Rheumatoid Arthritis: Joosten LA

Joosten LA, Netea MG. · No affiliation provided · Arthritis Rheum. · Pubmed #19479874 No free full text.

This publication has no abstract.

van den Berg WB, van Lent PL, Joosten LA, Abdollahi-Roodsaz S, Koenders MI. · Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Center, Geert Grooteplein 26, 6525 GA Nijmegen, The Netherlands. · Ann Rheum Dis. · Pubmed #17934095 No free full text.

Abstract: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by chronic joint inflammation and variable degrees of bone and cartilage erosion. Studies in animal models of arthritis provide insight into elements which can amplify destructive features. The presence of immune complexes in the joint makes arthritis more erosive. Although considerable bone erosion still occurs in the absence of FcgammaR triggering by immune complexes, through cytokine-induced RANKL and direct osteoclast activation, cartilage erosion is heavily dependent on the FcgammaR pathway. T cell factors such as IFNgamma and IL17 further amplify erosion through upregulation of the damaging FcgammaRI and stimulation of the influx of granulocytes, respectively. Apart from immune elements, environmental pressure and components of tissue damage contribute through innate pathways. Spontaneous T cell-dependent arthritis in IL1Ra-/- mice is absent under germ-free conditions, and markedly suppressed in TLR4-deficient mice. Moreover, TLR4 blocking with a receptor antagonist suppresses erosive arthritis.

Koenders MI, Joosten LA, van den Berg WB. · Radboud University Nijmegen Medical Center, Department of Rheumatology, Rheumatology Research and Advanced Therapeutics, 272, Geert Grooteplein 26, PO Box 9101, 6500 HB Nijmegen, the Netherlands. · Ann Rheum Dis. · Pubmed #17038468 No free full text.

Abstract: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by chronic joint inflammation and destruction. Interleukin (IL)-17 is a T cell cytokine expressed in the synovium and synovial fluid of patients with RA. IL-17 is a potent inducer of various cytokines such as tumour necrosis factor (TNF) and IL-1. IL-17 has been shown to have additive or even synergistic effects with TNF and IL-1 during the induction of cytokine expression and joint damage in vitro and in vivo. TNFalpha and IL-1 are considered powerful targets in the treatment of RA because of their leading role in driving the enhanced production of cytokines, chemokines, and degradative enzymes. Besides anti-TNF and anti-IL-1 therapies, whose clinical efficacy is now established, new targets have been proposed for RA which is not responding to conventional treatments. This paper discusses the role of IL-17 in experimental arthritis and its interrelationship with TNF and IL-1, currently the most targeted cytokines in the treatment of RA. IL-17 is involved in both initiation and progression of murine experimental arthritis. Studies have shown that IL-17 not only synergises with TNF, but also enhances inflammation and destruction independent of IL-1 and TNF. On the basis of these studies, the authors propose IL-17 as an interesting additional target in the treatment of RA.

van den Berg WB, Joosten LA, Lubberts E. · Department of Rheumatology, University Hospital Nijmegen, The Netherlands. · Curr Dir Autoimmun. · Pubmed #11791452 No free full text.

This publication has no abstract.

van den Berg WB, Joosten LA, van de Loo FA. · Department of Rheumatology, University Hospital Nijmegen, The Netherlands. · Clin Exp Rheumatol. · Pubmed #10589368 No free full text.

Abstract: Chronic arthritis is characterized by persistent joint inflammation and concomitant joint destruction. Using murine arthritis models and neutralizing antibodies as well as cytokine-specific knockout conditions, it was found that tumor necrosis factor alpha (TNF alpha) is important in joint swelling, whereas a direct role in tissue destruction is unlikely. Interleukin-1 (IL-1) is not a dominant cytokine in early joint swelling, but has a pivotal role in sustained cell infiltration and erosive cartilage damage. TNF alpha-independent IL-1 production is a prominent feature in murine arthritis models, implying that IL-1 as well as TNF alpha are appropriate targets for therapy. These observations provide evidence for the potential uncoupling of joint inflammation and erosive changes and underline the need for TNF alpha/IL-1 directed combination-therapy approaches.

den Broeder AA, Joosten LA, Saxne T, Heinegård D, Fenner H, Miltenburg AM, Frasa WL, van Tits LJ, Buurman WA, van Riel PL, van de Putte LB, Barrera P. · Department of Rheumatology, University Medical Centre Nijmegen, The Netherlands. · Ann Rheum Dis. · Pubmed #11874832 links to  free full text

Abstract: OBJECTIVES: To investigate the effect of prolonged neutralisation of tumour necrosis factor alpha (TNFalpha) on the radiological course in rheumatoid arthritis (RA). To assess whether the radiological course can be predicted by clinical variables or biological markers of cartilage and synovium turnover and of endothelial activation. PATIENTS AND METHODS: Forty seven patients with active RA enrolled at our centre in monotherapy trials with adalimumab (D2E7), a fully human anti-TNFalpha monoclonal antibody, were studied for two years. Radiographs of hands and feet obtained at baseline and after one and two years were scored in chronological order by a single, blinded observer using the modified Sharp method. Radiological course was classified as stable or progressive using the smallest detectable difference as cut off point. The relation between radiological course and serum markers of cartilage and synovium turnover (metalloproteinases (MMP-1 and MMP-3), cartilage oligomeric matrix protein (COMP), human cartilage glycoprotein-39 (HC gp-39)), endothelial activation (soluble E-selectin and intercellular adhesion molecule (ICAM-1)), and integrated measures of disease activity were assessed using univariate and multivariate analysis. RESULTS: Radiological evaluation was performed in 36 patients with paired sets of radiographs at baseline and two years. After two years a total of 15/36 (42%) presented no radiological progression. More patients with stable radiological course were still receiving anti-TNFalpha treatment after two years (13/15 (87%) v 11/21 (52%); p=0.03) and had lower baseline COMP and sICAM-1 levels (p=0.01 and 0.04, respectively) than those in the group with progressive disease. In a logistic regression model the combination of sustained TNF neutralisation and baseline COMP and sICAM-1 levels was predictive for radiological outcome (p=0.03). C reactive protein and disease activity score area under the curve were significantly correlated with changes in radiological scores after two years (r=0.40 and 0.37, p<0.05). Long term TNFalpha neutralisation decreased the levels of COMP, sICAM, MMPs, and HC gp-39, but not sE-selectin. CONCLUSION: The results suggest that long term monotherapy with anti-TNFalpha has a positive effect on radiological outcome and modulates cartilage and synovium turnover as measured by biological markers. Baseline serum sICAM-1 levels and COMP levels may be helpful to identify patients with progressive or non-progressive radiological outcome.

Barrera P, Joosten LA, den Broeder AA, van de Putte LB, van Riel PL, van den Berg WB. · Department of Rheumatology, University Hospital, Nijmegen, The Netherlands. · Ann Rheum Dis. · Pubmed #11406520 links to  free full text

Abstract: OBJECTIVES: To study the short term effects of a single dose of D2E7, a fully human anti-tumour necrosis factor (TNFalpha) monoclonal antibody (mAb), on the local and systemic homeostasis of interleukin 1beta (IL1beta) and TNFalpha in patients with rheumatoid arthritis (RA). METHODS: All patients with RA enrolled in a phase I, single dose, placebo controlled study with D2E7 in our centre were studied. Systemic cytokine levels, acute phase reactants, and leucocyte counts were studied at days 0, 1, and 14 after the first administration of anti-TNF mAb (n=39) or placebo (n=11). The cellularity and the expression of IL1 and TNFalpha in synovial tissue were studied in knee biopsy specimens obtained at baseline and at day 14 in 25 consenting patients. RESULTS: A single dose of anti-TNF mAb induced a rapid clinical improvement, a decrease in acute phase reaction, and increased lymphocyte counts in patients with active RA. The protein levels of IL1beta in the circulation were low and remained unchanged, but the systemic levels of IL1beta mRNA (p=0.002) and the concentrations of IL1 receptor antagonist (IL1ra) and IL6 (p=0.0001) had already dropped within 24 hours and this persisted up to day 14. Systemic levels of TNFalpha mRNA were low and remained unchanged, though total TNFalpha (free and bound) in the circulation increased after D2E7, probably reflecting the presence of TNF-antiTNF mAb complexes (p<0.005, at days 1 and 14). Both TNF receptors dropped below baseline levels at day 14 (p<0.005). Despite clinical improvement of arthritis, no consistent immunohistological changes were seen two weeks after anti-TNF administration. Endothelial staining for IL1beta tended to decrease in treated patients (p=0.06) but not in responders. The staining for IL1beta and TNFalpha in sublining layers and vessels was mutually correlated (r(s)=0.47 and 0.58 respectively, p<0.0005) and the microscopic scores for inflammation correlated with sublining TNFalpha and IL1beta scores (r(s)=0.65 and 0.54 respectively, p<0.0001), though none of these showed significant changes during the study. CONCLUSIONS: Blocking TNFalpha in RA results in down regulation of IL1beta mRNA at the systemic level and in reduction of the endogenous antagonists for IL1 and TNF and of other cytokines related to the acute phase response, such as IL6, within days. At the synovial level, anti-TNF treatment does not modulate IL1beta and TNFalpha in the short term. The synovial expression of these cytokines does not reflect clinical response to TNF neutralisation.

Abdollahi-Roodsaz S, Joosten LA, Helsen MM, Walgreen B, van Lent PL, van den Bersselaar LA, Koenders MI, van den Berg WB. · Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. · Arthritis Rheum. · Pubmed #19035506 No free full text.

Abstract: OBJECTIVE: Toll-like receptors (TLRs) may activate innate and adaptive immune responses in rheumatoid arthritis (RA) through recognition of microbial as well as endogenous ligands that have repeatedly been found in arthritic joints. The objective of this study was to investigate the involvement of TLR-2 and TLR-4 in the development of chronic destructive streptococcal cell wall (SCW)-induced arthritis, in which interleukin-1 (IL-1)/IL-17-dependent T cell-driven pathologic changes replace the macrophage-driven acute phase. METHODS: Chronic SCW arthritis was induced by 4 repeated intraarticular injections of SCW fragments in wild-type, TLR-2(-/-), and TLR-4(-/-) mice. Clinical, histopathologic, and immunologic parameters of arthritis were evaluated. RESULTS: The TLR-2 dependency of joint swelling during the acute phase was shifted to TLR-4 dependency during the chronic phase. Persistent joint inflammation in the latter phase of the model was significantly suppressed in TLR-4(-/-) mice. In the chronic phase, TLR-4 actively contributed to matrix metalloproteinase (MMP)-mediated cartilage destruction and to osteoclast formation, since the expression of the MMP-specific aggrecan neoepitope VDIPEN and the osteoclast marker cathepsin K was significantly reduced in TLR-4(-/-) mice. Furthermore, TLR-4(-/-) mice expressed less IL-1beta, tumor necrosis factor alpha, IL-6, and IL-23, cytokines that are implicated in IL-17 production. Accordingly, SCW-specific IL-17 production was found to be dependent on TLR-4 activation, since T cells from arthritic TLR-4(-/-) mice produced markedly less IL-17 upon SCW stimulation, whereas interferon-gamma production remained unaffected. CONCLUSION: These data indicate the involvement of TLR-4 in the chronicity and erosive character of arthritis coincident with the antigen-specific IL-17 response. The high position of TLR-4 in the hierarchy of erosive arthritis provides an interesting therapeutic target for RA.

Joosten LA, Heinhuis B, Abdollahi-Roodsaz S, Ferwerda G, Lebourhis L, Philpott DJ, Nahori MA, Popa C, Morre SA, van der Meer JW, Girardin SE, Netea MG, van den Berg WB. · Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands. · Proc Natl Acad Sci U S A. · Pubmed #18574154 links to  free full text

Abstract: The pathogenesis of chronic joint inflammation remains unclear, although the involvement of pathogen recognition receptors has been suggested recently. In the present article, we describe the role of two members of the NACHT-LRR (NLR) family, Nod1 (nucleotide-binding oligomerization domain) and Nod2 in a model of acute joint inflammation induced by intraarticular injection of Streptococcus pyogenes cell wall fragments. Here, we show that Nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, Nod1 gene-deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage, consistent with a model in which Nod1 controls the inflammatory reaction. To explore whether the different function of Nod1 and Nod2 occurs also in humans, we exposed peripheral blood mononuclear cells (PBMCs) carrying either Nod1ins/del or Nod2fs mutation with SCW fragments in vitro. Production of both TNFalpha and IL-1beta was clearly impaired in PBMCs carrying the Nod2fs compared with PBMCs isolated from healthy controls. In line with results in Nod1 gene-deficient mice, PBMCs from individuals bearing a newly described Nod1 mutation produced enhanced levels of proinflammatory cytokines after 24-h stimulation with SCW fragments. These data indicate that the NLR family members Nod1 and Nod2 have different functions in controlling inflammation, and that intracellular Nod1-Nod2 interactions may determine the severity of arthritis in this experimental model. Whether a distorted balance between the function of Nod1 and/or Nod2 is involved in the pathogenesis of human autoinflammatory or autoimmune disease, such as rheumatoid arthritis, remains to be elucidated.

Alten R, Gram H, Joosten LA, van den Berg WB, Sieper J, Wassenberg S, Burmester G, van Riel P, Diaz-Lorente M, Bruin GJ, Woodworth TG, Rordorf C, Batard Y, Wright AM, Jung T. · Department of Internal Medicine II, Rheumatology, Schlosspark-Klinik Teaching Hospital Charité University Medicine Berlin, Heubnerweg, D-14059 Berlin, Germany. · Arthritis Res Ther. · Pubmed #18534016 links to  free full text

Abstract: INTRODUCTION: IL-1beta is a proinflammatory cytokine driving joint inflammation as well as systemic signs of inflammation, such as fever and acute phase protein production. METHODS: ACZ885, a fully human monoclonal antibody that neutralizes the bioactivity of human IL-1beta, was generated to study the potent and long-lasting neutralization of IL-1beta in mechanistic animal models as well as in a proof-of-concept study in patients with rheumatoid arthritis (RA). RESULTS: The mouse IL-1 receptor cross-reacts with human IL-1beta, and it was demonstrated that ACZ885 can completely suppress IL-1beta-mediated joint inflammation and cartilage destruction in mice. This observation prompted us to study the safety, tolerability and pharmacodynamic activity of ACZ885 in RA patients in a small proof-of-concept study--the first to be conducted in humans. Patients with active RA despite treatment with stable doses of methotrexate were enrolled in this dose escalation study. The first 32 patients were split into four cohorts of eight patients each (six were randomly assigned to active treatment and two to placebo). ACZ885 doses were 0.3, 1, 3 and 10 mg/kg, administered intravenously on days 1 and 15. To explore efficacy within 6 weeks of treatment, an additional 21 patients were randomly assigned to the 10 mg/kg cohort, resulting in a total of 20 patients dosed with 10 mg/kg and 15 patients treated with placebo. There was clinical improvement (American College of Rheumatology 20% improvement criteria) at week 6 in the 10 mg/kg treatment group; however, this did not reach statistical significance (P = 0.085). A statistically significant reduction in disease activity score was observed after 4 weeks in the 10 mg/kg group. Onset of action was rapid, because most responders exhibited improvement in their symptoms within the first 3 weeks. C-reactive protein levels decreased in patients treated with ACZ885 within 1 week. ACZ885 was well tolerated. Three patients receiving ACZ885 developed infectious episodes that required treatment. No anti-ACZ885 antibodies were detected during the study. CONCLUSION: ACZ885 administration to methotrexate-refractory patients resulted in clinical improvement in a subset of patients. Additional studies to characterize efficacy in RA and to determine the optimal dose regimen appear warranted. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00619905.

Plater-Zyberk C, Joosten LA, Helsen MM, Koenders MI, Baeuerle PA, van den Berg WB. · Micromet AG, Munich, Germany. · Ann Rheum Dis. · Pubmed #18495731 No free full text.

Abstract: OBJECTIVE: A pathogenic role for granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)17 in rheumatoid arthritis (RA) has been suggested. In previously published work, the therapeutic potentials of GM-CSF and IL17 blockade in arthritis have been described. In the present study, the simultaneous blockade of both pathways in a mouse model for chronic arthritis was investigated to identify whether this double blockade provides a superior therapeutic efficacy. METHODS: A chronic relapsing arthritis was induced in C57Bl/6 wild type (WT) and C57Bl/6 genetically deficient for IL17 receptor (IL17R knockout (KO)) mice by intra-articular injection of Streptococcal cell wall (SCW) fragments into knees on days 0, 7, 14 and 21. Treatments (intraperitoneal) were given weekly starting on day 14. Animals were analysed for inflammation, joint damage and a range of inflammatory mediators. RESULTS: Joint swelling and cartilage damage were significantly reduced in the IL17R KO mice and in WT mice receiving anti-GM-CSF neutralising mAb 22E9 compared to isotype control antibodies. The therapeutic effect was significantly more pronounced in mice where IL17 and GM-CSF pathways were inhibited (eg, IL17R KO mice treated with 22E9 mAb). Tumour necrosis factor (TNF)alpha blockade had essentially no effect. CONCLUSION: Our data further support the therapeutic potentials of GM-CSF and IL17 blockade in a RA model that is no longer responsive to an established TNFalpha antagonist, moreover, our results suggest that concomitant inhibition of both pathways may provide the basis for a highly effective treatment of chronic RA in patients that are resistant to treatment by TNFalpha inhibitors.

Roelofs MF, Wenink MH, Toonen EJ, Coenen MJ, Joosten LA, van den Berg WB, van Riel PL, Radstake TR. · Department of Rheumatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. · J Rheumatol. · Pubmed #18381796 No free full text.

Abstract: OBJECTIVE: To investigate functional consequences of the Toll-like receptor 4 (TLR4) variant (Asp299Gly) in rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells from 28 patients with RA carrying or not carrying the TLR4 variant were incubated with lipopolysaccharide (LPS) and heat shock protein B8 (HSPB8). Concentrations of interleukin 6 (IL-6), tumor necrosis factor-alpha(TNF-alpha), and IL-10 were determined along with TLR4 and CD14 expression. RESULTS: TLR4 expression was similar in patients carrying or not carrying the variant. In contrast, both LPS and HSPB8 resulted in significantly lower secretion of IL-6, TNF-alpha, and IL-10 in those who carried the variant, whereas the frequency of CD14+ cells was higher in these individuals. CONCLUSION: TLR4 variant clearly reduces its potency to mediate signaling. Correction for CD14+ cells is necessary in comparable experiments.

Joosten LA, Abdollahi-Roodsaz S, Heuvelmans-Jacobs M, Helsen MM, van den Bersselaar LA, Oppers-Walgreen B, Koenders MI, van den Berg WB. · Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. · Arthritis Rheum. · Pubmed #18163514 links to  free full text

Abstract: OBJECTIVE: The pathogenesis of rheumatoid arthritis is often linked to bacterial infections. The present study was undertaken to develop a mouse model of chronic destructive arthritis induced by repeated intraarticular (IA) exposure to bacterial cell wall fragments and to investigate the cytokine dependence of this model. METHODS: Mice that were deficient in various cytokines were injected IA with cell wall fragments of Streptococcus pyogenes on days 0, 7, 14, and 21. The development of chronic destructive arthritis was compared between groups of mice lacking different cytokines, to assess which cytokines were crucial for development of chronic destructive arthritis. RESULTS: Repeated exposure of a joint to S pyogenes cell wall fragments resulted in the development of chronic destructive arthritis. In mice deficient in recombination-activating gene 2, streptococcal cell wall (SCW)-directed T cell reactivity was found and chronic arthritis did not develop, implicating T cells in the generation of chronic SCW-induced arthritis. Interleukin-17 (IL-17) receptor-deficient mice showed a reduction of joint destruction in the chronic stage, implicating a detrimental role of the recently discovered IL-17-producing T helper cells (Th17 cells). IL-23 expression was apparent during the late stages of arthritis. Joint swelling was no longer dependent on tumor necrosis factor alpha (TNFalpha) after the last flare, and pronounced cartilage damage was found after 28 days in TNFalpha-deficient mice. In contrast, IL-1beta-deficient mice were fully protected against joint swelling and cartilage and bone destruction during the late stages of disease. CONCLUSION: These findings indicate that the TNFalpha dependence of arthritis is lost during the erosive stage, when Th17 cells become crucial. IL-1beta dependence remains strong, consistent with its pivotal role in the generation of Th17 cells.

Abdollahi-Roodsaz S, Joosten LA, Koenders MI, Devesa I, Roelofs MF, Radstake TR, Heuvelmans-Jacobs M, Akira S, Nicklin MJ, Ribeiro-Dias F, van den Berg WB. · Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. · J Clin Invest. · Pubmed #18060042 links to  free full text

Abstract: TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.

Geurts J, Arntz OJ, Bennink MB, Joosten LA, van den Berg WB, van de Loo FA. · Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. · Gene Ther. · Pubmed #17851546 No free full text.

Abstract: The application of disease-regulated promoters in local gene therapy for rheumatoid arthritis potentiates the development of a sophisticated treatment that relies on a restricted and fine-tuned supply of biologicals. Although several studies have investigated regulated promoters for achieving effective transgene expression during arthritis, none have explored their potential for minimizing deleterious effects arising from constitutive overexpression of transgenes under naive conditions. Using naive and collagen-induced arthritic mice, we examined the applicability of a hybrid interleukin-1 enhancer/interleukin-6 proximal promoter for achieving efficacious murine interleukin-4 gene therapy under arthritic conditions, while minimizing interleukin-4-induced inflammation under naive conditions. We found strong upregulation of transgene expression in virally transduced knee joints under arthritic conditions compared to levels in naive animals. Besides its responsiveness, the promoter strength proved sufficient for generating therapeutically efficacious levels interleukin-4, as demonstrated by the successful protection against cartilage erosion in collagen-induced arthritis. Most importantly, promoter-mediated restriction of the potent chemotactic interleukin-4 in naive animals strongly reduced the amounts of inflammatory cell influx. This study suggests the suitability of the interleukin-1 enhancer/interleukin-6 proximal promoter for the development of a local gene therapy strategy for rheumatoid arthritis that requires fine-tuned and restricted expression of transgenes with a pleiotrophic nature.

Abdollahi-Roodsaz S, Joosten LA, Roelofs MF, Radstake TR, Matera G, Popa C, van der Meer JW, Netea MG, van den Berg WB. · Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. · Arthritis Rheum. · Pubmed #17763416 links to  free full text

Abstract: OBJECTIVE: Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll-like receptor 4 (TLR-4). The objective of this study was to investigate the involvement of TLR-4 activation in the development and progression of autoimmune destructive arthritis. METHODS: A naturally occurring TLR-4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen-induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin-1 receptor antagonist (IL-1Ra) gene deficiency were treated with TLR-4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL-1 in joints were evaluated after treatment. RESULTS: The TLR-4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR-4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR-4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti-type II collagen immunity crucial for this model. Treatment with TLR-4 antagonist strongly reduced IL-1beta expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL-1-mediated autoimmune arthritis in IL-1Ra(-/-) mice and protected the mice against cartilage and bone pathology. CONCLUSION: In the present study, we demonstrate for the first time that inhibition of TLR-4 suppresses the severity of experimental arthritis and results in lower IL-1 expression in arthritic joints. Our data suggest that TLR-4 might be a novel target in the treatment of rheumatoid arthritis.

Roelofs MF, Boelens WC, Joosten LA, Abdollahi-Roodsaz S, Geurts J, Wunderink LU, Schreurs BW, van den Berg WB, Radstake TR. · Department of Rheumatology Research and Advanced Therapeutics, Nijmegen Center for Molecular Life Sciences, The Netherlands. · J Immunol. · Pubmed #16709864 links to  free full text

Abstract: Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant alphaA crystallin, alphaB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both alphaA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP alphaA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.

Koenders MI, Lubberts E, van de Loo FA, Oppers-Walgreen B, van den Bersselaar L, Helsen MM, Kolls JK, Di Padova FE, Joosten LA, van den Berg WB. · Experimental Rheumatology and Advanced Therapeutics, Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB Nijmegen, the Netherlands. · J Immunol. · Pubmed #16670337 links to  free full text

Abstract: The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-alpha. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-alpha-deficient mice that TNF-alpha is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/BxN serum transfer arthritis to a similar degree in TNF-alpha-deficient mice and their wild-type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17-enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions.

Ostendorf B, Scherer A, Wirrwar A, Hoppin JW, Lackas C, Schramm NU, Cohnen M, Mödder U, van den Berg WB, Müller HW, Schneider M, Joosten LA. · Heinrich-Heine University Düsseldorf, Dusseldorf, Germany. · Arthritis Rheum. · Pubmed #16572444 links to  free full text

Abstract: OBJECTIVE: To image inflammatory arthritic lesions in experimental arthritis and in patients with arthritis, using a newly developed high-resolution multipinhole single-photon-emission computed tomography (MPH-SPECT) technique. METHODS: Six interleukin-1 receptor antagonist-deficient mice with arthritis of the front and back paws and 2 control BALB/c mice were imaged with MPH-SPECT and scored macroscopically for arthritis. SPECT imaging was performed with a conventional gamma camera upgraded with a pyramidal lead collimator affixed with MPH apertures. All images were reconstructed, and uptake in the paws was quantified in counts/weight and injected activity. To transfer the imaging technique to humans we examined the clinically dominant hand of 6 individuals (3 with established rheumatoid arthritis [RA], 1 with early RA, 1 with osteoarthritis, and 1 healthy control). RESULTS: MPH-SPECT images were high-resolution 3-dimensional tomographic images, which allowed exact localization and quantifiable observation of increased bone metabolism. MPH-SPECT counts of inflamed joints in mice correlated with macroscopic scoring and histologic joint analysis postmortem. In humans, MPH-SPECT images depicted a detailed visualization of tracer accumulation in bony structures of hand and finger joints, and were also capable of imaging increased bone metabolism that had appeared normal with other imaging modalities, e.g., magnetic resonance imaging. CONCLUSION: The MPH-SPECT technique represents a new diagnostic tool in the detection of bone pathology in small-animal arthritis research. Compared with macroscopic scoring, this new method provides a more objective and higher-precision quantifiable measurement of bone reaction, allowing visualization of inflammatory processes of the whole skeleton in vivo. These results suggest that MPH-SPECT may be useful as a diagnostic instrument for monitoring experimental arthritis, with further potential for use in human studies of RA.

Joosten LA, Netea MG, Kim SH, Yoon DY, Oppers-Walgreen B, Radstake TR, Barrera P, van de Loo FA, Dinarello CA, van den Berg WB. · Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, The Netherlands. · Proc Natl Acad Sci U S A. · Pubmed #16492735 links to  free full text

Abstract: IL-32 is a recently discovered cytokine that induces TNFalpha, IL-1beta, IL-6, and chemokines. We investigated whether IL-32 is expressed in the synovia of patients with rheumatoid arthritis (RA) and studied associations with disease severity and the presence of other cytokines. Immunohistochemistry revealed that IL-32 is highly expressed in RA synovial tissue biopsies, whereas IL-32 was not observed in synovial tissues from patients with osteoarthritis. Moreover, in synovial biopsies from 29 RA patients with active disease, the level of IL-32 staining correlated with erythrocyte sedimentation rate, a marker of systemic inflammation (R = 0.63 and P < 0.0003). Synovial staining of IL-32 also correlated with indices of synovial inflammation (R = 0.80 and P < 0.0001) as well as synovial presence of TNFalpha (R = 0.68 and P < 0.004), IL-1beta (R = 0.79 and P < 0.0001), and IL-18 (R = 0.82 and P < 0.001). IL-32 was a potent inducer of prostaglandin E(2) release in mouse macrophages and human blood monocytes, an important property for inflammation. After the injection of human IL-32gamma into the knee joints of naïve mice, joint swelling, with pronounced influx of inflammatory cells and cartilage damage, was observed. In TNFalpha-deficient mice, IL-32-driven joint swelling was absent and cell influx was markedly reduced, but loss of proteoglycan was unaffected, suggesting that IL-32 activity is, in part, TNFalpha-dependent. IL-32, strongly associated with TNFalpha, IL-1beta, and IL-18, appears to play a role in human RA and may be a novel target in autoimmune diseases.

Roelofs MF, Joosten LA, Abdollahi-Roodsaz S, van Lieshout AW, Sprong T, van den Hoogen FH, van den Berg WB, Radstake TR. · Radboud University Nijmegen Medical Centre, The Netherlands. · Arthritis Rheum. · Pubmed #16052591 links to  free full text

Abstract: OBJECTIVE: To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls. METHODS: Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam(3)Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients. RESULTS: TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2- and TLR-4-mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFalpha and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3-7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients. CONCLUSION: TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.

Koenders MI, Lubberts E, Oppers-Walgreen B, van den Bersselaar L, Helsen MM, Di Padova FE, Boots AM, Gram H, Joosten LA, van den Berg WB. · Department of Rheumatology, Experimental Rheumatology and Advanced Therapeutics, Radboud University Nijmegen Medical Center, 189, Geert Grooteplein Zuid 26-28, PO Box 9101, 6500 HB Nijmegen, The Netherlands. · Am J Pathol. · Pubmed #15972960 links to  free full text

Abstract: Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.

Radstake TR, Roelofs MF, Jenniskens YM, Oppers-Walgreen B, van Riel PL, Barrera P, Joosten LA, van den Berg WB. · University Medical Center Nijmegen, Nijmegen, The Netherlands. · Arthritis Rheum. · Pubmed #15593217 links to  free full text

Abstract: OBJECTIVE: To study the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals. METHODS: Synovial tissue specimens from 29 RA patients were stained for TLR-2, TLR-4, and proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-12, IL-17, IL-18, and tumor necrosis factor alpha [TNFalpha]). The expression of TLR-2, TLR-4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL-12 and IL-18, and TLR expression was assessed using fluorescence-activated cell sorter analysis. Production of TNFalpha and IL-6 was measured using Luminex bead array technology. RESULTS: In RA synovial tissue, the expression of TLR-2 was slightly higher than that of TLR-4. Interestingly, both TLR-2 and TLR-4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL-12 and IL-18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL-12 and IL-18 resulted in increased expression of both TLR-2 and TLR-4; this could be blocked with anti-interferon-gamma (anti-IFNgamma) antibodies, suggesting a role for IFNgamma. Lipopolysaccharide- or lipoteichoic acid-mediated triggering of PBMCs incubated with IL-12/IL-18 or IFNgamma led to an increased production of both TNFalpha and IL-6, indicating the functionality of TLR-2 and TLR-4. CONCLUSION: TLR-2 and TLR-4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL-12 and IL-18. The synergistic effect of IL-12 and IL-18 on T cell IFNgamma production seems to regulate expression of TLR-2 and TLR-4 in the synovial tissue of RA patients.

Joosten LA, Smeets RL, Koenders MI, van den Bersselaar LA, Helsen MM, Oppers-Walgreen B, Lubberts E, Iwakura Y, van de Loo FA, van den Berg WB. · Rheumatology Research Laboratory and Advanced Therapeutics, University Medical Center Nijmegen, Nijmegen, The Netherlands. · Am J Pathol. · Pubmed #15331419 links to  free full text

Abstract: Interleukin (IL)-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects and is a pivotal cytokine for the development of Th1 responses. The goal of the present study was to investigate whether IL-18 induces joint inflammation and joint destruction directly or via induction of other cytokines such as IL-1 and tumor necrosis factor (TNF). To this end we performed both in vitro and in vivo kinetic studies. For in vivo IL-18 exposure studies C57BL/6, TNF-deficient, and IL-1-deficient mice were injected intra-articularly with 1.10(7) pfu mIL-18 adenovirus followed by histopathological examination. Local overexpression of IL-18 resulted in pronounced joint inflammation and cartilage proteoglycan loss in control mice. Of high interest, IL-18 gene transfer in IL-1-deficient mice did not show cartilage damage, although joint inflammation was similar to that in wild-type animals. Overexpression of IL-18 in TNF-deficient mice showed that TNF was partly involved in IL-18-induced joint swelling and influx of inflammatory cells, but cartilage proteoglycan loss occurred independent of TNF. In vitro cartilage degradation by IL-18 was found after a 72-hour culture period. Blocking of IL-1 with IL-1Ra or an ICE-inhibitor resulted in complete protection against IL-18-mediated cartilage degradation. The present study demonstrated that IL-18 induces joint inflammation independently of IL-1. In addition, we showed that IL-1beta generation, because of IL-18 exposure, was essential for marked cartilage degradation both in vitro and in vivo. These findings implicate that IL-18, in contrast to TNF, contributes through separate pathways to joint inflammation and cartilage destruction.

Vossenaar ER, van Boekel MA, van Venrooij WJ, López-Hoyoz M, Merino J, Merino R, Joosten LA. · University of Nijmegen, Nijmegen, The Netherlands. · Arthritis Rheum. · Pubmed #15248238 links to  free full text

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