ARD Online First, published on August 11, 2005 as 10.1136/ard.2005.037929 - 1 - Ann Rheum Dis: first published as 10.1136/ard.2005.037929 on 11 August 2005. Downloaded from Expression of Jak3, STAT1, STAT4 and STAT6 in inflammatory arthritis: Unique Jak3 and STAT4 expression in dendritic cells in seropositive rheumatoid arthritis JG Walker MBBS, FRACP MJ Ahern MBBS, MD, FRACP M Coleman MBBS, FRACP H Weedon V Papangelis B.Appl.Sci. D Beroukas B.Sc. PJ Roberts-Thomson MBBS, PhD, FRACP MD Smith MBBS, PhD, FRACP Supported by the Daw Park Research Foundation. Dr Walker’s work is supported by the National Health and Medical Research Council of Australia and the Arthritis Foundation of Australia. Address reprint requests to Jennifer Walker MBBS, FRACP, Repatriation General Hospital, Daws Rd, Daw Park 5041 South Australia. E-mail: [email protected] Ph: 08 8276 9666 http://ard.bmj.com/ Keywords: dendritic cells, rheumatoid arthritis, transcription factors. "The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its on September 30, 2021 by guest. Protected copyright. Licensees to permit this article (if accepted) to be published in Annals of the Rheumatic Diseases editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://ard.bmjjournals.com/misc/ifora/licenceform.shtml)." Copyright Article author (or their employer) 2005. Produced by BMJ Publishing Group Ltd (& EULAR) under licence. - 2 - Ann Rheum Dis: first published as 10.1136/ard.2005.037929 on 11 August 2005. Downloaded from Objective. Modulation of Jak-STAT signalling may provide an effective therapeutic strategy in inflammatory arthritis (IA). We document Jak-STAT expression in a cohort of patients with active rheumatoid arthritis (RA), spondyloarthropathies (SpA) and osteoarthritis (OA) and compare these subsets with normal synovial tissue. Methods. Synovial tissue biopsies from patients with RA, OA and SpA and histologically normal tissue (n=10 in each arthritis group) were examined for the presence of Jak3, STAT1, STAT4 and STAT6 expression using immunohistochemistry. Phenotyping was performed using immunohistochemistry and immunofluorescence. Clinical and serological characteristics of RA patients expressing Jak3-STAT4 were assessed. Results. STAT1, STAT4 and Jak3 protein expression was generally increased in IA. In contrast, STAT6 expression was relatively heterogeneous. A subpopulation of CD1a positive dendritic cells unique to seropositive RA patients was detected. These cells showed intense protein expression for Jak3, STAT4 and STAT6. Conclusion. CD1a positive dendritic cells intensely express Jak3, STAT4 and STAT6 in seropositive RA tissue and may be an alternative marker for dendritic cells in their early stages of activation as well as providing a tool for identifying RA at the level of the synovium. Jak3 inhibition may be a potential therapeutic target to prevent dendritic cell maturation in RA. STAT1 expression is increased in inflammatory arthritis, suggesting that its pro-apoptotic and anti-inflammatory effects are unable to effectively counteract inflammation. STAT6 expression is heterogeneous in synovium, suggesting a possible homeostatic role in addition to any anti-inflammatory effects. http://ard.bmj.com/ on September 30, 2021 by guest. Protected copyright. - 3 - Ann Rheum Dis: first published as 10.1136/ard.2005.037929 on 11 August 2005. Downloaded from Rheumatoid arthritis (RA) is a chronic inflammatory disease, primarily of synovium[1]. Cytokines have important roles in perpetuating inflammation, particularly macrophage- derived tumour necrosis factor alpha (TNFα) and interleukin-1 (IL-1). Antibodies against TNFα and the use of IL-1 receptor antagonist have successfully suppressed inflammation in many RA patients but only 60% of patients will obtain a partial response and a minority experience no benefit[2]. The study of transcription factor pathways may provide an explanation for the variable responses to IL-1 and TNFα antagonists. The smaller molecules in these pathways make an attractive alternative therapeutic target because pharmaceuticals have the potential to be orally bioavailable. In addition, only a few inducible factors seem to play a pivotal role in the regulation of inflammatory genes (e.g. activator protein-1 (AP-1), CCAT/enhancer-binding proteins (C/EBP’s), STATs, NF-AT (nuclear factor of activated T cells) and NF-κΒ (nuclear factor-κΒ)[3]. The janus kinase and Signal Transducer and Activator of Transcription (Jak-STAT) pathway is the signalling target of a multitude of cytokines, including IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12 and IL-15, all of which are thought to have biologically significant roles in rheumatoid synovial inflammation[4, 5]. There are seven identified STATs and preliminary work in human synovial tissue suggests that 1. STAT1 expression and activity are increased in RA synovium, at least in those with active disease[6, 7], 2. STAT3 promotes survival of RA synovial fibroblasts[8], 3. STAT4 is expressed in RA synovium[9] and 4. IL-4 STAT mRNA (IL-4 primarily signals through STAT6) is increased in RA tissue[10]. In addition, murine arthritis knockout models suggest that IL-12, signalling through a http://ard.bmj.com/ STAT4 pathway is required for the development of proteoglycan-induced inflammatory arthritis while IL-4, signalling through STAT6, has an anti-inflammatory effect[11]. Generally STATs confer signalling specificity, while Jaks are more widely expressed but Jak3 expression is mostly limited to haematopoietic cell lines and as such, it is a potential on September 30, 2021 by guest. Protected copyright. therapeutic target in patients with autoimmune disease. There is no published work investigating Jak3 expression in human synovial tissue although it has been identified as a potential therapeutic target in autoimmune disease. The immunomodulatory effects of Jak3 extend beyond its role in STAT activation as it is (i) phosphorylated during dendritic cell activation through the CD40-CD154 ligand [12] and (ii) phosphorylated upon T cell receptor (TCR) stimulation in murine models and therefore may play a direct role in T cell activation[13]. With the exception of STAT1, most published research investigating Jak-STAT in inflammatory arthritis has been limited to work in synovial fibroblasts or tissue obtained at the time of joint replacement surgery, often without detailed information regarding clinical activity. Therefore, results must be interpreted with caution because the clinical and histological features of RA alter significantly depending on disease activity[14]. In addition, work in synovial fibroblasts cannot be extrapolated to macrophages or - 4 - Ann Rheum Dis: first published as 10.1136/ard.2005.037929 on 11 August 2005. Downloaded from lymphocytes, with both of these cell populations likely to have important pathogenic roles in RA. We hypothesized that Jak-STAT would be differentially expressed in RA compared with other forms of arthritis and now report on the protein expression of STAT1, STAT4, STAT6 and Jak3 in a series of patients with RA, spondyloarthritis (SpA), osteoarthritis (OA) and normal controls using synovial tissue taken from patients with clinically active disease. MATERIALS AND METHODS Patients. Synovial tissue was obtained from 38 subjects, including 10 patients with RA, 10 patients with spondyloarthritis(SpA), 10 patients with osteoarthritis (OA) and 8 control subjects. Control specimens were obtained from patients undergoing knee or ankle joint arthroscopy for undiagnosed joint pain, who were observed to have non- inflamed synovium at the time of arthroscopy and on subsequent histological analysis. SpA subjects consisted of 5 patients with undifferentiated SpA, 4 Psoriatic arthritis and 1 Ankylosing spondylitis. The diagnoses of RA and OA conformed to 1987 American College of Rheumatology criteria[15, 16]. Tables 1 and 2 summarise clinical details of individual RA and SpA patients, and provides conglomerate details of OA and normal control patients. All studies were approved by the Ethics Committee of the Repatriation General Hospital. Arthroscopic Biopsies. Synovial membrane samples were obtained from involved joints (knee, wrist or ankle) of patients with RA, SpA , and OA under direct vision using a 2.7mm mini-arthroscope (Dyonics, Andover, MA, USA) and standard approaches as previously described[17]. Normal specimens were obtained from patients undergoing http://ard.bmj.com/ arthroscopy for undiagnosed joint pain at a sports medicine facility who were observed to have non-inflamed synovium at the time of arthroscopy and on subsequent histological analysis. Samples were frozen in Tissue Tek O.C.T. compound (Miles Diagnostics) and stored at -80°C. Immunohistochemistry. Antibodies are listed in Table 3. Cryosections (4µm) were on September 30, 2021 by guest. Protected copyright. fixed in ice-cold acetone for 4 minutes. Sections were brought to room temperature, washed in phosphate buffered saline (PBS) and endogenous peroxidase activity was inhibited using 1%H202 and 0.1% Sodium Azide in PBS. Primary antibody was applied for 2 hours (1.5 hours for STAT1) at room temperature. (Control sections incubated with diluent PBS/1%BSA). HRP-conjugated goat anti-rabbit antibody was applied for 30 minutes, followed by HRP-conjugated swine anti-goat for 30