Rheumatoid Arthritis: Reedquist KA

Grabiec AM, Tak PP, Reedquist KA. · Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. · Arthritis Res Ther. · Pubmed #18983693 links to  free full text

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

Tas SW, Remans PH, Reedquist KA, Tak PP. · Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, 1100DD Amsterdam, The Netherlands. · Curr Pharm Des. · Pubmed #15720277 No free full text.

Abstract: Many chronic inflammatory diseases are associated with deregulated intracellular signal transduction pathways. Resultant pathogenic interactions between immune and stromal cells lead to changes in cell activation, proliferation, migratory capacity, and cell survival that all contribute to inflammation. Increasing efforts are now being made in the design of novel therapeutic compounds to interfere with signaling pathways in inflammatory diseases like rheumatoid arthritis (RA). In this review we will outline the major signal transduction pathways involved in the pathogenesis of RA. We will assess advances in targeting a number of key intracellular pathways, including nuclear factor-(kappa)B (NF-(kappa)B), mitogen-associated protein kinases (MAPKs), phosphoinositide 3-kinase (PI3K)/Akt, signal transducers and activators of transcription (STATs), and reactive oxygen species (ROS) production. Finally, we will discuss recently identified lead molecules and the progress of selected compounds towards becoming new drugs for the treatment of inflammatory diseases.

Abreu JR, Grabiec AM, Krausz S, Spijker R, Burakowski T, Maslinski W, Eldering E, Tak PP, Reedquist KA. · Academic Medical Center, University of Amsterdam, The Netherlands. · J Immunol. · Pubmed #19525395 No free full text.

Abstract: Genetic associations and the clinical success of compounds targeting TCR costimulatory proteins suggest an active role for TCR signaling in the initiation and perpetuation of rheumatoid arthritis (RA). Paradoxically, T cells isolated from affected joints in RA show impaired proliferative and cytokine responses following stimulation with mitogens and recall Ags attributed in part to chronic T cell exposure to oxidative stress and inflammatory cytokines. Therefore, it is uncertain how local autoreactive TCR signaling contributes to pathology in established RA. Using single-cell analysis, we show that in contrast to results obtained in bulk culture assays, T cells from the synovial fluid of RA patients proliferate and produce cytokines (IL-2, TNF-alpha, and IFN-gamma) as efficiently, if not more so, than T cells isolated from healthy donors and RA patient peripheral blood following TCR/CD28 stimulation. RA synovial fluid T cell hyporesponsiveness observed in bulk cultures can be attributed to spontaneous apoptosis ex vivo, which is associated with altered ratios of proapoptotic Noxa and anti-apoptotic Mcl-1 expression. The absence of RA synovial T cell proliferation and cytokine production in situ, despite the capacity of these cells to support productive TCR signaling, suggests that T cells contribute to local pathology in established RA by TCR-independent mechanisms.

Vergunst CE, Gerlag DM, Dinant H, Schulz L, Vinkenoog M, Smeets TJ, Sanders ME, Reedquist KA, Tak PP. · Division of Clinical Immunology and Rheumatology, Academic Medical Centre/University of Amsterdam, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. · Rheumatology (Oxford). · Pubmed #17965442 links to  free full text

Abstract: OBJECTIVES: All complement pathways lead to the formation of C5a, which is believed to contribute to the influx and activation of C5a-receptor (C5aR) bearing cells into the joints of patients with rheumatoid arthritis (RA). Studies in animal models of RA have suggested therapeutic potential of C5aR blockade. In this study, we examined the effects of the C5aR blockade on synovial inflammation in RA patients. METHODS: We performed a double-blind, placebo-controlled study using an orally administered C5aR-antagonist. Twenty-one patients with active RA were randomized 2:1 to treatment with a C5aR-antagonist AcF- (OpdChaWR) (PMX53) vs placebo for 28 days. Serum concentrations of PMX53 were determined. Synovial tissue was obtained at baseline and after 28 days of treatment for pharmacodynamic analysis using immunohistochemistry and digital image analysis. RESULTS: All patients completed the study. Areas under the curve (AUCs) of PMX53 in patients' blood samples showed a mean of 40.8 nmol h/l. There was neither decrease in cell infiltration, nor changes in key biomarkers associated with clinical efficacy after active treatment. In addition, there was no trend towards clinical improvement in the C5aR-antagonist-treated group compared with placebo nor was there a correlation between the AUC and clinical response. CONCLUSIONS: Treatment with PMX53 did not result in a reduction of synovial inflammation despite reaching serum levels of PMX53 that block C5aR-mediated cell activation in vitro. The data suggest that C5aR blockade does not result in reduced synovial inflammation in RA patients.

Ludikhuize J, de Launay D, Groot D, Smeets TJ, Vinkenoog M, Sanders ME, Tas SW, Tak PP, Reedquist KA. · Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. · Arthritis Rheum. · Pubmed #17599731 links to  free full text

Abstract: OBJECTIVE: Phosphatidylinositol 3-kinase-dependent activation of protein kinase B (PKB) has been observed in rheumatoid arthritis (RA) synovial tissue, and mechanisms that interfere with this process are protective in animal models of arthritis. PKB can regulate cell survival and proliferation via phosphorylation-dependent inactivation of forkhead box class O (FoxO) transcription factors. The present study was undertaken to examine whether FoxO transcription factors are differentially inactivated in RA synovial tissue, and whether this inactivation correlates with laboratory and clinical parameters of disease activity. METHODS: The expression and phosphorylation of FoxO family members were assessed in synovial biopsy tissue from 12 patients with RA and 9 patients with inflammatory osteoarthritis (OA), by immunohistochemistry and quantitative computer-assisted image analysis. Immunoblotting was used to assess the interleukin-1beta (IL-1beta)- and tumor necrosis factor alpha (TNFalpha)-induced phosphorylation of FoxO1 and FoxO4 in cultured fibroblast-like synoviocytes (FLS) and macrophages. RESULTS: FoxO1, FoxO3a, and FoxO4 were expressed and phosphorylated in synovial tissue from both RA patients and OA patients. In RA synovial tissue, phosphorylation of FoxO1 was observed in both FLS and macrophages, FoxO3a in T lymphocytes, and FoxO4 in macrophages alone. Following stimulation with IL-1beta and TNFalpha, FoxO1 and FoxO4 were phosphorylated in both RA and OA FLS and synovial macrophages, respectively. Inactivation of FoxO4 was significantly enhanced in the RA as compared with the OA synovial sublining. There was a strong negative correlation between inactivation of FoxO4 in RA synovial tissue and increased serum C-reactive protein levels and a raised erythrocyte sedimentation rate in RA patients. CONCLUSION: All 3 FoxO family members examined were phosphorylated in both RA and OA synovial tissue; in particular, inactivation of FoxO4 was significantly enhanced in macrophages from RA synovial tissue. Thus, cell-specific inactivation of FoxO family members appears to differentially regulate cell survival and proliferation in the RA synovium.

Reedquist KA, Ludikhuize J, Tak PP. · Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Room K0-140, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. · Biochem Soc Trans. · Pubmed #17052183 No free full text.

Abstract: Although the mechanisms leading to the induction of RA (rheumatoid arthritis) are poorly understood, improper activation, proliferation, survival and retention of neutrophils, macrophages, lymphocytes and other leucocytes contribute to perpetuation of inflammation and eventual joint destruction through activation of stromal fibroblast-like synoviocytes. Fundamental studies in developmental biology, cellular biology and immunology have established critical roles for PI3K (phosphoinositide 3-kinase) signal transduction pathways in cellular chemotactic responses, proliferation, apoptosis and survival. Despite profound alteration of these cellular processes in RA, involvement of PI3K signalling pathways in this chronic inflammatory disease, and their assessment as potential therapeutic targets, has until recently received scant attention. This review highlights recent advances in our understanding of PI3K signalling pathways, in particular regulation of FoxO (forkhead box O) transcription factors, and their relevance to RA.

Remans PH, Wijbrandts CA, Sanders ME, Toes RE, Breedveld FC, Tak PP, van Laar JM, Reedquist KA. · Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. · Arthritis Rheum. · Pubmed #17009234 links to  free full text

Abstract: OBJECTIVE: Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes. METHODS: ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS-dependent fluorescence of 6-carboxy-2',7'-dichlorofluorescein. Rap1 GTPase activation was assessed by activation-specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by 3H-thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA-4Ig fusion proteins. Short- and long-term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti-CD28 antibodies. RESULTS: T lymphocyte ROS production and Rap1 inactivation were mediated by cell-cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4-Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti-CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production. CONCLUSION: Cell-cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4-Ig fusion protein.

Remans PH, van Oosterhout M, Smeets TJ, Sanders M, Frederiks WM, Reedquist KA, Tak PP, Breedveld FC, van Laar JM. · Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. · Arthritis Rheum. · Pubmed #15986371 links to  free full text

Abstract: OBJECTIVE: To investigate the cellular and molecular sources of oxidative stress in patients with rheumatoid arthritis (RA) through analysis of the production of reactive oxygen species (ROS) in synovium. METHODS: Cytochemical procedures based on the 3,3'-diaminobenzidine (DAB)-Mn2+ deposition technique were used on unfixed cryostat sections of synovium from RA patients and rheumatic disease controls. For immunophenotyping, sections were incubated, fixed, and stained with fluorescein isothiocyanate-labeled antibodies. Fluorescence-activated cell sorter analysis of the ROS-reactive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) was used to measure intracellular ROS in T lymphocytes from peripheral blood and synovial fluid. To determine which enzymes produced ROS, different inhibitors were tested. RESULTS: Large quantities of DAB precipitated in the majority of RA synovial T lymphocytes, indicative of intracellular ROS production. These ROS-producing T lymphocytes were observed throughout the synovium. Polymerization of DAB was observed to a lesser extent in other forms of chronic arthritis, but was absent in osteoarthritis. DAB staining of cytospin preparations of purified RA synovial fluid T cells confirmed the presence of ROS-producing cells. One of the ROS involved appeared to be H2O2, since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H2O2, diphenyleneiodonium (an inhibitor of NADPH oxidase), N(G)-monomethyl-L-arginine (an inhibitor of nitric oxide synthesis), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and rotenone (an inhibitor of mitochondrial ROS production) failed to suppress ROS production. CONCLUSION: Our findings show that chronic oxidative stress observed in synovial T lymphocytes is not secondary to exposure to environmental free radicals, but originates from intracellularly produced ROS. Additionally, our data suggest that one of the intracellularly generated ROS is H2O2, although the oxidase(s) involved in its generation remains to be determined.

Remans PH, Gringhuis SI, van Laar JM, Sanders ME, Papendrecht-van der Voort EA, Zwartkruis FJ, Levarht EW, Rosas M, Coffer PJ, Breedveld FC, Bos JL, Tak PP, Verweij CL, Reedquist KA. · Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. · J Immunol. · Pubmed #15240679 links to  free full text

Abstract: Transient production of reactive oxygen species (ROS) plays an important role in optimizing transcriptional and proliferative responses to TCR signaling in T lymphocytes. Conversely, chronic oxidative stress leads to decreased proliferative responses and enhanced transcription of inflammatory gene products, and is thought to underlie the altered pathogenic behavior of T lymphocytes in some human diseases, such as rheumatoid arthritis (RA). Although the signaling mechanisms regulating ROS production in T lymphocytes has not been identified, activation of the small GTPase Ras has been shown to couple agonist stimulation to ROS production in other cell types. We find that Ras signaling via Ral stimulates ROS production in human T lymphocytes, and is required for TCR and phorbol ester-induced ROS production. The related small GTPase Rap1 suppresses agonist, Ras and Ral-dependent ROS production through a PI3K-dependent pathway, identifying a novel mechanism by which Rap1 can distally antagonize Ras signaling pathways. In synovial fluid T lymphocytes from RA patients we observed a high rate of endogenous ROS production, correlating with constitutive Ras activation and inhibition of Rap1 activation. Introduction of dominant-negative Ras into synovial fluid T cells restored redox balance, providing evidence that deregulated Ras and Rap1 signaling underlies oxidative stress and consequent altered T cell function observed in RA.