and SLE patients (n = 61) with different organ manifestations. CD4+CD25-Foxp3+T cells were correlated with clinical data, the immunosuppressive therapy and different disease activity indices. In patients with active glomerulonephritis, CD4+CD25-Foxp3+T cells were analyzed in urine sediment samples. Time course analyses of CD4+CD25-Foxp3+T cells were performed in patients with active disease activity before and after treatment with cyclophosphamide and prednisone. CD4+CD25-Foxp3+T cells were significantly increased in active SLE patients and the majority expressed Helios. Detailed analysis of this patient cohort revealed increased proportions of CD4+CD25-Foxp3+T cells in SLE patients with renal involvement. CD4+CD25-Foxp3+T cells were also detected in urine sediment samples of patients with active glomerulonephritis and correlated with the extent of proteinuria. CD4+CD25-Foxp3+T cells resemble regulatory rather than activated T cells. Comparative analysis of CD4+CD25-Foxp3+T cells in SLE patients revealed a significant association of this newly described cell population with active nephritis. Therefore CD4+CD25-Foxp3+T cells might serve as an important tool to recognize and monitor SLE patients with renal involvement.

Rheumatoid arthritis is a heterogeneous disease with respect to clinical manifestations, serologic abnormalities, joint damage and functional impairment. Predicting outcome in a reliable way to allow for strategic therapeutic decision-making as well as for prediction of the response to the various therapeutic modalities available today, especially biological agents, would provide means for optimization of care. In the present article, the current information on biological and clinical markers related to disease activity and joint damage as well as for predictive purposes is reviewed. It will be shown that the relationship of many biomarkers with disease characteristics is confounded by factors unrelated to the disease, and that only few biomarkers exist with some predictive value. Moreover, clinical markers appear of equal value as biomarkers for this purpose, although they likewise have limited capacity in these regards. The analysis suggests the search for better markers to predict outcomes and therapeutic responsiveness in rheumatoid arthritis needs to be intensified.

Ro ribonucleoprotein (RNP) complexes are composed of one molecule of a small noncoding cytoplasmic RNA, termed Y RNA, and the two proteins Ro60 and La. Additional proteins such as hnRNP I, hnRNP K, or nucleolin have recently been shown to be associated with subpopulations of Y RNAs. Ro RNPs appear to be localized in the cytoplasm of all higher eukaryotic cells but their functions have remained elusive. To shed light on possible functions of Ro RNPs, we tested protein components of these complexes for RNA chaperone properties employing two in vitro chaperone assays and additionally an in vivo chaperone assay. In these assays the splicing activity of a group I intron is measured. La showed pronounced RNA chaperone activity in the cis-splicing assay in vitro and also in vivo, whereas no activity was seen in the trans-splicing assay in vitro. Both hnRNP I and hnRNP K exhibited strong chaperone activity in the two in vitro assays, however, proved to be cytotoxic in the in vivo assay. No chaperone activity was observed for Ro60 in vitro and a moderate activity was detected in vivo. In vitro chaperone activities of La and hnRNP I were completely inhibited upon binding of Y RNA. Taken together, these data suggest that the Ro RNP components La, hnRNP K, and hnRNP I possess RNA chaperone activity, while Ro60-Y RNA complexes might function as transporters, bringing other Y RNA binding proteins to their specific targets.

Tumour necrosis factor (TNF) signalling molecules are considered as promising therapeutic targets of antirheumatic therapy. Among them, mitogen-activated protein kinases are thought to be of central importance. Herein, we investigate the role in vivo of TNF-α signalling through c-Jun N-terminal kinase (JNK)1 in destructive arthritis. Human TNF transgenic (hTNFtg) mice, which develop inflammatory arthritis, were intercrossed with JNK1-deficient (JNK1-/-) mice. Animals (n = 35) of all four genotypes (wild-type, JNK1-/-, hTNFtg, JNK1-/-hTNFtg) were assessed for clinical and histological signs of arthritis. Clinical features of arthritis (swelling and decreased grip strength) developed equally in hTNFtg and JNK1-/-hTNFtg mice. Histological analyses revealed no differences in the quantity of synovial inflammation and bone erosions or in the cellular composition of the synovial infiltrate. Bone destruction and osteoclast formation were observed to a similar degree in hTNFtg and JNK1-/-hTNFtg animals. Moreover, cartilage damage, as indicated by proteoglycan loss in the articular cartilage, was comparable in the two strains. Intact phosphorylation of JNK and c-Jun as well as expression of JNK2 in the synovial tissue of JNK1-/-hTNFtg mice suggests that signalling through JNK2 may compensate for the deficiency in JNK1. Thus, JNK1 activation does not seem to be essential for TNF-mediated arthritis.

Systemic lupus erythematosus (SLE) represents an autoimmune disease for which alterations of T cells, B cells as well as various antigen-presenting cell (APC) populations have been described. In order to better define APC-associated deficiencies, we analyzed morphologic, phenotypic and functional characteristics of in vitro-generated monocyte-derived dendritic cells (MoDC) from SLE patients as compared with healthy controls. Analysis of MoDC at different stages of maturation revealed substantial phenotypic and functional defects of MoDC from SLE patients as compared with healthy controls. In particular, we observed a significantly reduced up-regulation of MHC class II molecules on MoDC upon activation which correlated with disease activity scores and functional deficiencies in mixed lymphocyte reaction experiments. Our data imply a crucial role of APC in the immunological imbalance in SLE for foreign and self-antigen reactivity.

Arthritis can destroy the cortical bone barrier and expose bone marrow to synovial tissue. This study examines bone marrow changes in arthritis and its effects on cortical bone remodeling. Bone marrow next to arthritic lesions exhibits B‐lymphocyte‐rich infiltrates, which express BMPs and stimulate endosteal bone formation. Thus, bone marrow actively participates in the arthritic process.Introduction:Imaging studies have shown that bone marrow changes occur in patients with rheumatoid arthritis (RA). To examine whether bone marrow is affected during arthritis, human TNF transgenic (hTNFtg) mice, which constitute an established animal model of human RA, were examined for bone marrow changes.Materials and Methods:The hind paws (tarsal area) of 22 untreated hTNFtg mice, 5 hTNFtg mice treated with anti‐TNF (infliximab), and 5 wildtype (WT) mice were examined histologically, immunohistochemically, and by means of mRNA in situ hybridization.Results and Conclusions:All untreated hTNFtg mice with moderate (n= 10) and severe (n= 7) disease developed inflammatory bone marrow lesions during the course of disease, whereas no such lesions appeared in hTNFtg mice with mild disease (n= 5) and WT mice. Bone marrow infiltrates were almost exclusively composed of lymphocytes, and the overwhelming proportion (>80%) was B‐cells. Presence and extent of bone marrow infiltrates were closely linked to severity of arthritis. In addition, blockade of TNF effectively reduced bone marrow inflammation. Interestingly, osteoblast numbers were increased at the endosteal surface in the vicinity of these lesions. Moreover, osteoid deposition; expression of bone matrix proteins, such as osteocalcin and osteopontin; and mineralization were enhanced, suggesting that inflammatory bone marrow infiltrates induce bone formation. Indeed, B‐lymphocytes of these lesions expressed bone morphogenetic protein (BMP)‐6 and −7, which are important stimulators of new bone formation. Thus, we conclude that bone marrow actively participates in destructive arthritis by generating B‐lymphocyte‐rich bone marrow lesions and inducing endosteal bone formation.

Arthritis can destroy the cortical bone barrier and expose bone marrow to synovial tissue. This study examines bone marrow changes in arthritis and its effects on cortical bone remodeling. Bone marrow next to arthritic lesions exhibits B‐lymphocyte‐rich infiltrates, which express BMPs and stimulate endosteal bone formation. Thus, bone marrow actively participates in the arthritic process.

Local bone erosion and systemic bone loss are hallmarks of rheumatoid arthritis and cause progressive disability. Tumor necrosis factor (TNF) is a key mediator of arthritis and acts catabolically on bone by stimulating bone resorption and inhibiting bone formation. We hypothesized that the concerted action of anti-TNF, which reduces inflammation and parathyroid hormone (PTH), which stimulates bone formation, or osteoprotegerin (OPG), which blocks bone resorption and could lead to repair of local bone erosions and reversal of systemic bone loss. To test this, human TNF-transgenic mice with established erosive arthritis and systemic bone loss were treated with PTH, OPG, and anti-TNF, alone or in combination. Local bone erosions almost fully regressed, on combined treatment with anti-TNF and PTH and/or OPG, suggesting repair of inflammatory skeletal lesions. In contrast, OPG and anti-TNF alone led to arrest of bone erosions but did not achieve repair. Treatment with PTH alone had no influence on the progression of bone erosions. Local bone erosions all showed signs of new bone formation such as the presence of osteoblasts, osteoid formation, and mineralization. Furthermore, systemic bone loss was completely reversed on combined treatment and this effect was mediated by osteoblast stimulation and osteoclast blockade. In summary, we conclude that local joint destruction and systemic inflammatory bone loss because of TNF can regress and that repair requires a combined approach by reducing inflammation, blocking bone resorption, or stimulating bone formation.

Die Diagnostik der rheumatoiden Arthritis (RA) (bzw. chronischen Polyarthritis) wurde bislang durch das Fehlen krankheitsspezifischer serologischer Marker erschwert. So werden nach wie vor Rheumafaktoren (RF) trotz ihrer begrenzten Spezifität als einzig anerkannte Markerantikörper verwendet und stellen unter den diagnostischen Kriterien des „American College of Rheumatology” das einzige serologische dar. In den letzten Jahren sind allerdings einige neue Autoantikörper beschrieben worden, denen eine im Vergleich zu RF weit höhere Krankheitsspezifität zuzukommen scheint. Dies gilt insbesondere für Autoantikörper gegen citrullinierte Antigene (Anti-Keratin bzw. Anti-Filaggrin-Antikörper sowie Antikörper gegen synthetisch hergestellte citrullinierte Peptide) die weitgehend spezifisch für die RA zu sein scheinen. Als weitere Antikörper von diagnostischem Interesse sind Anti-RA33-Antikörper sowie Antikörper gegen das Stressprotein BiP zu nennen. Abgesehen von ihrer möglichen Anwendung als diagnostische Marker könnten diese Antikörper bzw. die ihnen zu Grunde liegenden zellulären Autoimmunreaktionen auch eine Rolle im pathologischen Geschehen der RA spielen. The diagnosis of rheumatoid arthritis has been hampered by the lack of a truely disease-specific serologic marker. Thus, despite its moderate specificity rheumatoid factor (RF) is still the only established marker antibody for RA and among the diagnostic criteria of the American College of Rheumatology the only serologic one. However, in recent years, several newly characterized autoantibodies have been described that may have the potential to become diagnostic indicators for RA. In particular, antibodies to citrullinated targets (anti-keratin or anti-filaggrin antibodies, respectively, antibodies to synthetic citrullinated peptides) appear to be highly specific for RA. Other potentially useful antibodies include anti-RA33 autoantibodies and antibodies to the stress protein BiP which seem to have higher specificity for RA than RF. Apart from being promising diagnostic markers these autoantibodies or the underlying cellular autoimmune reactions, respectively, may also play a role in the pathogenesis of RA.

Autoantibodies are proven useful diagnostic tools for a variety of rheumatic and non-rheumatic autoimmune disorders. However, a highly specific marker autoantibody for rheumatoid arthritis (RA) has not yet been determined. The presence of rheumatoid factors is currently used as a marker for RA. However, rheumatoid factors have modest specificity (~70%) for the disease. In recent years, several newly characterized autoantibodies have become promising candidates as diagnostic indicators for RA. Antikeratin, anticitrullinated peptides, anti-RA33, anti-Sa, and anti-p68 autoantibodies have been shown to have >90% specificity for RA. These autoantibodies are reviewed and the potential role of the autoantibodies in the pathogenesis of RA is briefly discussed.

To study the effects of osteoclast-targeted therapies, such as osteoprotegerin (OPG) and pamidronate, on joint inflammation and bone destruction using a tumor necrosis factor α (TNFα)-transgenic mouse model. Mice were placed into 5 groups that received either OPG, pamidronate, a combination of both agents, infliximab as a positive control, or phosphate buffered saline as a negative control. Treatment was initiated at the onset of arthritis, continued over 6 weeks, and thereafter, the clinical, radiologic, and histologic outcomes were assessed. A significant improvement in clinical symptoms, as assessed by the reduction of paw swelling, was only found in the infliximab group, whereas all other treatment groups failed to show significant improvement. However, when assessing structural damage with radiographic analysis, a significant retardation of joint damage was evident in animals treated with OPG (55% reduction of erosions), pamidronate (50% reduction of erosions) the combination therapy of OPG and pamidronate (64% reduction of erosions), and with infliximab (66% reduction of erosions). Confirming these data, quantitative histologic analysis revealed a significant reduction in the size of bone erosions in all treatment groups (OPG 56%, pamidronate 53%, OPG and pamidronate 81%, and infliximab 46%) compared with the control group. Furthermore, a significant reduction of osteoclast numbers was seen in animals treated with OPG alone or in combination with pamidronate as well as in animals treated with infliximab. These data suggest that OPG alone or in combination with bisphosphonates is an effective therapeutic tool for the prevention of TNFα-mediated destruction of bone by reducing the number of bone-resorbing cells in the inflammatory tissue.

Rheumatoid arthritis is a prototype of a destructive inflammatory disease. Inflammation triggered by the overexpression of tumor necrosis factor α (TNFα) is a driving force of this disorder and mediates tissue destruction. Since matrix metalloproteinases (MMPs) are among the molecules activated by TNFα, we hypothesized that overexpression of their natural inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), in TNFα transgenic mice could inhibit the development of destructive arthritis. Systemic treatment was carried out by replication-defective adenoviral vectors for TIMP-1, β-galactosidase, or phosphate buffered saline (PBS), which were applied once at the onset of arthritis. Clinical, serologic, radiologic, and histologic outcomes were assessed 18 days after the treatment. The AdTIMP-1 group showed significantly reduced paw swelling and increased grip strength compared with the 2 control groups, whereas total body weight, TNFα, and interleukin-6 levels were similar in all 3 groups. Radiographic assessment revealed a significant reduction of joint destruction in the AdTIMP-1 group; this was confirmed by histologic analyses showing reduced formation of pannus and erosions in the AdTIMP-1 group compared with the AdLacZ and PBS control groups. The formation of arthritis-specific autoantibodies to heterogeneous nuclear RNP A2 was not observed in the AdTIMP-1 group but was present in the 2 control groups. These results indicate a central role of MMPs in TNFα-mediated tissue damage in vivo and a promising therapeutic role for TIMP-1.

To investigate the expression of the transcription factor Ets‐1 in synovial tissue and cultured synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to study the regulation of Ets‐1 expression and activation in synovial fibroblasts by proinflammatory cytokines.In situ expression of Ets‐1 in synovial tissue from RA and OA patients was examined by double immunohistochemistry. The effects of interleukin‐1 (IL‐1) or tumor necrosis factor α (TNFα) on Ets‐1 expression and activation (DNA binding) in cultured synovial fibroblasts were analyzed by Western blotting and DNA gel shift assay, respectively. In addition, the intracellular location of Ets‐1 in synovial fibroblasts was determined by immunofluorescence.Pronounced expression of Ets‐1 was detected in synovial tissues from all RA patients evaluated, particularly in the synovial lining layer and the sublining areas. Ets‐1 was expressed by both fibroblasts and macrophages as well as by endothelial cells, while only a few T cells stained positive for Ets‐1. In synovial specimens from OA patients, Ets‐1 expression was much less frequently observed and was largely restricted to vascular cells. Ets‐1 was expressed to a similar degree in cultured synovial fibroblasts from RA and OA patients, as demonstrated by reverse transcriptase–polymerase chain reaction and Western blotting. Both IL‐1 and TNFα induced pronounced up‐regulation of Ets‐1 in synovial fibroblasts. Moreover, binding of Ets‐1 to its specific DNA binding site was induced by both cytokines, although with different time courses. Immunofluorescence staining revealed a dominant nuclear localization of Ets‐1 in IL‐1– or TNFα‐stimulated synovial fibroblasts.The overexpression of Ets‐1 observed in RA synovial tissue appears to be caused by TNFα and IL‐1, suggesting that Ets‐1 may be an important factor in the cytokine‐mediated inflammatory and destructive cascade characteristic of RA.

The hY RNAs are a group of four small cytoplasmic RNAs of unknown function that are stably associated with at least two proteins, Ro60 and La, to form Ro ribonucleoprotein complexes. Here we show that the heterogeneous nuclear ribonucleoproteins (hnRNP) I and K are able to associate with a subset of hY RNAs in vitroand demonstrate these interactions to occur also in vivoin a yeast three-hybrid system. Experiments performed in vitroand in vivowith deletion mutants of hY1 RNA revealed its pyrimidine-rich central loop to be involved in interactions with both hnRNP I and K and clearly showed their binding sites to be different from the Ro60 binding site. Both hY1 and hY3 RNAs coprecipitated with hnRNP I in immunoprecipitation experiments performed with HeLa S100 extracts and cell extracts from COS-1 cells transiently transfected with VSV-G-tagged hnRNP-I, respectively. Furthermore, both anti-Ro60 and anti-La antibodies coprecipitated hnRNP I, whereas coprecipitation of hnRNP K was not observed. Taken together, these data strongly suggest that hnRNP I is a stable component of a subpopulation of Ro RNPs, whereas hnRNP K may be transiently bound or interact only with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions related to translation regulation have been assigned to both proteins and also to La, our findings may provide novel clues toward understanding the role of Y RNAs and their respective RNP complexes.

To investigate whether stress- and mitogen-activated protein kinases (SAPK/MAPK), such as extracellular signal–regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, are significantly activated in rheumatoid arthritis (RA) synovial tissue compared with their activation in degenerative joint disease; to assess the localization of SAPK/MAPK activation in rheumatoid synovial tissue; and to search for the factors leading to stress kinase activation in human synovial cells. Immunoblotting and immunohistology by antibodies specific for the activated forms of SAPK/MAPK were performed on synovial tissue samples from patients with RA and osteoarthritis (OA). In addition, untreated and cytokine-treated human synovial cells were assessed for SAPK/MAPK activation and downstream signaling by various techniques. ERK, JNK, and p38 MAPK activation were almost exclusively found in synovial tissue from RA, but not OA, patients. ERK activation was localized around synovial microvessels, JNK activation was localized around and within mononuclear cell infiltrates, and p38 MAPK activation was observed in the synovial lining layer and in synovial endothelial cells. Tumor necrosis factor α, interleukin-1 (IL-1), and IL-6 were the major inducers of ERK, JNK, and p38 MAPK activation in cultured human synovial cells. Signaling through SAPK/MAPK pathways is a typical feature of chronic synovitis in RA, but not in degenerative joint disease. SAPK/MAPK signaling is found at distinct sites in the synovial tissue, is induced by proinflammatory cytokines, and could lead to the design of highly targeted therapies.

To compare and investigate antihistone and antichromatin antibody responses as well as clinical variables in patients with systemic lupus erythematosus (SLE) who were either positive (LEC+) or negative (LEC−) for the lupus erythematosus (LE) cell phenomenon. The binding properties of LEC+ and LEC− SLE sera to chromatin-associated nuclear antigens (histones H1, H2A, H2B, H3, H4; complexes of H2A–H2B, [H2A–H2B]–DNA, H1–DNA; total and H1-stripped chromatin; native and denatured DNA) were investigated. In addition, sera from patients with drug-induced lupus (by procainamide, hydralazine, or quinidine), as well as from patients with rheumatoid arthritis and osteoarthritis, were assessed. Enzyme-linked immunosorbent assay was used to detect specific antibody binding. Mirroring the important role of histone H1 in the formation of LE cells, anti–histone H1 reactivity was 8-fold higher in LEC+ sera than in LEC− sera. In addition, reactivities to most of the other antigens tested, i.e., other histones and histone–DNA complexes as well as chromatin and DNA, were significantly higher in LEC+ sera than in LEC− sera. All but 1 serum sample from the patients with drug-induced lupus were negative for LE cell formation as well as for anti–histone H1 reactivity, but displayed high antibody reactivities to histone–DNA complexes, including chromatin. Sera from patients with rheumatoid arthritis and osteoarthritis did not show significant binding to these antigens. When comparing the clinical features of LEC+ and LEC− SLE patients, severe organ involvement, including nephritis and central nervous system involvement, was common in the LEC+ group, but rare in the LEC− group. A positive LE cell phenomenon not only correlated with the presence of high anti–histone H1 antibody levels in SLE, but also indicated serologically and clinically active disease with major organ involvement.

To investigate the expression of the stroma cell product stem cell factor (SCF) in synovial fibroblasts (SFB) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and to analyze the capacity of SFB to induce mast cell (MC) chemotaxis. Synovial tissue was obtained from 29 patients with RA and 25 patients with OA. Tissue was dispersed by enzymatic digestion using collagenase. SFB were grown in serial passage and exposed to tumor necrosis factor α (TNFα) or control medium. Expression of SCF in cultured SFB was analyzed by reverse transcription–polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunostaining. The ability of SFB (supernatants) to induce MC migration was analyzed using a double-chamber chemotaxis assay and the human mast cell line HMC-1. In situ expression of SCF in synovial tissue from patients with RA (n = 6) and OA (n = 6) was examined by double immunohistochemistry using antibodies against SCF and the fibroblast-specific antibody ASO2. In both RA and OA, cultured SFB were found to express SCF messenger RNA, as assessed by RT-PCR. In addition, the SCF protein was detectable in cell lysates and supernatants of SFB by ELISA. Incubation of SFB with TNFα resulted in an increased expression and release of SCF. Recombinant human SCF (rHuSCF) and SFB supernatants induced significant migration of HMC-1 cells above control levels. In addition, exposure of SFB to TNFα led to an increased migration of HMC-1, and a blocking anti-SCF antibody inhibited the rHuSCF- and SFB-induced migration of HMC-1. In situ double immunostaining revealed expression of SCF in ASO2-positive SFB in the synovium of patients with RA. Our results show that SFB (in RA and OA) express SCF and induce MC chemotaxis. Furthermore, TNFα was found to augment SCF expression in SFB. It is hypothesized that these cellular interactions play an important role in MC accumulation and related events in RA.

Background: There is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers. Methods: Using protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. Results: HnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CNDL-Index) was identified as a new value for an “individual window of treatment success” in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24–46%). Negative CNDL-index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients. Conclusion: CNDL-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.

Activation of heat shock factor (HSF) 1‐DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane‐bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each other's activation. Induction of FAS‐mediated signaling was followed by a rapid decrease in HSF1‐DNA binding and inducible hsp70 expression. Inhibition of HSF1‐DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE‐mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas‐mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1‐DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas‐mediated apoptosis and vice versamay facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.—Schett, G., Steiner, C.‐W., Gröger, M., Winkler, S., Graninger, W., Smolen, J., Xu, Q., Steiner, G. Activation of Fas inhibits heat‐induced activation of HSF1 and up‐regulation of hsp70. FASEB J.13, 833–842 (1999)

No abstract.

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are major constituents of the spliceosome. They are composed of approximately 30 different proteins which can bind to nascent pre-mRNA. Among these, the hnRNP-A/B proteins form a subgroup of highly related proteins consisting of two adjacent RNA binding domains (RBD) within the N-terminal parts, whereas the C-terminal halves contain almost 50% glycine residues. These proteins, in particular A2/RA33, are targeted by autoantibodies from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). In SLE anti-hnRNP antibodies frequently occur together with antibodies to U1 small nuclear RNP (U1-snRNP) and Sm, other proteins of the spliceosome. Preliminary epitope mapping studies have revealed major antibody binding sites in the RNA binding regions for all three diseases. Nevertheless, there is some indication of disease specific epitope recognition. Studies in animal models have demonstrated anti-RA33/hnRNP-A/B antibodies in lupus-prone mouse strains.

The characteristics of 3 patients with longstanding rheumatoid arthritis (RA) and consecutive evolution of limited cutaneous systemic sclerosis (lcSSc) were evaluated and compared with those of patients with lcSSc alone (n = 20) or with RA alone (n = 120). Clinical features of the different patient populations were compared. Serologic analyses included tests for antinuclear antibodies (ANA) and ANA subsets, in particular anticentromere antibodies (ACA) and anti‐heterogeneous nuclear RNP (hnRNP)‐A2/RA33 (anti‐A2/RA33). The 3 patients with RA developed lcSSc 11, 29, or 50 years after the onset of RA. Features of lcSSc were Raynaud's phenomenon, sclerodactyly, and telangiactasias in all 3 patients, and esophageal dysmotility in 1 patient. Rheumatoid factor (RF) and anti‐A2/RA33 were each found in 2 patients, and 1 of these patients was seropositive for both RF and anti‐A2/RA33. ACA titers were positive in all cases. However, similar to the development of RA prior to lcSSc, the occurrence of autoantibodies typical of RA preceded the occurrence of ACA, at least in 2 of the patients. Using affinity‐purified antibodies, cross‐reactivities between anti‐centromere protein A (CENP‐A) and anti‐CENP‐B antibodies with anti‐A2/RA33 antigens were seen in the 2 anti‐A2/RA33‐positive patients. Such cross‐reactivities were not found in lcSSc patients without concomitant RA. Epitope mapping revealed that both autoantibody specificities recognized the known major epitopes: anti‐CENP‐B reacted with the C‐terminal region and anti‐A2/RA33 with the second RNA binding domain in the N‐terminal region of hnRNP‐A2. The RA‐lcSSc overlap syndrome in these 3 patients with longstanding RA was characterized by an incomplete CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome. The study demonstrated the presence of autoantibodies typical of both diseases and cross‐reactivity of ACA with hnRNP‐A2/RA33 in the sera of these patients.

Aims: To determine the diagnostic value of anti-acetylated peptide antibodies (AAPA) in patients with rheumatoid arthritis (RA).Methods: Three acetylated peptides (ac-lysine, ac-lysine.inv and ac-ornithine) derived from vimentin were employed to measure AAPA by enzyme-linked immunosorbent assay (ELISA) in sera of 120 patients with early RA (eRA), 195 patients with established RA (est RA), 99 healthy controls (HC), and 216 patients with other inflammatory rheumatic diseases. A carbamylated and a citrullinated version of the vimentin peptide were used additionally. Receiver operating characteristics and logistic regression analyses were used to assess the discriminative capacity of AAPA.Results: AAPA were detected in 60% of eRA and 68.7% of estRA patients, 22.2% of HC, and 7.1– 30.6% of patients with other rheumatic diseases. Importantly, AAPA were also present in 40% of seronegative RA patients, while antibodies to the carbamylated peptide were detected less frequently. Diagnostic sensitivity of individual peptides for eRA was 28.3%, 35.8%, and 34% for ac-lysine, ac-ornithine, and ac-lysine.inv, respectively. Positive likelihood ratios (LR+) for eRA versusHC were 14.0, 7.1, and 2.1. While the presence of a single AAPA showed varying specificity (range: 84–98%), the presence of two AAPA increased specificity considerably since 26.7% of eRA, as compared with 6% of disease controls, were double positive. Thus, double positivity discriminated eRA from axial spondyloarthritis with a LR+ of 18.3. Remarkably, triple positivity was 100% specific for RA, being observed in 10% of eRA and 21.5% of estRA patients, even in the absence of RF and ACPA.Conclusion: AAPA are highly prevalent in early RA and occur also independently of RF and ACPA, thereby reducing the gap of seronegativity. Furthermore, multiple AAPA reactivity increased the specificity for RA, suggesting high diagnostic value of AAPA testing.

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No abstract.

Pristane-induced arthritis (PIA) in the rat has been described as an animal model of inflammatory arthritis which exhibits features similar to rheumatoid arthritis in humans, such as a chronic, destructive, and symmetrical involvement of peripheral joints. However, so far little is known about the earliest inflammatory events and their influence on locomotor behaviour during the course of PIA. To investigate this issue a detailed analysis of the pathologic changes occurring during the prodromal and early stages of PIA was performed.

Tumour necrosis factor (TNF) signalling molecules are considered as promising therapeutic targets of antirheumatic therapy. Among them, mitogen-activated protein kinases are thought to be of central importance. Herein, we investigate the role in vivo of TNF-α signalling through c-Jun N-terminal kinase (JNK)1 in destructive arthritis. Human TNF transgenic (hTNFtg) mice, which develop inflammatory arthritis, were intercrossed with JNK1-deficient (JNK1-/-) mice. Animals (n = 35) of all four genotypes (wild-type, JNK1-/-, hTNFtg, JNK1-/-hTNFtg) were assessed for clinical and histological signs of arthritis. Clinical features of arthritis (swelling and decreased grip strength) developed equally in hTNFtg and JNK1-/-hTNFtg mice. Histological analyses revealed no differences in the quantity of synovial inflammation and bone erosions or in the cellular composition of the synovial infiltrate. Bone destruction and osteoclast formation were observed to a similar degree in hTNFtg and JNK1-/-hTNFtg animals. Moreover, cartilage damage, as indicated by proteoglycan loss in the articular cartilage, was comparable in the two strains. Intact phosphorylation of JNK and c-Jun as well as expression of JNK2 in the synovial tissue of JNK1-/-hTNFtg mice suggests that signalling through JNK2 may compensate for the deficiency in JNK1. Thus, JNK1 activation does not seem to be essential for TNF-mediated arthritis.

This review focuses on the mechanisms of stress response in the synovial tissue of rheumatoid arthritis. The major stress factors, such as heat stress, shear stress, proinflammatory cytokines and oxidative stress, are discussed and reviewed, focusing on their potential to induce a stress response in the synovial tissue. Several pathways of stress signalling molecules are found to be activated in the synovial membrane of rheumatoid arthritis; of these the most important examples are heat shock proteins, mitogen-activated protein kinases, stress-activated protein kinases and molecules involved in the oxidative stress pathways. The expression of these pathways in vitroand in vivoas well as the consequences of stress signalling in the rheumatoid synovium are discussed. Stress signalling is part of a cellular response to potentially harmful stimuli and thus is essentially involved in the process of synovitis. Stress signalling pathways are therefore new and promising targets of future anti-rheumatic therapies.

An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinically and followed prospectively for 1 year to determine the clinical significance of a number of rheumatoid arthritis (RA) associated autoantibodies. Serum samples collected at the time of the initial evaluation were tested for rheumatoid factor (RF) and antibodies to Sa (anti-Sa), RA-33, (pro)filaggrin [antifilaggrin antibody (AFA)], cyclic citrullinated peptide (anti-CCP), calpastatin, and keratin [antikeratin antibody (AKA)]. RF had a sensitivity of 66% and a specificity of 87% for RA. Anti-Sa, AFA, and anti-CCP all had a specificity of more than 90%, but a sensitivity of less than 50% for this diagnosis. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P< 0.001). Of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one. Finally, anti-SA identified a subset of predominantly male RA patients with severe, erosive disease. Anti-SA, AFA and anti-CCP are all specific for early RA but, overall, have little additional diagnostic value over RF alone. Although these antibodies may preferentially recognize citrullinated antigens, the modest degree of concordance between them in individual patient sera suggests that it is unlikely a single antigen is involved in generating these responses.

Wybutinep(Ywye) , situated next to the 3′-side of the anti-codon of tRNAPhe from Saccharomyces cerevisiae, can be photo-crosslinked to mRNA when bound to Escherichia coli ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)6. In order to identify the site of reaction on the mRNA,5′-[32P]-labelledh pAUGUUU was crosslinked by irradiation at 320 nm with Phe-tRNAPhe from yeast bound to the acceptor-site. The photoproduct was subsequently digested with P1-nuclease and analyzed by electrophoresis followed by homochromatography in the second dimension. As a result of the photoreaction the wybutine was found to be cross-linked to the U at the 5′-position of the corresponding UUU-co-don.