Experimental studies on signal transduction pathways in rheumatoid arthritis

Reumatoïde artritis (RA) is een chronische ontstekingsziekte, die met name gelocaliseerd is in de gewrichten. De ziekte komt voor bij 0.5 - 1% van de bevolking, en komt meer voor bij vrouwen dan bij mannen. Door de ontsteking in de gewrichten raken kraakbeen en bot beschadigd en dit kan leiden tot handicaps en verminderde kwaliteit van leven. De behandeling van RA is er ten eerste op gericht om de verschijnselen van ontsteking (pijn en koorts) te bestrijden en in de tweede plaats om de schade aan de gewrichten te beperken. Uit onderzoek is gebleken dat de zogenaamde cytokines tumor necrosis factor (TNF-) en interleukine-1 (IL-1) een centrale rol spelen in het ontstekingsproces. De laatste jaren is gebleken dat het blokkeren van deze cytokines met antilichamen een goede therapie is voor RA. Helaas reageren niet alle patiënten op deze therapie en komen ook incidenteel ernstige bijwerkingen van deze therapie voor. Een andere manier om de produktie en activiteit van deze cytokines te remmen is door het blokkeren van intracellulaire signaal transductieroutes. Deze routes zijn mechanismen waarbij een prikkel buiten de cel omgezet wordt in een reactie in de cel, meestal in de vorm van de produktie van ontstekingseiwitten. In deze signaal transductie routes spelen de zogenaamde kinases een belangrijke rol, doordat ze het signaal in de vorm van een fosfaatgroep aan elkaar doorgeven. In dit proefschrift is onderzocht of het remmen van een belangrijke intracellulaire signaal transductie route (de p38 mitogen-activated protein kinase (MAPK) route) een goede therapie zou kunnen zijn voor RA. In ons onderzoek is gekeken naar de gevolgen van remming met een specifieke p38 MAPK remmer (RWJ 67657) in cellen, die een rol spelen bij ontsteking

High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus

Introduction: High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity. Methods: The study population consisted of 70 SLE patients and 35 age-and sex-matched HC. Thirty-three SLE patients had quiescent disease, the other 37 patients were selected for having active disease. Nineteen of these had lupus nephritis. HMGB1 levels were measured with both Western blot and ELISA. Anti-HMGB1 levels were measured by ELISA. Clinical and serological parameters were assessed according to routine procedures. Results: HMGB1 levels in SLE patients could be measured reliably by Western blotting only, and were significantly increased compared to HC. During active disease HMGB1 levels increased, in particular in patients with renal involvement. Serum HMGB1 levels correlated with SLEDAI, proteinuria, and anti-dsDNA levels, and showed a negative correlation with complement C3. Anti-HMGB1 levels were significantly increased in SLE patients compared to HC, and positively correlated with HMGB1 levels. Conclusions: Levels of HMGB1 in the sera of SLE patients, in particular in those with active renal disease, are increased. Serum HMGB1 levels are related to SLEDAI scores and proteinuria, as well as to levels of anti-HMGB1 antibodies. These findings suggest that besides HMGB1, HMGB1-anti-HMGB1 immune complexes play a role in the pathogenesis of SLE, in particular in patients with renal involvement

Advanced glycation endproducts are increased in rheumatoid arthritis patients with controlled disease

Introduction: Advanced glycation end products (AGEs) are produced and can accumulate during chronic inflammation, as might be present in patients with rheumatoid arthritis (RA). AGEs are involved in the development of cardiovascular disease. The aim of this study is to evaluate whether AGEs are increased in patients with long-standing RA and whether AGE accumulation is related to disease activity, disease severity and measures of (premature) atherosclerosis, such as endothelial activation, endothelial dysfunction and intima media thickness (IMT). Methods: In a cross-sectional study, 49 consecutive RA patients with longstanding disease (median disease duration of 12.3 years (range 9.3 to 15.1)), receiving standard of care, were included and compared with 49 age-and sex-matched healthy controls (HC). AGEs were determined by skin autofluorescence. Disease activity was evaluated by the Disease Activity Score of 28 joints (DAS-28) score and joint damage by modified Sharp-v.d. Heijde score. Endothelial activation (soluble vascular cellular adhesion molecule-1) sVCAM-1, von Willebrand factor (vWF), thrombomodulin), endothelial dysfunction (determined by small artery elasticity (SAE)) and IMT were measured and related to AGE accumulation. Results: AGEs were increased in RA patients (median 2.4 arbitrary units (a.u.), range 1.6 to 4.2) compared to HC (2.2, 1.3 to 3.8). RA patients had a DAS-28 score of 2.9 (0.8 to 6.9) and a modified Sharp-v.d. Heijde score of 19 (0 to 103). sVCAM-1 and vWF levels were higher in RA patients. SAE was significantly decreased in RA (3.9 ml/mmHg (1.4 to 12.2) vs. 6.1 in HC (1.7 to 12.9). IMT did not differ between the two groups. Combining both groups' AGEs correlated with vWF, sVCAM-1 and IMT, and was inversely related to SAE. In RA, AGEs had an inverse relation with SAE, but did not relate to disease activity or radiological damage. In multivariate analysis for both groups, smoking, glucose levels, vWF, SAE and male gender were significantly related to the formation of AGEs. Conclusions: AGEs were increased in RA patients with long-standing disease and without signs of premature atherosclerosis. AGEs were related to endothelial activation and endothelial dysfunction. This supports the hypothesis that in RA AGEs may be an early marker of cardiovascular disease

Is serum HMGB1 a biomarker in ANCA-associated vasculitis?

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic inflammatory disorders that include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Churg-Strauss syndrome and renal limited vasculitis (RLV). Extracellular high-mobility group box 1 (HMGB1) acts as an alarmin and has been shown to be a biomarker of disease activity as well as an autoantigen in systemic lupus erythematosus (SLE) and, possibly, in AAV. This study aims to assess antibodies against HMGB1 and HMGB1 levels as biomarkers for AAV disease activity and predictors of relapsing disease.Methods: AAV patients with active disease and healthy controls (HC) were evaluated for anti-HMGB1 antibodies while serum HMGB1 levels were measured longitudinally in AAV patients at presentation, during remission, prior to and at relapses.Results: HMGB1 levels were similar between AAV patients at presentation (n = 52) and HC (n = 35) (2.64 +/- 1.80 ng/ml vs. 2.39 +/- 1.09 ng/ml; P = 0.422) and no difference regarding HMGB1 levels could be found among AAV disease subsets (GPA: 2.66 +/- 1.83 ng/ml vs. MPA: 3.11 +/- 1.91 ng/ml vs. RLV: 1.92 +/- 1.48 ng/ml; P = 0.369). AAV patients with renal involvement had lower HMGB1 levels than patients without renal involvement at presentation (2.35 +/- 1.48 ng/ml vs. 3.52 +/- 2.41 ng/ml; P = 0.042). A negative correlation was observed between HMGB1 levels and 24-hour proteinuria (rho = -0.361, P = 0.028). Forty-nine AAV patients were evaluated for HMGB1 levels during follow-up and no differences were observed between relapsing and nonrelapsing patients (P = 0.350). No significant increase in HMGB1 levels was observed prior to a relapse compared with the remission period and changes in HMGB1 levels were not associated with an increased risk for relapse in AAV. Positivity for anti-HMGB1 antibodies was low in patients with active AAV (three out of 24 patients).Conclusions: Serum HMGB1 levels at presentation are not increased and are lower in patients with renal involvement. Relapses are not preceded or accompanied by significant rises in HMGB1 levels and changes in HMGB1 levels are not related to ensuing relapses. Anti-HMGB1 antibodies are present in only a few patients in AAV. in contrast to SLE, HMGB1 is not a useful biomarker in AAV.Univ Groningen, Univ Med Ctr Groningen, Dept Rheumatol & Clin Immunol, NL-9713 GZ Groningen, NetherlandsUniversidade Federal de São Paulo, Escola Paulista Med Unifesp EPM, Div Rheumatol, BR-04023900 São Paulo, BrazilUniv Groningen, Univ Med Ctr Groningen, Dept Lab Med, NL-9713 GZ Groningen, NetherlandsUniv Groningen, Univ Med Ctr Groningen, Div Nephrol, Dept Internal Med, NL-9713 GZ Groningen, NetherlandsMartini Hosp, Dept Rheumatol & Internal Med, NL-9728 NT Groningen, NetherlandsUniversidade Federal de São Paulo, Escola Paulista Med Unifesp EPM, Div Rheumatol, BR-04023900 São Paulo, BrazilWeb of Scienc

Effect of Anti-Rheumatic Treatment on the Periodontal Condition of Rheumatoid Arthritis Patients

Periodontitis, a bacterial-induced infection of the supporting soft and hard tissues of the teeth (the periodontium), is common in patients with rheumatoid arthritis (RA). As RA and periodontitis underlie common inflammatory pathways, targeting the progression of RA might mediate both periodontitis and RA. On the other hand, patients with RA on immunosuppressive medication have an increased risk of infection. Therefore, the objective of this longitudinal observation study was to assess the effect of methotrexate (MTX) and anti-tumor necrosis factor-alpha (anti-TNF, etanercept) treatment on the periodontal condition of RA patients. Overall, 14 dentate treatment-naive RA patients starting with MTX and 12 dentate RA patients starting with anti-TNF therapy in addition to MTX were included. Follow-up was scheduled matching the routine protocol for the respective treatments. Prior to the anti-rheumatic treatment with MTX or the anti-TNF therapy in addition to MTX, and during follow-up, i.e., 2 months for MTX, and 3 and 6 months for the anti-TNF therapy in addition to MTX, the periodontal inflamed surface area (PISA) was measured. The efficacy of the anti-rheumatic treatment was assessed by determining the change in RA disease activity (DAS28-ESR). Furthermore, the erythrocyte sedimentation rates were determined and the levels of C-reactive protein, IgM-rheumatoid factor, anti-cyclic citrullinated protein antibodies, and antibodies to the periodontal pathogen Porphyromonas gingivalis, were measured. Subgingival sampling and microbiological characterization of the subgingival microflora was done at baseline. MTX or anti-TNF treatment did not result in an improvement of the periodontal condition, while both treatments significantly improved DAS28 scores (both p <0.01), and reduced C-reactive protein levels and erythrocyte sedimentation rates (both p <0.05). It is concluded that anti-rheumatic treatment (MTX and anti-TNF) has negligible influence on the periodontal condition of RA patients

Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations

INTRODUCTION: Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity. METHODS: The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies. RESULTS: Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4. CONCLUSION: Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation

Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus

INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disease accompanied by a disturbed T-cell balance skewed towards effector T-cells, in particular Th17-cells. The novel cytokine interleukin-21 (IL-21) is suggested to be crucial for triggering T-cell responses towards IL-17 producing cells. Thus, we aimed to investigate the ability of T-cells to produce IL-21 and IL-17 in SLE patients. METHODS: Peripheral blood of 34 SLE patients and 18 healthy controls (HC) was stimulated with phorbol myristate acetate (PMA) and calcium ionophore (Ca-Io). Percentages of IL-21- and IL-17A expressing T-cells were analysed by flow cytometry. The expression levels of the transcription factors B-cell lymphoma-6 (BCL-6) and factors retinoid-related orphan receptor (ROR-γt) were assessed in T-cells by real-time RT-PCR and flow cytometry. Additionally, IL-21 receptor (IL-21R) expression on B- and T-cells of patients and HC was analyzed. RESULTS: Significantly increased percentages of IL-21 expressing CD4(+ )T-cells and CD8(+ )T-cells were found in SLE patients as compared to HC. The percentages of IL-21(+ )CD4(+ )T-cells and CD8(+ )T-cells correlated significantly with the percentages of IL-17A(+ )CD4(+ )T-cells and CD8(+ )T-cells, respectively. The relative expression of BCL-6 and ROR-γt did not differ between SLE patients and HC. IL-21R expression occurred mainly on B-cells and was not different comparing SLE patients and HC. CONCLUSIONS: This study demonstrates an increased proportion of IL-21(+ )T-cells in SLE patients correlating with the proportion of IL-17(+ )T-cells. This suggests a pivotal role of IL-21 in the pathogenesis of SLE

High mobility group box 1 levels in large vessel vasculitis are not associated with disease activity but are influenced by age and statins

Introduction: Takayasu arteritis (TA) and giant cell arteritis (GCA) are large vessel vasculitides (LVV) that usually present as granulomatous inflammation in arterial walls. High mobility group box 1 (HMGB1) is a nuclear protein that acts as an alarmin when released by dying or activated cells. This study aims to evaluate whether serum HMGB1 can be used as a biomarker in LVV. Methods: Twenty-nine consecutive TA patients with 29 healthy controls (HC) were evaluated in a cross-sectional study. Eighteen consecutive GCA patients with 16 HC were evaluated at the onset of disease and some of them during follow-up. Serum HMGB1 levels were measured by enzyme-linked immunosorbent assay. Results: In GCA patients at disease onset mean serum HMGB1 levels did not differ from HC (5.74 +/- 4.19 ng/ml vs. 4.17 +/- 3.14 ng/ml; p = 0.230). No differences in HMGB1 levels were found between GCA patients with and without polymyalgia rheumatica (p = 0.167), ischemic manifestations (p = 0.873), systemic manifestations (p = 0.474) or relapsing disease (p = 0.608). During follow-up, no significant fluctuations on serum HMGB1 levels were observed from baseline to 3 months (n = 13) (p = 0.075), 12 months (n = 6) (p = 0.093) and at the first relapse (n = 4) (p = 0.202). Serum HMGB1 levels did not differ between TA patients and HC [1.19 (0.45-2.10) ng/ml vs. 1.46 (0.89-3.34) ng/ml; p = 0.181] and no difference was found between TA patients with active disease and in remission [1.31 (0.63-2.16) ng/ml vs. 0.75 (0.39-2.05) ng/ml; p = 0.281]. HMGB1 levels were significantly lower in 16 TA patients on statins compared with 13 patients without statins [0.59 (0.29-1.46) ng/ml vs. 1.93 (0.88-3.34) ng/ml; p = 0.019]. Age was independently associated with higher HMGB1 levels regardless of LVV or control status. Conclusions: Patients with TA and GCA present similar serum HMGB1 levels compared with HC. Serum HMGB1 is not useful to discriminate between active disease and remission. In TA, use of statins was associated with lower HMGB1 levels. HMGB1 is not a biomarker for LVV