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1 UvA-DARE (Digital Academic Repository) Clinical studies and tissue analyses in the earliest phases of rheumatoid arthritis: In search of the transition from being at risk to having clinically apparent disease de Hair, M.J.H. Link to publication Citation for published version (APA): de Hair, M. J. H. (2013). Clinical studies and tissue analyses in the earliest phases of rheumatoid arthritis: In search of the transition from being at risk to having clinically apparent disease General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 22 Dec 2017

2 CLINICAL STUDIES AND TISSUE ANALYSES IN THE EARLIEST PHASES OF RHEUMATOID ARTHRITIS: IN SEARCH OF THE TRANSITION FROM BEING AT RISK TO HAVING CLINICALLY APPARENT DISEASE Maria Johanna Helena de Hair

3 ISBN: Layout and Printing: Off Page, Cover: Rosemieke de Hair, Copyright 2013 by M.J.H. de Hair. All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without prior permission of the author.

4 CLINICAL STUDIES AND TISSUE ANALYSES IN THE EARLIEST PHASES OF RHEUMATOID ARTHRITIS: IN SEARCH OF THE TRANSITION FROM BEING AT RISK TO HAVING CLINICALLY APPARENT DISEASE ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op vrijdag 17 mei 2013, te 12:00 uur door Maria Johanna Helena de Hair geboren te Eindhoven

5 PROMOTIECOMMISSIE: Promotor: Prof. dr. P.P. Tak Copromotor: Dr. E.G.M. van Baarsen Overige leden: Prof. dr. S. Florquin Prof. dr. J.M.W. Hazes Prof. dr. B.O. Roep Prof. dr. J.A. Romijn Prof. dr. C.J.M. de Vries Faculteit der Geneeskunde

6 CONTENTS Chapter 1 General introduction 7 PART I Chapter 2 Chapter 3 PART II Chapter 4 Chapter 5 Chapter 6 PART III Chapter 7 Chapter 8 Chapter 9 CLINICAL ASPECTS DURING THE EARLIEST STAGES OF RA 21 The clinical picture of rheumatoid arthritis according to the 2010 ACR/EULAR criteria: Is this still the same disease? 23 Smoking and overweight determine the likelihood of developing rheumatoid arthritis 35 THE SYNOVIUM DURING THE EARLIEST STAGES OF RA 49 Experience of patients undergoing mini-arthroscopy compared to MRI in the earliest phases of arthritis 51 Features of the synovium of individuals at risk of developing rheumatoid arthritis: implications for understanding preclinical rheumatoid arthritis 61 Expression of prostaglandin E 2 enzymes in the synovium of arthralgia patients at risk of rheumatoid arthritis and in early arthritis patients 77 THE IMMUNE RESPONSE IN DIFFERENT COMPARTMENTS DURING THE EARLIEST STAGES OF RA 93 Hunting for the pathogenesis of rheumatoid arthritis: Core-needle biopsy of inguinal lymph nodes as a new research tool 95 The cellular composition of lymph nodes in the earliest phase of inflammatory arthritis 101 Inflamed target tissue provides a specific niche for highly expanded T-cell clones in early human autoimmune disease 111 Chapter 10 General discussion 137 Addendum English summary 157 Nederlandse samenvatting 161 Curriculum vitae 167 Dankwoord 169 List of publications 173

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8 GENERAL INTRODUCTION 1 Parts of this chapter have been adapted from: Maria J.H. de Hair*, Leonard C. Harty*, Danielle M. Gerlag, Constantino Pitzalis, Douglas J. Veale, and Paul P. Tak *Both authors contributed equally Synovial tissue analysis for the discovery of diagnostic and prognostic biomarkers in patients with early arthritis J Rheumatol 2011;38:

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10 GENERAL INTRODUCTION RHEUMATOID ARTHRITIS Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by pain, swelling and stiffness of the joints due to synovial inflammation. Inflammation of the synovium is the hallmark of the disease. In socioeconomic terms, RA is the most common and most important of the inflammatory arthritides. The inflamed synovium expands into and destroys the underlying cartilage and bone resulting in irreversible erosion of the bone and eventually in loss of normal joint architecture and disability 1. It is a debilitating chronic erosive disease that affects 1-2% of the population worldwide and affects females three times more than males. It is associated with an increased incidence of cardiovascular morbidity and mortality 2. The management of RA includes both drug therapy and non-pharmacological measures, such as physiotherapy, occupational therapy and, in case of destructive disease, joint surgery. Drug therapy consists of (a combination of) non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids and disease-modifying antirheumatic drugs (DMARDs). During the last few decades, treatment options for RA have largely improved, not only by an increase in the availability of DMARDs, but also by increased availability of biological targeted therapies, such as tumor necrosis factor (TNF) inhibitors, B-cell depleting therapy, CTLA4-Ig treatment, and interleukin-6 (IL-6) receptor antibody treatment 3, 4. Patientspecific treatment should be initiated early after disease onset, as there is a therapeutic window of opportunity during which antirheumatic treatments are more effective and joint destruction can be reduced or halted 5. To allow initiation of appropriate treatment, RA patients should be diagnosed in an early stage of the disease. However, a subset of the early arthritis patients cannot be diagnosed during early disease due to the heterogeneity of the disease and the lack of definitive diagnostic markers, and are diagnosed as undifferentiated arthritis patients. Accordingly, in this group of patients proper treatment may not always be initiated. 1 DISEASE HETEROGENEITY AND DIAGNOSIS OF RA Although RA is most typically characterized by symmetrical pain and swelling in the small joints of hands and feet, it is a heterogeneous disease, both on a clinical and a molecular level 6. Clinically this is characterized for example by variability in joint involvement, which can be symmetric or asymmetric and can include small joints only, large joints only, or a combination. Patients can present with or without extra-articular manifestations, such as rheumatoid nodules, pleuritis and vasculitis. Moreover, there is large variation with respect to response to treatment regimens. In addition, the prognostic outcome can be variable, ranging from self-limiting disease to persistent disease with or without joint destruction. On a molecular level, the heterogeneity of the disease is characterized for example by the presence or absence of RA-specific autoantibodies like IgM rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA), which are present in up to 80% of the RA patients 7. When we look into the synovial tissue, there is heterogeneity in patterns of cell infiltration and gene expression signatures 8. Together, this suggests that the phenotype described as RA may be the result of different pathogenetic pathways 9. 9

11 Due to heterogeneity of the disease it is difficult to diagnose patients based on a single diagnostic test. For research purposes, RA patients have been classified for many years based on mostly clinical features, according to the 1987 American College of Rheumatology (ACR) criteria for RA 10 (Table 1). Of importance, these criteria were developed to differentiate RA patients from arthritis patients having other established rheumatologic disorders. One of the criteria, for example, is radiographic changes typical of RA, which is generally a feature of relatively late disease. The 1987 ACR criteria for RA were developed to classify RA patients during a late stage of the disease. More recently, new classification criteria focusing on early RA were developed by a joint venture of the ACR and the European League Against Rheumatism (EULAR), resulting in the 2010 ACR/EULAR criteria for RA 11, 12 (Table 2). An important difference with the 1987 ACR criteria is the omission of radiographic changes from the criteria. Secondly, the more recent discovery of ACPA being highly specific for RA 7 led to the inclusion of ACPA in the criteria. The advent of the new criteria leads to new questions. Could the 2010 ACR/EULAR criteria result in false positive classification of patients with self-limiting disease as having RA? What would be the effect on the disease heterogeneity when these classification criteria are applied? Table ACR criteria for RA 10 Criterion Definition 1. Morning stiffness Morning stiffness in and around the joints, lasting at least 1 hour before maximal improvement 2. Arthritis of 3 or more joint areas At least 3 joint areas simultaneously have had soft tissue swelling or fluid (not bony overgrowth alone) observed by a physician. The 14 possible areas are right or left PIP, MCP, wrist, elbow, knee, ankle, and MTP joints 3. Arthritis of hand joints At least 1 area swollen (as defined above) in a wrist, MCP, or PIP joint 4. Symmetric arthritis Simultaneous involvement of the same joint areas (as defined in 2) on both sides of the body (bilateral involvement of PIPs, MCPs, or MTPs is acceptable without absolute symmetry) 5. Rheumatoid nodules Subcutaneous nodules, over bony prominences, or extensor surfaces, or in juxtaarticular regions, observed by a physician 6. Serum rheumatoid factor Demonstration of abnormal amounts of serum rheumatoid factor by any method for which the result has been positive in <5% of normal control subjects 7. Radiographic changes Radiographic changes typical of rheumatoid arthritis on posteroanterior hand and wrist radiographs, which must include erosions or unequivocal bony decalcification localized in or most marked adjacent to the involved joints (osteoarthritis changes alone do not qualify) For classification purposes, a patient shall be said to have rheumatoid arthritis if he/she has satisfied at least 4 of these 7 criteria. Criteria 1 through 4 must have been present for at least 6 weeks. Patients with 2 clinical diagnoses are not excluded. 10

12 GENERAL INTRODUCTION 11, 12 Table ACR/EULAR criteria for RA Target population (Who should be tested?): Patients who 1. have at least 1 joint with definite clinical synovitis (swelling) * 2. with the synovitis not better explained by another disease Score Classification criteria for RA (score-based algorithm: add score of categories A D; a score of 6/10 is needed for classification of a patient as having definite RA) A. Joint involvement 1 large joint large joints small joints (with or without involvement of large joints) # small joints (with or without involvement of large joints) 3 >10 joints (at least 1 small joint) ** 5 B. Serology (at least 1 test result is needed for classification) Negative RF and negative ACPA 0 Low-positive RF or low-positive ACPA 2 High-positive RF or high-positive ACPA 3 C. Acute-phase reactants (at least 1 test result is needed for classification) Normal CRP and normal ESR 0 Abnormal CRP or abnormal ESR 1 D. Duration of symptoms <6 weeks 0 6 weeks 1 1 * The criteria are aimed at classification of newly presenting patients. In addition, patients with erosive disease typical of rheumatoid arthritis (RA) with a history compatible with prior fulfilment of the 2010 criteria should be classified as having RA. Patients with longstanding disease, including those whose disease is inactive (with or without treatment) who, based on retrospectively available data, have previously fulfilled the 2010 criteria should be classified as having RA. Differential diagnoses vary among patients with different presentations, but may include conditions such as systemic lupus erythematosus, psoriatic arthritis, and gout. If it is unclear about the relevant differential diagnoses to consider, an expert rheumatologist should be consulted. Although patients with a score of <6/10 are not classifiable as having RA, their status can be reassessed and the criteria might be fulfilled cumulatively over time. Joint involvement refers to any swollen or tender joint on examination, which may be confirmed by imaging evidence of synovitis. Distal interphalangeal joints, first carpometacarpal joints, and first metatarsophalangeal joints are excluded from assessment. Categories of joint distribution are classified according to the location and number of involved joints, with placement into the highest category possible based on the pattern of joint involvement. Large joints refers to shoulders, elbows, hips, knees, and ankles. # Small joints refers to the metacarpophalangeal joints, proximal interphalangeal joints, second through fifth metatarsophalangeal joints, thumb interphalangeal joints, and wrists. ** In this category, at least 1 of the involved joints must be a small joint; the other joints can include any combination of large and additional small joints, as well as other joints not specifically listed elsewhere (e.g., temporomandibular, acromioclavicular, sternoclavicular, etc.). Negative refers to IU values that are less than or equal to the upper limit of normal (ULN) for the laboratory and assay; low-positive refers to IU values that are higher than the ULN but 3 times the ULN for the laboratory and assay; high-positive refers to IU values that are >3 times the ULN for the laboratory and assay. Where rheumatoid factor (RF) information is only available as positive or negative, a positive result should be scored as low-positive for RF. ACPA = anti-citrullinated protein antibody. Normal/abnormal is determined by local laboratory standards. CRP = C-reactive protein; ESR = erythrocyte sedimentation rate. Duration of symptoms refers to patient self-report of the duration of signs or symptoms of synovitis (e.g., pain, swelling, tenderness) of joints that are clinically involved at the time of assessment, regardless of treatment status. 11

13 PATHOGENESIS OF RA The etiology of RA is largely unknown, but several risk factors associated with RA have been recognized. Genetic risk factors as well as environmental risk factors and the interaction between these play a key role. However, the factors leading to the onset of synovial inflammation are currently unknown. Risk factors Besides the presence of RA-specific autoantibodies, several other risk factors have been associated with RA. HLA-DRB1 and PTPN22 (the latter in Caucasian populations) have the largest genetic contribution to RA susceptibility 13, 14, but many other genes have been associated with RA as well, such as CTLA4, CD40 15, PADI4 13, 14, 16 and CD2/CD58 17, although with much smaller contributory effect. The genetic loci which have currently been associated with RA have mainly been associated with ACPA-positive RA. In addition, these genetic factors probably contribute for not more than 50% to the genetic susceptibility, and overall explain only a part of the susceptibility to RA. Environmental factors appear to contribute to the pathogenesis of RA as well. Examples are smoking 18, 19, obesity 20-22, low vitamin D levels 23, viral infections 24 and a history of periodontitis 25. The exact mechanisms of how these factors contribute to the development of RA are unknown, although an important interaction between autoimmune, genetic and environmental factors has recently been shown 26. Collectively, an accumulation of several risk factors and their interaction may lead to a breach of immune tolerance. Inflammation of the synovium The synovium is a soft tissue layer lining non-cartilagenous joint surfaces. Its physiologic function is to secrete synovial fluid which lubricates the joint and nourishes the avascular cartilage. In the healthy state the synovial tissue is composed of one to three layers of specialized columnar cells called fibroblast-like synoviocytes (FLS) with interspersed macrophages 27. Lymphatic and blood vessels as well as nerve fibres are present in the healthy synovium and it can contain adipocytes 28. It is divided into an intimal lining layer without an underlying basement membrane, and a synovial sublining layer which is continuous with the joint capsule. As synovitis is the primary pathogenic event underlying signs and symptoms of arthritis in RA, we endeavour to better understand its pathology. Autoimmune activation, coupled with up-regulation of pro-inflammatory cytokines and mobilization of inflammatory cells to the synovium play considerable roles but the precise etiology of the disease is as yet unclear. Microscopic analysis of the synovium has given us some insight into the pathogenesis of RA. Rheumatoid synovial tissue is hypertrophic and edematous and is characterized by marked intimal lining hyperplasia and by accumulation of T lymphocytes, plasma cells, macrophages, B lymphocytes, neutrophils, mast cells, natural killer cells, and dendritic cells (DC) in the synovial sublining 29. Villous projections of synovial tissue protrude into the synovial cavity and erode into the underlying cartilage and bone. Neo-angiogenesis, the development of new blood vessels, within the inflamed synovium facilitates the migration of leukocytes and contributes to the perpetuation of this chronic disease. 12

14 GENERAL INTRODUCTION As already mentioned, the factors leading to synovial inflammation are currently unknown. Presumably, antigens in close contact to the synovium are presented to antigen presenting cells (APCs) such as DCs and FLS 30, leading to T- and B-lymphocyte activation. Upon activation T lymphocytes produce pro-inflammatory cytokines and adhesion molecules, and B lymphocytes start to produce antibodies which may lead to deposition of immune complexes in the joint and subsequent cytokine production. In addition, activated B lymphocytes may play a role in co-stimulation of T-lymphocytes 31. Together, the immune activation may result in recruitment of other inflammatory cells into the synovium. The presence of immune cells in a pro-inflammatory environment may trigger an inflammatory cascade, resulting in severe synovial inflammation. At current, RA-specific antigens have as yet not been detected, and it is believed that self-antigen, recognized by autoreactive T- and B lymphocytes 31, may play an important role. Candidate antigens are citrullinated peptides, to which ACPAs are directed, which have been demonstrated in synovial tissue 32. However, antigens initiating this immune response are not specific for RA 32 and may not be joint-specific, since the presence of citrullinated proteins is also found in other forms of inflammation 33. It has been hypothesized that citrullination of peptides in the lungs, due to cigarette smoking 34, or in the gingiva, due to periodontitis 25, may be important for triggering the immune activation cascade, leading to systemic autoimmunity. What determines the transition from the phase characterized by circulating ACPAs to chronic synovitis? We have previously proposed that a second hit to the synovium may be necessary to induce citrullination of peptides in the synovium 35. This might, for example, be a minor trauma or a viral infection. In the presence of pre-existing immunity against citrullinated antigens at sites other than the synovium, citrullination of peptides in the synovial tissue due to an inflammatory response to this second hit, might lead to epitope spreading and autonomous progression of synovitis. Clearly, this hypothesis still needs to be proven. There has been an enormous upsurge of studies on the synovial tissue response to treatment during the last 20 years. Synovial cell infiltration, particularly by CD68 positive macrophages, and macrophage-derived cytokine expression are reduced after prednisolone therapy with a significant correlation with beneficial clinical effect 36. This effect is not restricted to corticosteroid treatment, as several experiments have consistently shown that the quantity of CD68 positive macrophages in the synovial sublining is reduced concurrent with a reduction in disease activity 37. It has also been shown that when therapy has failed and inflammation persists, CD68 positive macrophages do not decrease in number, further supporting its use as an accurate biomarker that can be used on the group level to distinguish effective from ineffective treatment 38. Accordingly, the expression of pro-inflammatory cytokines and inflammatory cells is reduced in the synovial membrane of treated RA patients with low disease activity 39. Synovial tissue analysis has also provided insight into biomarkers predictive of the response to treatment in individual patients 6. Pre-treatment synovial inflammation levels, TNF expression and the presence of lymphocyte aggregates all correlate with the therapeutic response to infliximab The clinical response to rituximab treatment is related to the change in B cell derived plasma cells in the synovium 43, 44. Together, these findings illustrate that 1 13

15 descriptive studies of the rheumatoid synovium help us to understand the events that take place in vivo and complement experimental animal studies as well as in vitro studies. PRECLINICAL RA RF and ACPA can be present years before the development of RA Besides the presence of RA-specific autoantibodies before the onset of arthritis, other signs of systemic immune activation have been observed during that phase, such as increased C-reactive protein 46 and monocyte chemoattractant protein-1 levels 48. In addition, broadening of the ACPA repertoire due to epitope spreading towards the onset of arthritis in ACPA-positive RA patients has been reported 49. Together, these observations suggest the presence of an RA-specific adaptive immune response before clinical signs of arthritis are present. However, only a subset of the individuals who are positive for RF and/or ACPA will develop RA over time 50. At current, it is unknown which subset of these autoantibody-positive individuals will make the transition to the development of clinically apparent RA and which subset will not, and why. Detection of RF and ACPA has enabled us to identify individuals having systemic autoimmunity associated with RA who are at risk of the development of the disease 51. This has greatly facilitated research on the earliest stages of the disease. The synovium during preclinical RA Despite growing insight into the characteristics of the synovial inflammation in RA, which has led to better directed therapies targeting specific molecules and pathways, not much is known about the local processes at play before the development of RA. Studies comparing characteristics of the inflamed synovial tissue of early arthritis patients with those of patients with established disease have shown that there are no major differences with respect to the cellular infiltrate 29, 52, 53, cytokines 29, or adhesion molecules 57, suggesting that early arthritis already reflects chronic disease and that, before arthritis becomes clinically apparent, a period of subclinical synovial inflammation might exist. Firstly, this is supported by the observation that a subset of early onset arthritis patients presents with erosions 58, 59. Secondly, several studies analyzing unaffected joints of RA patients have revealed comparable infiltration of the synovium by the major inflammatory cell types and expression of cytokines 60 to affected joints. Thirdly, using ultrasound imaging in patients with early oligoarthritis 63 and in autoantibody-positive individuals who are at risk of developing RA 64 signs of synovitis were observed in joints without clinically apparent arthritis. Lastly, using a collagen induced arthritis model in rhesus monkeys, increased infiltration of the synovial tissue by T cells and macrophages was observed after immunization with type II collagen before the onset of clinical arthritis 60. In contrast, the first analyses of the synovial tissue in a small group of autoantibody-positive individuals at risk of developing RA have not revealed clear inflammation when compared to the synovium of healthy controls 35. To be more conclusive about possible subclinical synovial inflammation preceding the development of arthritis in autoantibody-positive individuals, a prospective follow-up study is needed in a larger group of individuals. 14

16 GENERAL INTRODUCTION AIM AND OUTLINE OF THIS THESIS In this thesis we studied the earliest stages of RA to get more insight into the mechanisms behind the transition from a phase in which individuals are at risk for developing RA, characterized by systemic autoimmunity associated with RA, to having clinically apparent disease. Part I of this thesis focuses on clinical aspects, part II focuses on the main target tissue of RA, the synovium, and part III focuses on the immune response in different compartments during the earliest stages of RA. 1 PART I: CLINICAL ASPECTS DURING THE EARLIEST STAGES OF RA In chapter 2 we investigate the impact of application of the 2010 ACR/EULAR criteria instead of the 1987 ACR criteria for RA on the number and the time frame of classification of early arthritis patients as RA as well as on the clinical characteristics of patients classified as RA. In chapter 3 we address the contributory role of smoking and high body mass index (BMI) to the development of RA in a prospective cohort of autoantibody-positive individuals with an increased risk of developing the disease. PART II: THE SYNOVIUM DURING THE EARLIEST STAGES OF RA In chapter 4 we investigate the experience of patients undergoing mini-arthroscopy in comparison to MRI in a research setting. In chapter 5 we perform synovial tissue analysis in autoantibody-positive individuals with an increased risk of developing RA in relation to the development of the disease, to get more insight into the pathogenic events in the synovial tissue which may lead to clinically apparent disease. In addition, since these autoantibody-positive individuals may suffer from arthralgia, we investigate the prostaglandin (PG) E 2 pathway, a major pathway in pain sensation, in the synovial tissue of these individuals in chapter 6. In parallel, since PGE 2 is also a proinflammatory mediator, we investigate this pathway in the synovial tissue of early arthritis patients in relation to several disease characteristics. PART III: THE IMMUNE RESPONSE IN DIFFERENT COMPARTMENTS DURING THE EARLIEST STAGES OF RA Besides analysing the synovial tissue in studying the earliest phases of RA, it might be important to investigate other compartments of the immune system which may be involved in early immune activation. In chapter 7 we describe the goals and the procedure of inguinal lymph node biopsy sampling for studies investigating the earliest phases of RA pathogenesis and in chapter 8 we describe the cellular composition of these lymph node biopsies. In chapter 9 we investigate the T-cell receptor repertoire in peripheral blood and synovial tissue of early and established RA patients, to evaluate the possible role of expanded T-cell clones in the pathogenesis of RA. 15

17 REFERENCE LIST 1. Tak PP, Bresnihan B. The pathogenesis and prevention of joint damage in rheumatoid arthritis: advances from synovial biopsy and tissue analysis. Arthritis Rheum 2000;43(12): Avina-Zubieta JA, Abrahamowicz M, De Vera MA et al. Immediate and past cumulative effects of oral glucocorticoids on the risk of acute myocardial infarction in rheumatoid arthritis: a population-based study. Rheumatology (Oxford) 2013;52(1): Smolen JS, Aletaha D, Koeller M, Weisman MH, Emery P. New therapies for treatment of rheumatoid arthritis. Lancet 2007;370(9602): Tak PP, Kalden JR. Advances in rheumatology: new targeted therapeutics. Arthritis Res Ther 2011;13 Suppl 1:S5. 5. Smolen JS, Landewe R, Breedveld FC et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs. Ann Rheum Dis 2010;69(6): Tak PP. A personalized medicine approach to biologic treatment of rheumatoid arthritis: a preliminary treatment algorithm. Rheumatology (Oxford) 2012;51(4): Schellekens GA, Visser H, de Jong BA et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 2000;43(1): Bugatti S, Manzo A, Bombardieri M et al. Synovial tissue heterogeneity and peripheral blood biomarkers. Curr Rheumatol Rep 2011;13(5): Tak PP. Analyzing synovial tissue samples. What can we learn about early rheumatoid arthritis, the heterogeneity of the disease, and the effects of treatment? J Rheumatol Suppl 2005;72: Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31(3): Aletaha D, Neogi T, Silman AJ et al Rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010;62(9): Aletaha D, Neogi T, Silman AJ et al rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010;69(9): Barton A, Worthington J. Genetic susceptibility to rheumatoid arthritis: an emerging picture. Arthritis Rheum 2009;61(10): Gregersen PK. Susceptibility genes for rheumatoid arthritis - a rapidly expanding harvest. Bull NYU Hosp Jt Dis 2010;68(3): Raychaudhuri S, Remmers EF, Lee AT et al. Common variants at CD40 and other loci confer risk of rheumatoid arthritis. Nat Genet 2008;40(10): Eyre S, Bowes J, Diogo D et al. High-density genetic mapping identifies new susceptibility loci for rheumatoid arthritis. Nat Genet 2012;44(12): Raychaudhuri S, Thomson BP, Remmers EF et al. Genetic variants at CD28, PRDM1 and CD2/CD58 are associated with rheumatoid arthritis risk. Nat Genet 2009;41(12): Klareskog L, Stolt P, Lundberg K et al. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA-DR (shared epitope)-restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum 2006;54(1): Sugiyama D, Nishimura K, Tamaki K et al. Impact of smoking as a risk factor for developing rheumatoid arthritis: a metaanalysis of observational studies. Ann Rheum Dis 2010;69(1): Crowson CS, Matteson EL, Davis JM, III, Gabriel SE. Contribution of obesity to the rise in incidence of rheumatoid arthritis. Arthritis Care Res (Hoboken ) 2013;65(1): Symmons DP, Bankhead CR, Harrison BJ et al. Blood transfusion, smoking, and obesity as risk factors for the development of rheumatoid arthritis: results from a primary care-based incident case-control study in Norfolk, England. Arthritis Rheum 1997;40(11): Voigt LF, Koepsell TD, Nelson JL, Dugowson CE, Daling JR. Smoking, obesity, alcohol consumption, and the risk of rheumatoid arthritis. Epidemiology 1994;5(5):

18 GENERAL INTRODUCTION 23. Song GG, Bae SC, Lee YH. Association between vitamin D intake and the risk of rheumatoid arthritis: a meta-analysis. Clin Rheumatol 2012;31(12): Jorgensen KT, Wiik A, Pedersen M et al. Cytokines, autoantibodies and viral antibodies in premorbid and postdiagnostic sera from patients with rheumatoid arthritis: case-control study nested in a cohort of Norwegian blood donors. Ann Rheum Dis 2008;67(6): Farquharson D, Butcher JP, Culshaw S. Periodontitis, Porphyromonas, and the pathogenesis of rheumatoid arthritis. Mucosal Immunol 2012;5(2): Lundberg K, Bengtsson C, Kharlamova N et al. Genetic and environmental determinants for disease risk in subsets of rheumatoid arthritis defined by the anticitrullinated protein/peptide antibody fine specificity profile. Ann Rheum Dis Smith MD, Barg E, Weedon H et al. Microarchitecture and protective mechanisms in synovial tissue from clinically and arthroscopically normal knee joints. Ann Rheum Dis 2003;62(4): Smith MD. The normal synovium. Open Rheumatol J 2011;5: Tak PP, Smeets TJ, Daha MR et al. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum 1997;40(2): Tran CN, Davis MJ, Tesmer LA et al. Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes. Arthritis Rheum 2007;56(5): McQueen F. A B cell explanation for autoimmune disease: the forbidden clone returns. Postgrad Med J 2012;88(1038): Vossenaar ER, Smeets TJ, Kraan MC, Raats JM, van Venrooij WJ, Tak PP. The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum 2004;50(11): Makrygiannakis D, af KE, Lundberg IE et al. Citrullination is an inflammation-dependent process. Ann Rheum Dis 2006;65(9): Klareskog L, Malmstrom V, Lundberg K, Padyukov L, Alfredsson L. Smoking, citrullination and genetic variability in the immunopathogenesis of rheumatoid arthritis. Semin Immunol 2011;23(2): van de Sande MG, de Hair MJ, van der Leij C et al. Different stages of rheumatoid arthritis: features of the synovium in the preclinical phase. Ann Rheum Dis 2011;70(5): Gerlag DM, Haringman JJ, Smeets TJ et al. Effects of oral prednisolone on biomarkers in synovial tissue and clinical improvement in rheumatoid arthritis. Arthritis Rheum 2004;50(12): Haringman JJ, Gerlag DM, Zwinderman AH et al. Synovial tissue macrophages: a sensitive biomarker for response to treatment in patients with rheumatoid arthritis. Ann Rheum Dis 2005;64(6): Wijbrandts CA, Vergunst CE, Haringman JJ, Gerlag DM, Smeets TJ, Tak PP. Absence of changes in the number of synovial sublining macrophages after ineffective treatment for rheumatoid arthritis: Implications for use of synovial sublining macrophages as a biomarker. Arthritis Rheum 2007;56(11): Gerlag DM, Tak PP. Novel approaches for the treatment of rheumatoid arthritis: lessons from the evaluation of synovial biomarkers in clinical trials. Best Pract Res Clin Rheumatol 2008;22(2): Klaasen R, Thurlings RM, Wijbrandts CA et al. The relationship between synovial lymphocyte aggregates and the clinical response to infliximab in rheumatoid arthritis: a prospective study. Arthritis Rheum 2009;60(11): van der Pouw Kraan TC, Wijbrandts CA, van Baarsen LG et al. Responsiveness to antitumour necrosis factor alpha therapy is related to pre-treatment tissue inflammation levels in rheumatoid arthritis patients. Ann Rheum Dis 2008;67(4): Wijbrandts CA, Dijkgraaf MG, Kraan MC et al. The clinical response to infliximab in rheumatoid arthritis is in part dependent on pretreatment tumour necrosis factor alpha expression in the synovium. Ann Rheum Dis 2008;67(8): Teng YK, Levarht EW, Toes RE, Huizinga TW, van Laar JM. Residual inflammation after rituximab treatment is associated with sustained synovial plasma cell infiltration and enhanced B cell repopulation. Ann Rheum Dis 2009;68(6): Thurlings RM, Vos K, Wijbrandts CA, Zwinderman AH, Gerlag DM, Tak PP. Synovial tissue response to rituximab: mechanism of action and identification of biomarkers of response. Ann Rheum Dis 2008;67(7):

19 45. Aho K, Heliovaara M, Maatela J, Tuomi T, Palosuo T. Rheumatoid factors antedating clinical rheumatoid arthritis. J Rheumatol 1991;18(9): Nielen MM, van SD, Reesink HW et al. Increased levels of C-reactive protein in serum from blood donors before the onset of rheumatoid arthritis. Arthritis Rheum 2004;50(8): Rantapaa-Dahlqvist S, de Jong BA, Berglin E et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003;48(10): Rantapaa-Dahlqvist S, Boman K, Tarkowski A, Hallmans G. Up regulation of monocyte chemoattractant protein-1 expression in anticitrulline antibody and immunoglobulin M rheumatoid factor positive subjects precedes onset of inflammatory response and development of overt rheumatoid arthritis. Ann Rheum Dis 2007;66(1): van de Stadt LA, van der Horst AR, de Koning MH et al. The extent of the anti-citrullinated protein antibody repertoire is associated with arthritis development in patients with seropositive arthralgia. Ann Rheum Dis 2011;70(1): Bos WH, Wolbink GJ, Boers M et al. Arthritis development in patients with arthralgia is strongly associated with anti-citrullinated protein antibody status: a prospective cohort study. Ann Rheum Dis 2010;69(3): Gerlag DM, Raza K, van Baarsen LG et al. EULAR recommendations for terminology and research in individuals at risk of rheumatoid arthritis: report from the Study Group for Risk Factors for Rheumatoid Arthritis. Ann Rheum Dis 2012;71(5): Baeten D, Demetter P, Cuvelier C et al. Comparative study of the synovial histology in rheumatoid arthritis, spondyloarthropathy, and osteoarthritis: influence of disease duration and activity. Ann Rheum Dis 2000;59(12): Zvaifler NJ, Boyle D, Firestein GS. Early synovitis- -synoviocytes and mononuclear cells. Semin Arthritis Rheum 1994;23(6 Suppl 2): Muller-Ladner U, Judex M, Ballhorn W et al. Activation of the IL-4 STAT pathway in rheumatoid synovium. J Immunol 2000;164(7): Smeets TJ, Dolhain RJEM, Miltenburg AM, De KR, Breedveld FC, Tak PP. Poor expression of T cell-derived cytokines and activation and proliferation markers in early rheumatoid synovial tissue. Clin Immunol Immunopathol 1998;88(1): Thurkow EW, van der Heijden IM, Breedveld FC et al. Increased expression of IL-15 in the synovium of patients with rheumatoid arthritis compared with patients with Yersinia-induced arthritis and osteoarthritis. J Pathol 1997;181(4): Tak PP, Thurkow EW, Daha MR et al. Expression of adhesion molecules in early rheumatoid synovial tissue. Clin Immunol Immunopathol 1995;77(3): Mottonen TT. Prediction of erosiveness and rate of development of new erosions in early rheumatoid arthritis. Ann Rheum Dis 1988;47(8): van der Heijde DM. Joint erosions and patients with early rheumatoid arthritis. Br J Rheumatol 1995;34 Suppl 2: Kraan MC, Versendaal H, Jonker M et al. Asymptomatic synovitis precedes clinically manifest arthritis. Arthritis Rheum 1998;41(8): Pando JA, Duray P, Yarboro C, Gourley MF, Klippel JH, Schumacher HR. Synovitis occurs in some clinically normal and asymptomatic joints in patients with early arthritis. J Rheumatol 2000;27(8): Soden M, Rooney M, Cullen A, Whelan A, Feighery C, Bresnihan B. Immunohistological features in the synovium obtained from clinically uninvolved knee joints of patients with rheumatoid arthritis. Br J Rheumatol 1989;28(4): Wakefield RJ, Green MJ, Marzo-Ortega H et al. Should oligoarthritis be reclassified? Ultrasound reveals a high prevalence of subclinical disease. Ann Rheum Dis 2004;63(4): van de Stadt LA, Bos WH, Meursinge RM et al. The value of ultrasonography in predicting arthritis in auto-antibody positive arthralgia patients: a prospective cohort study. Arthritis Res Ther 2010;12(3):R98. 18

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22 PART CLINICAL ASPECTS DURING THE EARLIEST STAGES OF RA I

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24 THE CLINICAL PICTURE OF RHEUMATOID ARTHRITIS ACCORDING TO THE 2010 ACR/EULAR CRITERIA: IS THIS STILL THE SAME DISEASE? M.J.H. de Hair 1, MD, K.A. Lehmann 1, Drs, M.G.H. van de Sande 1, MD, K.I. Maijer 1, MD, D.M. Gerlag 1, MD, PhD, P.P. Tak 1, MD, PhD. 1 Division of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, the Netherlands 2 Arthritis Rheum Feb;64(2):389-93

25 ABSTRACT Objective We examined the implication of the use of the new classification criteria for rheumatoid arthritis (RA) in clinical practice in a very early arthritis cohort. Methods We included 301 disease-modifying antirheumatic drug naïve early arthritis patients. Baseline diagnosis was assessed applying 1987 ACR and 2010 ACR/EULAR criteria for RA as well as established diagnostic criteria for other rheumatic diseases. Diagnostic and prognostic data after 2 years follow up were collected. We evaluated fulfilment of the 2010 ACR/EULAR criteria in the subset of patients diagnosed as undifferentiated arthritis (UA) when the 1987 ACR criteria were applied, and tested fulfilment of RA criteria over time applying the two different criteria sets. Results Median arthritis duration at inclusion was 3 months (range 0-12). At baseline 28% fulfilled the 1987 ACR criteria and 45% the 2010 ACR/EULAR criteria for RA. Of the patients classified as UA at baseline using 1987 ACR criteria, 36% fulfilled the 2010 ACR/EULAR criteria already at baseline. Of the patients classified as UA at baseline but who fulfilled 1987 ACR criteria after 2 years follow up, 85% fulfilled the 2010 ACR/EULAR criteria at baseline. Patients fulfilling 2010 ACR/EULAR criteria during early disease were less likely to be autoantibody positive and more likely to have monoarthritis at presentation than those fulfilling 1987 ACR criteria. Conclusion Use of the 2010 ACR/EULAR criteria clearly allows earlier diagnosis of RA, although the clinical picture is slightly different on the group level, and some patients with self-limiting disease may be falsely diagnosed with RA. 24

26 2010 ACR/EULAR CRITERIA FOR RA IN EARLY ARTHRITIS PATIENTS Rheumatoid arthritis (RA) is a symmetric, polyarticular chronic inflammatory disease leading to joint destruction, deformity and disability with heterogeneous manifestations 1, 2. In 1987 the revised criteria for the classification of RA were formulated by the American College of Rheumatology (ACR) 3. These criteria were derived by discriminating established RA from other rheumatic diseases. Thus, the 1987 ACR criteria define patients with longstanding disease and have not been designed to diagnose RA patients early after arthritis onset. This explains in part the relatively large proportion of early arthritis patients who cannot be classified during early disease and are diagnosed as undifferentiated arthritis (UA). Since early treatment has proven to reduce or halt joint destruction 4, RA patients should be classified in an early disease phase to allow timely initiation of disease-modifying therapy. To facilitate the study of persons in an earlier disease stage and to be able to start early and effective intervention, new criteria were developed by a joint working group of the ACR and the European League Against Rheumatism (EULAR) in 2010, the 2010 ACR/ EULAR criteria for RA 5, 6. After the introduction of the 1987 ACR criteria there was a debate about the wide spectrum of clinical characteristics of patients who were all classified as having RA 7. Conceivably, with the introduction of the 2010 ACR/EULAR criteria heterogeneity might increase even more, as patients who will ultimately have self-limiting disease could be classified as having RA as well. Therefore, we examined the implications of the use of the new criteria in clinical practice in our early arthritis cohort. 2 Patients and methods Disease-modifying antirheumatic drug (DMARD) naïve early arthritis patients with disease duration less than one year were enrolled in the early arthritis cohort of the Academic Medical Center (AMC) in Amsterdam. Disease onset was defined as clinically evidence of arthritis observed by an experienced rheumatologist. In case of suspicion of arthritis, patients are directly referred by the general practitioners in the context of an early and rapid referral protocol to our early arthritis clinic, which has a waiting list of < 2 weeks. This protocol has been developed for rapid referral of patients who are suspected of having arthritis and for standardized treatment of arthritis patients. All patients were followed prospectively according to a stringent protocol. At baseline and after 2 years follow up the following clinical parameters were obtained: 68 tender (TJC) and 66 swollen (SJC) joint count, erythrocyte sedimentation rate (ESR), C-reactive protein levels (CRP), 28-joint disease activity score (DAS28) using ESR, and IgM rheumatoid factor (IgM-RF) and levels of anti-citrullinated protein antibodies (ACPA) were measured using IgM-RF ELISA (Sanquin, Amsterdam, the Netherlands) and anti-ccp2 ELISA (CCPlus, Eurodiagnostica, Nijmegen, the Netherlands), respectively. X-rays of hands and feet were obtained to evaluate erosive disease, as assessed by an experienced radiologist together with the rheumatologist. In addition, diagnosis was assessed applying the 1987 ACR criteria for RA as well as established diagnostic criteria for other rheumatic diseases 3, In retrospect, the 2010 ACR/EULAR criteria for RA were applied. Patients who did not fulfill any of the classification criteria were classified as UA patients. When patients were lost to follow up 25

27 after classification with an established disease at baseline or at an interim study visit, then diagnosis of this observation was carried forward. After 2 years follow up patients were classified according to outcome: self-limiting, persistent non-erosive or persistent erosive disease as described by Visser et al. 11. After enrolment and during follow up, patients received standard of care according to their treating physician; for RA the treatment algorithm was consistent with the recently published EULAR guidelines 12. The study was approved by the Medical Ethics Committee of the AMC/University of Amsterdam and performed according to the Declaration of Helsinki. All patients gave written informed consent. Statistical analyses We evaluated in the subset of patients classified as UA when applying the 1987 ACR criteria the fulfilment of the 2010 ACR/EULAR criteria and tested fulfilment of RA criteria over time applying the two different criteria sets. We tested the sensitivity, specificity and positive and negative predictive value of the 2010 ACR/EULAR criteria at baseline using RA diagnosis according to the 1987 ACR criteria after 2 years follow up as gold standard. In addition, we compared the clinical picture using the two criteria sets at baseline. We restricted ourselves to descriptive analysis in light of the partial overlap of patients in the diagnostic groups, resulting in the semi-paired character of the data. Analysis was performed using SPSS V.18.0 (Chicago, IL) software. RESULTS Cohort description Between August 2002 and November patients were included in our early arthritis cohort. 239 patients were followed for 2 years; of 186 patients prognostic outcome data were available (50% of the patients lost to follow up were classified as UA according to the 1987 ACR criteria). Of all patients included 189 were female (63%). Median age was 50 years (range 18-88). Median disease duration at baseline was 3 months (range 0-12). Median 68 tender joint count was 5 (range 0-64) and 66 swollen joint count was 3 (range 1-41). 68% of the patients was negative for both IgM-RF and ACPA, 3% was low positive (5;6) and 29% was high positive for IgM-RF and/or ACPA. Median ESR was 19 mm/hr (range 2-130), median CRP was 7 mg/l (range 0-255), and median DAS28 was 4.3 (range ). Use of 2010 ACR/EULAR criteria allows earlier diagnosis of RA At baseline 60 patients could be classified as having a rheumatologic disease other than RA (7 inflammatory osteoarthritis (joint effusion in the absence of rheumatologic disease other than osteoarthritis), 1 systemic lupus erythematosis, 17 psoriatic arthritis, 1 ankylosing spondylitis, 5 undifferentiated spondyloarthritis, 16 gout, 13 other diagnoses such as sarcoidosis, Behçet s disease and Lyme disease). Eighty-three patients (28%) fulfilled the 1987 ACR criteria for RA at baseline, compared to 134 patients (45%) fulfilling the 2010 ACR/EULAR criteria for RA. After 2 years follow up 26

28 2010 ACR/EULAR CRITERIA FOR RA IN EARLY ARTHRITIS PATIENTS this percentage increased to 36% and 55%, respectively. 159 patients (53%) were classified as UA at baseline when applying the 1987 ACR criteria compared to 107 patients (36%) when applying the 2010 ACR/EULAR criteria. After 2 years follow up the proportion of UA patients in the cohort decreased to 44% when applying the 1987 ACR criteria and to 22% when applying the 2010 ACR/EULAR criteria. See Figure 1 for diagnostic outcome after 2 years follow up in all UA and RA patients. Of the 159 patients classified as UA at baseline when the 1987 ACR criteria were applied, 57 fulfilled the 2010 ACR/EULAR criteria already at baseline. Of the 20 patients initially classified as UA at baseline when the 1987 ACR criteria were applied, but who could be classified as RA according to the 1987 ACR criteria after 2 years follow up, 17 already fulfilled the 2010 ACR/EULAR criteria at baseline. Of importance, 93% of the RA patients fulfilling the 1987 ACR criteria at baseline also fulfilled the 2010 ACR/EULAR criteria. Next, we analyzed the sensitivity, specificity and predictive value of the new criteria in this cohort when using as gold standard the fulfilment of 1987 ACR criteria after 2 years follow up in the absence of another rheumatologic diagnosis explaining the signs and symptoms. Eighty-eight per cent of the patients fulfilling the 1987 ACR criteria for RA after 2 years follow up fulfilled the 2010 ACR/EULAR criteria at baseline, with a specificity of The positive and negative predictive values were 0.77 and 0.91, respectively. Patient groups and characteristics of patients who were false positively or false negatively diagnosed with RA are shown in Supplementary Tables 1 and 2. In addition, five patients could be classified with an established other disease over time of which 4 were initially classified as UA and 1 as RA, both when applying the 1987 ACR and 2010 ACR/EULAR criteria. 2 The 2010 ACR/EULAR criteria may define a slightly different clinical picture To assess the clinical signs and symptoms of patients with RA according to the 2010 ACR/ EULAR criteria compared to the 1987 ACR criteria, we evaluated the clinical expression of the disease when the different sets of criteria were used at baseline. See Table 1 for patient characteristics. Figure 1. Diagnostic outcome after 2 years follow up when applying either 1987 American College of Rheumatology (ACR) or 2010 ACR/European League Against Rheumatism (EULAR) criteria. % of all UA and RA patients after 2 years follow up; RA>>RA, RA already at baseline; UA>>RA, UA at baseline but fulfilling RA criteria after 2 years follow up; UA>>UA, UA at baseline and after 2 years follow up. 27

29 Joint involvement The 68TJC and 66SJC were lower in RA patients fulfilling the 2010 ACR/EULAR criteria than in those fulfilling the 1987 ACR criteria (median TJC 11 (range 0-64) compared to 13 (range 0-64) and median SJC 6 (range 0-41) compared to 9 (range 0-41), respectively). Of the RA patients fulfilling the 1987 ACR criteria at baseline 3 patients (4%) presented with monoarthritis. The number of RA patients according to the 2010 ACR/EULAR criteria presenting with monoarthritis at baseline was markedly higher: 10% (13 patients). In these patients disease duration was similar to the RA patients presenting with more than one arthritic joint. However, they showed higher ESR levels and had a higher frequency of positive serology and, if positive, they had higher serum levels of RA-specific autoantibodies. Autoantibody status Of the patients with RA according to the 1987 ACR criteria, 67% was IgM-RF and/or ACPA positive compared to 62% for RA patients fulfilling the 2010 ACR/EULAR criteria. In Table 1. Patient characteristics of RA patients fulfilling 1987 ACR or 2010 ACR/EULAR criteria at baseline 1987 RA (n=83) 2010 RA (n=134) Female (%) Age, years (Median (range)) 52 (18-82) 51 (18-82) ESR, mm/hr (Median (range)) 18 (2-117) 21 (2-130) CRP, mg/l (Median (range)) 9 (1-162) 8 (0-218) DAS28 (Median (range)) 5.3 ( ) 5.1 ( ) Joint involvement 68 TJC (Median (range)) 13 (0-64) 11 (0-64) 66 SJC (Median (range)) 9 (0-41) 6 (0-41) Monoarthritis (%) 4 10 Autoantibody status IgM-RF and ACPA negative (%) IgM-RF and/or ACPA low positive (%) 4 4 IgM-RF and/or ACPA high positive (%) Duration of symptoms Arthritis duration, months (median (range)) 4 (0-12) 4 (0-12) Outcome after 2 years follow up* Self-limiting (%) 2 12 Persistent non-erosive (%) Persistent erosive (%) RA, rheumatoid arthritis; ESR, erythrocyte sedimentation rate; CRP, c-reactive protein; 68 TJC, 68 tender joint count; 66 SJC, 66 swollen joint count; IgM-RF, IgM-rheumatoid factor; ACPA, anticitrullinated protein antibodies; low positive: value higher than upper limit of normal but 3 times; high positive: value > 3 times upper limit of normal; * In the 1987 cohort, 55 patients were evaluated; in the 2010 cohort, 83 patients were evaluated. 28

30 2010 ACR/EULAR CRITERIA FOR RA IN EARLY ARTHRITIS PATIENTS addition, fewer patients fulfilling the 2010 ACR/EULAR criteria had high positive values for IgM-RF and/or ACPA (58% compared to 63%). Acute phase response ESR and CRP were comparable between RA patients fulfilling either the 1987 ACR or 2010 ACR/EULAR criteria (median ESR was 18 (range 2-117) and 21 (range 2-130), respectively; median CRP was 9 (range 1-162) and 8 (range 0-218), respectively). 2 Disease duration Disease duration at baseline was comparable for patients fulfilling either the 1987 ACR criteria for RA or the 2010 ACR/EULAR criteria, with in both groups a median of 4 months (range 1-12). Disease activity DAS28 was comparable between RA patients fulfilling the 2010 ACR/EULAR criteria and those fulfilling the 1987 ACR criteria with a median of 5.1 (range ) and 5.3 (range ), respectively. Outcome Of the 55 patients according to the 1987 ACR criteria for RA at baseline who could be classified according to outcome after 2 years follow up 1 patient had self-limiting disease. In contrast, this proportion was 12% when applying the 2010 ACR/EULAR criteria, where 10 of the 83 patients who could be classified according to outcome after 2 years follow up had self-limiting disease. Only 5 of these patients had been started on DMARD therapy or prednisolone, which was 71% in the overall group of RA patients fulfilling the 2010 ACR/EULAR criteria. Remarkably, these 10 patients had lower ESR and CRP levels at baseline and were less likely to be autoantibody positive. The proportion of RA patients who developed erosive disease over time decreased from 38% to 25% (Figure 2). Figure 2. Prognostic outcome after 2 years follow up when applying either 1987 ACR or 2010 ACR/EULAR criteria. % of all RA patients after 2 years follow up. 29

31 DISCUSSION The results presented here show that by introduction of the 2010 ACR/EULAR criteria for RA significantly more early arthritis patients are diagnosed with RA in an earlier phase than when using the 1987 ACR criteria. Thirty-six percent of the patients previously classified as UA were diagnosed as RA according to the new criteria at baseline. Eighty-five percent of the patients who were previously diagnosed with UA when applying the 1987 ACR criteria in the diagnostic workup at baseline, but who did fulfil the 1987 ACR criteria after 2 years follow up, could already be classified as RA during (very) early disease when applying the new ACR/EULAR criteria. Therefore, the main goal of the development of a new set of criteria to classify RA has been reached: to diagnose RA patients in an earlier phase. In our cohort, the sensitivity of the 2010 ACR/EULAR criteria was 0.88, with a specificity of Of importance, no additional patients with other established rheumatologic disease were initially false positively diagnosed by introduction of the 2010 ACR/EULAR criteria. These results confirm and extend the findings of a very recent comparison of the 1987 ACR and 2010 ACR/EULAR criteria from Leiden, the Netherlands 13, and Birmingham, UK 14. The difference between our cohort and the Leiden cohort is the median disease duration, which is markedly shorter in our cohort. Our study population is comparable to that from Birmingham in terms of disease duration, but our cohort is larger and we have slightly longer follow up. Together, the studies provide a highly consistent picture. The proportion of RA patients with self-limiting disease after 2 years follow up increased from 2 to 12% when applying the 2010 ACR/EULAR criteria. A similar increase was seen in the Birmingham cohort, even though in this cohort a higher number of RA patients with self-limiting disease could already be observed in patients according to the 1987 ACR criteria. In addition, we found that the proportion of patients who develop erosive disease over time is somewhat lower when applying the 2010 ACR/EULAR criteria. Use of the 2010 ACR/EULAR criteria resulted in more patients previously being classified as UA now diagnosed with RA. On the other hand, more patients with selflimiting disease were classified as RA as well. It should also be noted that on the group level, the clinical characteristics of patients with RA according to the 2010 ACR/EULAR criteria differ on some important aspects from RA patients fulfilling the 1987 ACR criteria; generally the disease is less severe, more often mono- or oligoarticular and less frequently autoantibody positive. Taken together, use of the 2010 ACR/EULAR criteria allows earlier diagnosis of RA, although the clinical picture is somewhat different on the group level, which may be important for clinical research. Furthermore, some patients with self-limiting disease may be falsely diagnosed with RA. ACKNOWLEDGEMENTS This study was supported by the Reumafonds (Dutch Arthritis Association). 30

32 2010 ACR/EULAR CRITERIA FOR RA IN EARLY ARTHRITIS PATIENTS REFERENCE LIST 1. Thurlings RM, Wijbrandts CA, Mebius RE, Cantaert T, Dinant HJ, van der Pouw-Kraan TC et al. Synovial lymphoid neogenesis does not define a specific clinical rheumatoid arthritis phenotype. Arthritis Rheum 2008; 58: van Baarsen LG, Wijbrandts CA, Timmer TC, van der Pouw Kraan TC, Tak PP, Verweij CL. Synovial tissue heterogeneity in rheumatoid arthritis in relation to disease activity and biomarkers in peripheral blood. Arthritis Rheum 2010; 62: Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31: Finckh A, Liang MH, van Herckenrode CM, de Pablo P. Long-term impact of early treatment on radiographic progression in rheumatoid arthritis: A meta-analysis. Arthritis Rheum 2006; 55: Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO, III et al Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62: Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO, III et al rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010; 69: MacGregor AJ. Classification criteria for rheumatoid arthritis. Baillieres Clin Rheumatol 1995; 9: Dougados M, van der Linden S, Juhlin R, Huitfeldt B, Amor B, Calin A et al. The European Spondylarthropathy Study Group preliminary criteria for the classification of spondylarthropathy. Arthritis Rheum 1991; 34: Taylor W, Gladman D, Helliwell P, Marchesoni A, Mease P, Mielants H. Classification criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum 2006; 54: Wallace SL, Robinson H, Masi AT, Decker JL, McCarty DJ, Yu TF. Preliminary criteria for the classification of the acute arthritis of primary gout. Arthritis Rheum 1977; 20: Visser H, le CS, Vos K, Breedveld FC, Hazes JM. How to diagnose rheumatoid arthritis early: a prediction model for persistent (erosive) arthritis. Arthritis Rheum 2002; 46: Smolen JS, Landewe R, Breedveld FC, Dougados M, Emery P, Gaujoux-Viala C et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs. Ann Rheum Dis 2010; 69: van der Linden MP, Knevel R, Huizinga TW, van der Helm-van Mil AH. Classification of rheumatoid arthritis: comparison of the 1987 American College of Rheumatology criteria and the 2010 American College of Rheumatology/European League Against Rheumatism criteria. Arthritis Rheum 2011; 63: Cader MZ, Filer A, Hazlehurst J, de PP, Buckley CD, Karim R. Performance of the 2010 ACR/EULAR criteria for rheumatoid arthritis: comparison with 1987 ACR criteria in a very early synovitis cohort. Ann Rheum Dis 2011; 70:

33 SUPPLEMENTARY TABLES Supplementary Table 1. Classification according to 2010 ACR/EULAR criteria in all RA and UA patients after 2 years follow up when applying 1987 ACR criteria 2010 ACR/EULAR criteria at baseline 1987 ACR criteria after 2 years follow up RA UA RA, rheumatoid arthritis; UA, undifferentiated arthritis. RA UA Supplementary Table 2. Patient characteristics of patients with a false positive or false negative diagnosis of RA when applying 2010 ACR/EULAR criteria using RA according to 1987 ACR criteria after 2 years follow up as gold standard False positive (n=25) False negative (n=10) Female (%) Age, years (Median (range)) 45 (18-77) 61 (41-82) ESR, mm/hr (Median (range)) 15 (3-117) 8 (3-77) CRP, mg/l (Median (range)) 8 (0-64) 6 (1-34) DAS28 (Median (range)) 4.7 ( ) 4.3 ( ) Joint involvement 68 TJC (Median (range)) 11 (0-56) 5 (1-11) 66 SJC (Median (range)) 3 (0-19) 3 (0-12) Monoarthritis (%) Autoantibody status IgM-RF and ACPA negative (%) IgM-RF and/or ACPA low positive (%) 4 10 IgM-RF and/or ACPA high positive (%) 28 0 Duration of symptoms Arthritis duration, months (median (range)) 5 (0-12) 3 (0-12) Outcome after 2 years follow up Self-limiting (%) 50 0 Persistent non-erosive (%) Persistent erosive (%) 0 56 RA, rheumatoid arthritis; ESR, erythrocyte sedimentation rate; CRP, c-reactive protein; 68 TJC, 68 tender joint count; 66 SJC, 66 swollen joint count; IgM-RF, IgM-rheumatoid factor; ACPA, anticitrullinated protein antibodies; low positive: value higher than upper limit of normal but 3 times; high positive: value > 3 times upper limit of normal. 32

34

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36 SMOKING AND OVERWEIGHT DETERMINE THE LIKELIHOOD OF DEVELOPING RHEUMATOID ARTHRITIS Maria J.H. de Hair, M.D. 1, Prof. Robert B. Landewé, M.D., Ph.D. 12, Marleen G.H. van de Sande, M.D., Ph.D. 1, Dirkjan van Schaardenburg, M.D., Ph.D. 3, Lisa G.M. van Baarsen Ph.D. 1, Danielle M. Gerlag, M.D., Ph.D. 1, Prof. Paul P. Tak, M.D., Ph.D. 1* 1 Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands, 2 Atrium Medical Center, Heerlen, the Netherlands, 3 Department of Rheumatology, Jan van Breemen Research Institute/Reade, Amsterdam, the Netherlands 3 Ann Rheum Dis Nov 17 [Epub ahead of print]

37 ABSTRACT Objectives Rheumatoid arthritis (RA) is a prototypic chronic inflammatory disease with a debilitating course if untreated. A genetic predisposition for RA is known, and its occurrence is associated with the presence of autoantibodies in the serum as well as environmental factors. It is unknown if smoking and overweight are contributory factors for developing RA in individuals with RA-specific autoantibodies in the serum. Methods Fifty-five individuals at risk for developing RA, based on the presence of RA-specific autoantibodies in the serum, who never had any evidence of arthritis upon physical examination, were followed over time. Smoking was assessed as being never or ever smoker, and body mass index (BMI) as <25 (normal) or 25kg/m 2 (overweight). Clinical endpoint was the occurrence of arthritis. Proportional hazard regression analysis was performed to investigate the potential of (combinations of) variables in predicting the onset of arthritis over time. Results After a median follow up time of 13 (IQR 6-27) months, 15 individuals (27%) developed arthritis. Smoking was associated with the development of arthritis (hazard ratio (HR) (95%CI): 9.6 (1.3 to 73.0); p=0.029). Overweight was, independently of smoking, associated with arthritis (HR (95%CI): 5.6 (1.3 to 25.0); p=0.023). The overall arthritis risk of 28% after a median of 27 months follow up increased to 60% in individuals with a smoking history combined with overweight. Conclusions This is the first prospective study showing that smoking and overweight increase the risk of development of arthritis in a cohort of autoantibody-positive individuals at risk for developing RA. These results show the importance of life style factors in development of RA and should be critically evaluated in future clinical research aimed at disease prevention. 36

38 SMOKING AND OVERWEIGHT DETERMINE THE LIKELIHOOD OF DEVELOPING RA Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease characterized by inflammation of synovial joints, often resulting in degradation of articular cartilage and bone, ultimately leading to joint deformities. If untreated, RA leads to disability, loss of quality of life, and work loss. RA causes premature death due to cardiovascular disease, analogous to diabetes mellitus 1 and the impact of RA on costs for society is huge 2. The treatment of established RA is promising but expensive; therefore the need for prevention of RA, if possible, is obvious. The etiology of RA, though largely unknown, is considered multifactorial: a family history of RA and the presence of MHC class II genes 3 and PTPN22 4 increase the susceptibility of RA; the presence of rheumatoid factor (RF) and anti-citrullinated protein-antibodies (ACPA) point to a contribution of autoimmunity mechanisms, and environmental factors such as smoking 5 and obesity 6-8, and their interactions with genetic factors, have been considered important 9. Recent research has discovered that circulating autoantibodies and increased acute phase reactants 13 can precede the clinical onset of RA, but only a minority of individuals with RA-specific autoantibodies actually develops clinically manifest RA 14. The detection of these autoantibodies, however, may define patients with systemic auto-immunity associated with RA without clinical evidence of arthritis, who are at risk of developing RA 15. In this prospective observational study the contributory role of the modifiable factors smoking and overweight on the development of arthritis in autoantibody-positive individuals at risk for developing RA was investigated. 3 METHODS Study subjects Individuals with either arthralgia and/or a positive family history for RA, but without any evidence of arthritis upon thorough physical examination, who were positive for IgM-RF and/or ACPA, were included in the study between June 2005 and August IgM-RF was measured using IgM-RF ELISA (Sanquin, Amsterdam, the Netherlands (upper limit of normal (ULN) 12.5 IU/mL)) until December 2009 and thereafter using IgM-RF ELISA (Hycor Biomedical, Indianapolis, IN (ULN 49 IU/mL)). ACPA was measured using anti-ccp2 ELISA CCPlus (Eurodiagnostica, Nijmegen, the Netherlands (ULN 25 kau/l)). These individuals were recruited via the outpatient clinic of the department of Clinical Immunology and Rheumatology of the AMC, Amsterdam, via the Rheumatology outpatient clinic of Reade, Amsterdam, or via testing family members of RA patients seen at the outpatient clinic or at public fairs across the Netherlands. The study was performed according to the principles of the Declaration of Helsinki, approved by the institutional review board, and all study subjects gave written informed consent. Study design At baseline, demographic parameters were obtained as well as environmental and clinical parameters. Overweight was defined as a body mass index (BMI) 25kg/m 2, according to the World Health Association (fact sheet n 311). A detailed smoking history and smokingstatus using number of pack years was assessed, and smoking status was assigned 37

39 as being a never smoker or an ever smoker. One pack year was defined as smoking 20 cigarettes per day for one year. Study subjects were followed over time until January Annual study visits were performed. The development of arthritis, defined as a painful and swollen joint, was the endpoint of this study. In individuals with suspected arthritis an extra visit was performed, at which the presence of arthritis was confirmed by two independent investigators (MS and DG or MH and DG). Statistical analysis Differences between study groups in continuous parameters were analyzed using t-test or Mann-Whitney U-test if appropriate. Categorical data were analyzed using Chi-square test. Prediction analysis for arthritis-onset was performed using proportional hazard regression analysis (Cox-regression). Follow-up duration was defined as the time between inclusion in the cohort and the onset of clinically manifest arthritis, or between inclusion and January 2012 (censored). After univariate analysis, variables with p-value <0.2 were selected for a multivariable analysis (forward and backward selection procedures). Meaningful statistical interactions were excluded upfront. Statistical analysis was performed using PASW Statistics 18 (SPSS Inc, Chicago, Il). RESULTS Cohort description Fifty-five autoantibody-positive individuals were included in the study (51 were referred to rheumatology outpatient clinics because of arthralgia, and 4 subjects without arthralgia were tested because of a positive family history for RA). Nineteen individuals were solely IgM-RF-positive, 22 were solely ACPA-positive and 14 were positive for both autoantibodies. Individuals were followed for a median duration of 27 (IQR: 14-47; range 1-75) months. Fifteen of the 55 individuals (27%) developed arthritis during follow up after a median duration of 13 (IQR: 6-27, range ) months. Of the 15 patients who developed arthritis, 11 fulfilled the 2010 ACR/EULAR criteria for RA 17, 18 at arthritis onset and 3 were initially diagnosed as having undifferentiated arthritis (UA) but fulfilled the RA classification criteria later on (6 fulfilled the 1987 ACR criteria for RA [20] at arthritis onset and 1 later on). For clinical parameters at arthritis onset as well as baseline see online supplementary Table S1. One patient who developed arthritis fulfilled the ACR classification criteria for osteoarthritis (OA) of the hand 19, but not for RA. This patient received non-steroidal anti-inflammatory drugs resulting in a permanent remission of clinical signs and symptoms of arthritis. Of the 14 patients ultimately fulfilling the classification criteria for RA, 2 did not receive diseasemodifying antirheumatic drugs (DMARDs). One patient refused conventional treatment and used alternative medicines and the other patient refused treatment because of transient mono-arthritis for which he was closely monitored. Seven RA patients were treated with DMARD monotherapy, 2 with a combination of two DMARDs, 1 with a combination of a DMARD and anti-tnf therapy and 1 was included in a clinical trial evaluating the effects of anti-tnf therapy in early arthritis. After a median follow up of 18 (range 0-60) months 38

40 SMOKING AND OVERWEIGHT DETERMINE THE LIKELIHOOD OF DEVELOPING RA 3 RA patients developed RA-erosions on X-ray (the 2 patients who did not receive DMARD therapy and one patient treated with DMARD monotherapy). Increased percentage of smoking and overweight in autoantibody-positive individuals who develop arthritis after follow up The percentage of ever smokers and individuals with BMI 25 kg/m 2 was higher in the group that developed arthritis than in the group that did not (93% vs 53%; p=0.005 for ever vs never smoking and 87% vs 45%; p=0.006 for individuals with a BMI 25 kg/m 2 vs those with a BMI <25 kg/m 2 ). The percentage of individuals who developed arthritis was 38% in past smokers (n=21) versus 43% in current smokers (n=14) (p=0.778 for the comparison between past and current smokers), compared to 5% in the never smokers (n=21) (p=0.019 for the comparison between never smokers and past/current smokers). IgM-RF- and ACPA-status and levels were comparable between individuals who developed arthritis versus those who did not (see Table 1). 3 Smoking and overweight are predictors for the development of clinically manifest arthritis In the proportional hazard regression analysis, ever smoking was associated with the development of clinically manifest arthritis (HR (95%CI): 9.6 (1.3 to 73.0); p=0.029) (Table 2). Table 1. Baseline demographic and clinical parameters Characteristic No arthritis developed Arthritis developed N=40 # N=15 P-value Sex, female (n (%)) 28 (70) 9 (60) Age (years) (mean (SD)) 44 (12) 47 (8) Smoking history, ever * (n (%)) 21 (53) 14 (93) Pack years ** (median (IQR)) 10 (4-20) 15 (10-24) BMI 25, kg/m 2 (n (%)) 18 (45) 13 (87) IgM-RF positive (n (%)) 24 (57) 9 (60) IgM-RF level low positive (n (%)) *** 15 (62) 4 (44) IgM-RF level high positive (n (%)) *** 9 (38) 5 (56) $ ACPA positive (n (%)) 24 (60) 12 (80) ACPA level *** (kau/l) (median (IQR)) ( ) ( ) IgM-RF and ACPA both pos. (n (%)) 8 (20) 6 (40) # 7 Individuals have been at risk for arthritis development for a shorter period of time: two patients have started with hydroxychloroquine for arthralgias, and two with anti-tnf therapy because of a diagnosis of ankylosing spondylitis (n=1) or Crohn s disease (n=1). Three individuals were lost to follow up after some time. * Smoking history: never smoked: 0 pack-years; ever smoked: >0 pack-years BMI: body mass index; IgM-RF: IgM-rheumatoid factor; ** Only in smokers *** only in positive individuals: low positive: 3 times upper limit of normal (ULN), high positive: >3 times ULN (according to the 2010 ACR/EULAR criteria for RA 17, 18 ); $ Using Chi-square test for frequencies of low and high positivity of IgM-RF in the two outcome groups; ACPA: anti-citrullinated protein antibodies P-values < 0.05 in bold. 39

41 Table 2. Multivariable regression analysis for smoking and BMI Variables in model Hazard ratio (95% confidence interval) P-value Model 1: Smoking (ever vs never) 9.6 (1.3 to 72.9) Model 2: BMI ( 25 vs <25 kg/m 2 ) 5.6 (1.3 to 25.0) Model 3: Smoking (ever vs never) BMI ( 25 vs <25 kg/m 2 ) BMI: body mass index. 8.2 (1.1 to 62.6) 4.8 (1.1 to 21.4) Only one individual who never smoked developed arthritis over time. There was no difference between past and current smokers with regard to the onset of arthritis (p=0.677). The relationship between smoking and the development of arthritis was not confounded by any of the other variables measured (data not shown). Besides smoking, BMI 25 kg/m 2 at baseline was associated with the development of arthritis (HR (95%CI): 5.6 (1.3 to 25.0); p=0.023). In the proportional hazard regression analysis smoking and BMI independently conferred an increased risk on clinically manifest arthritis. Of importance, BMI did not significantly correlate with smoking status (p=0.472) and within the group of ever smokers BMI was significantly higher in the subgroup of individuals who developed arthritis compared to the group who did not (28.1 ( ) (median (IQR)) vs ( ): p=0.010). In this cohort IgM-RF positivity was not associated with the development of arthritis, and ACPA-positivity showed a trend towards a predictive association (HR (95%CI): 2.7 (0.8 to 9.7); p=0.119), but did not have predictive value over and above smoking and/or BMI. Of note, subjects were selected based on being positive for IgM-RF and/or ACPA, which limits the predictive interpretability of IgM-RF and ACPA in this cohort. Other clinical parameters were not associated with the development of arthritis (data not shown). When combining smoking and BMI the frequency of arthritis development was significantly higher in ever smokers with BMI 25 kg/m 2 compared to the other 3 categories (p=0.001) (see Table 3). In conclusion, when performing proportional hazard regression analysis the combination of being an ever smoker with BMI 25 kg/m 2 resulted in an increase of the Table 3. Frequency table for arthritis development in subgroups of smoking and BMI Never smoker Ever smoker BMI <25 kg/m 2 (N=10) BMI 25 kg/m 2 (N=10) BMI <25 kg/m 2 (N=14) BMI 25 kg/m 2 (N=21) Follow-up time, months, median (IQR) 40 (24-71) 28 (17-51) 23 (17-41) 26 (9-42) Arthritis developed, n (%) 0 (0) 1 (10) 2 (14) 12 (57) BMI: body mass index 40

42 SMOKING AND OVERWEIGHT DETERMINE THE LIKELIHOOD OF DEVELOPING RA risk for development of clinically manifest arthritis from 28% to 60% after median follow up in this cohort of 27 months ( Figure 1). 3 Figure 1. Cumulative hazard for arthritis development in subgroups of smoking and body mass index. X-axis: follow up time in months; Y-axis: cumulative hazard for arthritis development DISCUSSION The results presented here show that modifiable lifestyle factors such as smoking and overweight may importantly contribute to the onset of clinically manifest arthritis in autoantibody-positive individuals at risk of developing RA. The first important observation in this prospective study is the association between smoking and development of arthritis. Smoking has recently been suggested to be a risk factor for developing RA 5, but so far only association studies have been performed. Our study is the first analyzing the impact of smoking on the development of arthritis by prospectively following individuals with RA-specific autoantibodies without signs of arthritis who are at risk of developing the disease. Recent studies have suggested that the lung may be an early site of RA-related auto-immunity, supposedly by the effect of smoking on the citrullination of peptides 20. Interestingly, in a comparable cohort of individuals at risk for RA with elevated ACPA levels and 2 RF isotypes without arthritis airway abnormalities were observed similar to those found in RA patients, and significantly more than in autoantibody-negative controls 21. In contrast to what has been suggested previously 22, the high risk for smoking on arthritis development was not dependent on ACPA-status in our study. In addition, we were unable to define ACPA-status itself as risk factor, in contrast to what was observed in another cohort studying 147 autoantibody-positive individuals without clinically 41

43 apparent arthritis 14. This is likely due to the relatively small number of study participants and differences in inclusion criteria between the current study and the previous. Second, overweight appeared to be a predicting factor for arthritis onset, independently of smoking. Thus far, the results of association studies of overweight/obesity and RA have been variable 6-8, 23-26, with different results for ACPA positive and negative RA 25, 26 and males and females 6, 26. More in general, an association between obesity and inflammation has been clearly demonstrated 27. Adiposity is characterized by adipocyte hypertrophy, leading to release of stress signals, such as endoplasmic reticulum stress 28, and production of reactive oxygen species. As a result, inflammatory pathways are activated and inflammatory cells, such as macrophages and CD8+ T-cells, recruited into the adipose tissue. Adiposity is associated with increased production of pro-inflammatory adipocytokines, including leptin, TNFα, interleukin-1 (IL-1), IL-6 and monocyte chemotactic protein-1 (MCP-1), and decreased production of the anti-inflammatory adiponectin. How this systemic proinflammatory state would lead to inflammation in the joint,needs to be elucidated. It is also possible that pro-inflammatory activity of adipose tissue in the synovial sublining of the joint might be involved. Previous work has shown that articular adipose tissue obtained from RA patients produced pro- and anti-inflammatory (adipo-) cytokines upon activation, with stimulating effects on fibroblast-like synoviocytes 29. In our cohort the overall arthritis risk of 28% after a median of 27 months follow up increased to 60% in individuals with a smoking history combined with overweight. This observation was made in a relatively small cohort, suggesting that the effects of smoking and overweight are strong. In comparison, the risk for developing arthritis in never smokers with normal weight was only 2%, showing that life style modification might have important consequences for arthritis development in RA-prone individuals. The importance of our findings is emphasized by the increased incidence of RA over the last decades, which cannot be explained by genetic factors, but is most likely influenced by environmental factors 8. Although this study does not demonstrate that smoking cessation and/or weight reduction are effective in reducing the risk of RA, it is obvious that smoking and increased body weight are inherently modifiable lifestyle factors which are also important in other diseases and that modification leads to decreased health risks in general. An intensive prevention program in Finland aiming at dietary changes and smoking cessation has resulted in long-term prevention of cardiovascular diseases 30 which was accompanied by a similar decline in the incidence of RA 31. Such programs may contribute to the prevention of RA and related co-morbidities and to a decreased socio-economic burden. The results of this study, if confirmed in larger, independent cohorts, may also help to better define a population at high risk of developing RA based on the presence of RAspecific autoantibodies, smoking history, and overweight. Improved prediction models may facilitate studies aimed at prevention of RA by targeted intervention during the preclinical phase 32. A limitation of this study is the relatively small sample size. The effects observed in our cohort provide the rationale for larger studies in independent cohorts to validate the results presented here. Currently we are unable to determine whether smoking and 42

44 SMOKING AND OVERWEIGHT DETERMINE THE LIKELIHOOD OF DEVELOPING RA obesity are crucial for development of autoantibody positive RA in most patients, or whether these factors merely advance the onset of arthritis. We will continue to follow these individuals to address this issue over time. It should also be noted that we chose to define the presence of arthritis based on clinical assessment by two experienced investigators rather than by imaging, consistent with clinical practice and evaluation in most clinical trials. Of note, the implications of synovitis shown by ultrasound or MRI in the absence of clinical evidence of arthritis are currently still incompletely understood. In conclusion, the results presented here suggest that preventable factors, such as smoking and overweight, may increase the risk of developing RA in RF and/or ACPA positive individuals. 3 ACKNOWLEDGEMENTS We thank our study subjects for participating in the study. The study was supported by the Dutch Arthritis Foundation (grant and ), the Netherlands Organisation for Health Research and Development (ZonMw) grant , and the IMI EU funded project BeTheCure n

45 REFERENCE LIST 1. Bisoendial RJ, Stroes ES, Tak PP. Critical determinants of cardiovascular risk in rheumatoid arthritis. Curr Pharm Des 2011;17(1): Boonen A, Severens JL. The burden of illness of rheumatoid arthritis. Clin Rheumatol 2011;30 Suppl 1:S3-S8. 3. Stastny P. Association of the B-cell alloantigen DRw4 with rheumatoid arthritis. N Engl J Med 1978;298(16): Begovich AB, Carlton VE, Honigberg LA et al. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004;75(2): Sugiyama D, Nishimura K, Tamaki K et al. Impact of smoking as a risk factor for developing rheumatoid arthritis: a metaanalysis of observational studies. Ann Rheum Dis 2010;69(1): Symmons DP, Bankhead CR, Harrison BJ et al. Blood transfusion, smoking, and obesity as risk factors for the development of rheumatoid arthritis: results from a primary care-based incident case-control study in Norfolk, England. Arthritis Rheum 1997;40(11): Voigt LF, Koepsell TD, Nelson JL, Dugowson CE, Daling JR. Smoking, obesity, alcohol consumption, and the risk of rheumatoid arthritis. Epidemiology 1994;5(5): Crowson CS, Matteson EL, Davis JM, III, Gabriel SE. Obesity fuels the upsurge in rheumatoid arthritis. Arthritis Care Res (Hoboken ) Scott IC, Steer S, Lewis CM, Cope AP. Precipitating and perpetuating factors of rheumatoid arthritis immunopathology: linking the triad of genetic predisposition, environmental risk factors and autoimmunity to disease pathogenesis. Best Pract Res Clin Rheumatol 2011;25(4): Aho K, Heliovaara M, Maatela J, Tuomi T, Palosuo T. Rheumatoid factors antedating clinical rheumatoid arthritis. J Rheumatol 1991;18(9): Nielen MM, van SD, Reesink HW et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum 2004;50(2): Rantapaa-Dahlqvist S, de Jong BA, Berglin E et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003;48(10): Nielen MM, van SD, Reesink HW et al. Increased levels of C-reactive protein in serum from blood donors before the onset of rheumatoid arthritis. Arthritis Rheum 2004;50(8): Bos WH, Wolbink GJ, Boers M et al. Arthritis development in patients with arthralgia is strongly associated with anti-citrullinated protein antibody status: a prospective cohort study. Ann Rheum Dis 2010;69(3): Gerlag DM, Raza K, van Baarsen LG et al. EULAR recommendations for terminology and research in individuals at risk of rheumatoid arthritis: report from the Study Group for Risk Factors for Rheumatoid Arthritis. Ann Rheum Dis 2012;71(5): van de Sande MG, de Hair MJ, van der Leij C et al. Different stages of rheumatoid arthritis: features of the synovium in the preclinical phase. Ann Rheum Dis 2011;70(5): Aletaha D, Neogi T, Silman AJ et al Rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010;62(9): Aletaha D, Neogi T, Silman AJ et al rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010;69(9): Altman R, Alarcon G, Appelrouth D et al. The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the hand. Arthritis Rheum 1990;33(11): Klareskog L, Malmstrom V, Lundberg K, Padyukov L, Alfredsson L. Smoking, citrullination and genetic variability in the immunopathogenesis of rheumatoid arthritis. Semin Immunol 2011;23(2): Demoruelle MK, Weisman MH, Simonian PL et al. Brief report: airways abnormalities and rheumatoid arthritis-related autoantibodies in subjects without arthritis: early injury or initiating site of autoimmunity? Arthritis Rheum 2012;64(6): Lundstrom E, Kallberg H, Alfredsson L, Klareskog L, Padyukov L. Gene-environment interaction between the DRB1 shared epitope and smoking in the risk of anti-citrullinated 44

46 SMOKING AND OVERWEIGHT DETERMINE THE LIKELIHOOD OF DEVELOPING RA protein antibody-positive rheumatoid arthritis: all alleles are important. Arthritis Rheum 2009;60(6): Cerhan JR, Saag KG, Criswell LA, Merlino LA, Mikuls TR. Blood transfusion, alcohol use, and anthropometric risk factors for rheumatoid arthritis in older women. J Rheumatol 2002;29(2): Hernandez AM, Liang MH, Willett WC et al. Reproductive factors, smoking, and the risk for rheumatoid arthritis. Epidemiology 1990;1(4): Pedersen M, Jacobsen S, Klarlund M et al. Environmental risk factors differ between rheumatoid arthritis with and without auto-antibodies against cyclic citrullinated peptides. Arthritis Res Ther 2006;8(4):R Wesley A, Bengtsson C, Elkan AC, Klareskog L, Alfredsson L, Wedren S. Association between overweight, obesity, ACPA positive and ACPA negative Rheumatoid Arthritis-, results from the EIRA case-control study. Arthritis Care Res (Hoboken ) Ouchi N, Parker JL, Lugus JJ, Walsh K. Adipokines in inflammation and metabolic disease. Nat Rev Immunol 2011;11(2): Guri AJ, Bassaganya-Riera J. Systemic effects of white adipose tissue dysregulation and obesity-related inflammation. Obesity (Silver Spring) 2011;19(4): Kontny E, Plebanczyk M, Lisowska B, Olszewska M, Maldyk P, Maslinski W. Comparison of rheumatoid articular adipose and synovial tissue reactivity to proinflammatory stimuli: contribution to adipocytokine network. Ann Rheum Dis 2012;71(2): Puska P, Vartiainen E, Tuomilehto J, Salomaa V, Nissinen A. Changes in premature deaths in Finland: successful long-term prevention of cardiovascular diseases. Bull World Health Organ 1998;76(4): Kaipiainen-Seppanen O, Kautiainen H. Declining trend in the incidence of rheumatoid factor-positive rheumatoid arthritis in Finland J Rheumatol 2006;33(11): Tak PP. Are we ready to change the pace of arthritis treatment? Treating pre-arthritis and very early arthritis. Acta Reumatol Port 2011;36(1):

47 Supplementary table S1. Disease activity at baseline and arthritis onset Disease activity parameter No arthritis developed; Baseline N=40 Arthritis developed; Baseline N=15 Arthritis developed; Arthritis onset N=15 68TJC (n) (median (IQR)) 2 (0-8) 2 (0-8) 8 (3-16) * 66SJC (n) (median (IQR)) 0 (0) 0 (0) 5 (2-7) ** ESR (mm/hr) (median (IQR)) 9 (2-20) 7 (5-15) 12 (6-17)** CRP (mg/l) (median (IQR)) 2.1 ( ) 4.0 ( ) 5.2 ( ) *** 68TJC: tender joint count of 68 joints; 66SJC: swollen joint count of 66 joints; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; * missing n=5; ** missing n=1; *** missing n=3. 46

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50 PART THE SYNOVIUM DURING THE EARLIEST STAGES OF RA II

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52 EXPERIENCE OF PATIENTS UNDERGOING MINI-ARTHROSCOPY COMPARED TO MRI IN THE EARLIEST PHASES OF ARTHRITIS Maria J.H. de Hair M.D. 1, Marleen G.H. van de Sande M.D. Ph.D. 1, Mario Maas M.D. Ph.D. 2, Danielle M. Gerlag M.D. Ph.D. 1, Prof. Paul P. Tak M.D. Ph.D. 1 1 Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, the Netherlands, 2 Department of Radiology, Academic Medical Center/University of Amsterdam, Amsterdam, the Netherlands 4 Submitted for publication

53 ABSTRACT Objectives To evaluate the expectations and experience of patients undergoing mini-arthroscopy compared to contrast enhanced MRI for research purposes. Methods Seventeen patients with early, active arthritis (group A) and 21 autoantibody-positive individuals without any evidence of arthritis upon physical examination (group B) were included. All subjects underwent both contrast enhanced MRI and synovial biopsy sampling by mini-arthroscopy of the same joint within one week. At inclusion and after both procedures, subjects filled in questionnaires with items about expectations and experience with regard to the procedures. Results Before procedures subjects in group B had higher fear of and reluctance to undergo miniarthroscopy compared to MRI (p< and p=0.001, respectively). Before procedures 42% of the subjects preferred MRI, 11% of the subjects preferred mini-arthroscopy and 47% had no preference for either procedure. After both procedures subjects preferences changed to 39% for MRI, 32% for mini-arthroscopy and 29% for no preference for one or the other procedure. When comparing group A with group B, there were no significant differences in preference before and after the procedures. Conclusions Synovial biopsy sampling by mini-arthroscopy for analysis of synovial inflammation is a well-experienced procedure when compared to contrast enhanced MRI. These results support the use of mini-arthroscopy in a research setting from a patient perspective. 52

54 PATIENT TOLERABILITY OF MINI-ARTHROSCOPY COMPARED TO MRI Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by inflammation of synovial tissue leading to joint destruction and deformity 1. Since the synovium is the main target tissue affected in RA, analysis of the features of the synovial inflammation is of major importance for pathogenetic studies. For analysis of synovial inflammation imaging but also histologic studies can be used. Analysis of the synovial tissue can be used to give insight into disease pathogenesis and to evaluate the effects of new treatments and the mechanisms of action of therapeutic compounds. Mini-arthroscopy, performed under local anaesthetics at the outpatient clinic, is a feasible means of synovial biopsy sampling. It has been used for research purposes and is generally well tolerated with low complication rates 2-4. Still, mini-arthroscopy is regarded as a rather invasive procedure and only performed in a few specialised centres. Imaging of synovial inflammation can be done making use of MRI. MRI gives information about the degree of synovial inflammation, and additionally the compartment surrounding the synovium can be evaluated, including the bone (marrow) and cartilage. In the current project we performed dynamic contrast enhanced (DCE) MRI, by injecting a contrast agent intravenously during and after which time-dependent changes in MRI signal can be registered. DCE-MRI clearly visualizes the degree of synovial inflammation 5, enables to study physiologic characteristics of the inflamed synovium, such as vessel permeability, and has been shown to be a sensitive tool to detect changes after treatment At international scientific meetings, when results from studies using synovial biopsy sampling by mini-arthroscopy are presented by researchers of our Department of Clinical Immunology and Rheumatology of the Academic Medical Center (AMC) Amsterdam, questions are raised concerning patient s experience of synovial biopsy sampling by miniarthroscopy, especially in individuals without arthritis. It seems that there is a general idea that mini-arthroscopy is an invasive procedure and a burden for patients, which seems to hamper the use of mini-arthroscopic synovial biopsy sampling in some research centres. Patient expectations and experience of mini-arthroscopy have never been studied. Therefore, we investigated patient s expectations before and experience after mini-arthroscopic synovial biopsy sampling and compared those to expectations before and experience after undergoing dynamic contrast-enhanced MRI, which is generally seen as a non-invasive procedure. 4 METHODS Study subjects Group A consisted of early arthritis patients (arthritis duration less than 1 year) with an inflamed knee, ankle or wrist, who were disease modifying antirheumatic drug naive (AMC s Synoviomics program) 12. Group B consisted of individuals at risk for developing RA, defined by the presence of IgM-rheumatoid factor and/or anti-citrullinated protein antibodies, but no evidence of arthritis upon physical examination 13 (AMC s PreSynoviomics program) 14. The study was performed according to the principles of the Declaration of Helsinki, approved by the institutional review board, and all study subjects gave written informed consent. 53

55 MRI All study subjects underwent DCE-MRI as previously described 6. In group A, a clinically inflamed (swollen and painful) wrist, knee or ankle joint was examined and in group B an arbitrarily chosen knee joint was examined in all cases. Briefly, images were acquired on either a closed (1.5 Tesla GE Signa Horizon Echospeed, LX9.0, General Electric Medical Systems, Milwaukee, Wisconsin, USA) or open (Panorama 1 Tesla Open, Philips, Best, the Netherlands) MRI scanner, depending on the availability of the machine. Three scans were performed after which a contrast agent gadolinium (Magnevist, Schering, Berlin, Germany) was injected intravenously and 2 additional scans were performed. Total duration of the procedure was 60 minutes. Synovial biopsy sampling by mini-arthroscopy Within one week after the MRI, synovial biopsy sampling was performed at the outpatient clinic by means of mini-arthroscopy under local anaesthetics, as previously described 2, 6, 15. The same joint was chosen for both procedures. For each study group 24 up to 32 synovial tissue biopsies were obtained during one procedure. The duration of the total procedure was 45 to 60 minutes. Questionnaires Before and after both procedures, subjects filled in questionnaires with items about expectations and the experience they had with regard to the procedures. Questions asked were 1. Do you have preference for MRI or mini-arthroscopy or do you have no preference? 2. Please mark how well you think you are prepared for (a) MRI and (b) mini-arthroscopy 3. Please mark the level of fear you experience of (a) MRI and (b) mini-arthroscopy? 4. Please mark if you are reluctant to undergo (a) MRI and (b) mini-arthroscopy. The first question was multiple choice; the latter three questions were depicted on a visual analogue scale (VAS) of mm. In addition, study subjects could comment their choice of preference for one of the procedures. The first questionnaire was completed and handed in before and the second questionnaire was filled in after both procedures. Statistical analysis We describe preference for either of the procedures before and after the procedures and compared preference in group A with group B using Chi-square test. In addition, differences in baseline emotional aspects with respect to both procedures and differences in preference after procedures compared to baseline were analysed using Wilcoxon signed rank test for related samples. P-value <0.5 was considered statistically significant. Statistical analysis was performed using PASW Statistics 18 (SPSS Inc, Chicago, Il). RESULTS Of 38 subjects baseline and follow-up questionnaires were available: 17 from group A and 21 from group B. Table 1 shows the disposition of study subjects with regard to type of joint examined and MRI machine used. 54

56 PATIENT TOLERABILITY OF MINI-ARTHROSCOPY COMPARED TO MRI Emotional aspects With respect to emotional aspects subjects generally felt well prepared for both procedures. In group B scores for fear and reluctance were higher for mini-arthroscopy compared to MRI, see Table 2. This was not the case for group A. However, there were no statistically significant differences between group A and B at baseline for individual emotional aspects (Table 3). 4 Experience of mini-arthroscopy and DCE-MRI In the total study population, before undergoing both procedures 42% of the subjects preferred MRI, 11% preferred mini-arthroscopy and 47% had no preference for either procedure. After both procedures subjects preference changed to 39% preferring MRI, 32% mini-arthroscopy and 29% having no preference for one of the procedures for studying synovitis. This shows that there was not a clear preference for one of the procedures. In addition, preference after both procedures was not significantly different from baseline preference (p=0.602). Table 1. Disposition of study subjects Group A* Open MRI Closed MRI Wrist 0 1 Knee 2 10 Ankle 1 3 Group B* Open MRI Closed MRI Knee 6 15 *Group A represents early arthritis patients, group B represents autoantibody-positive individuals without arthritis at risk for developing RA. Table 2. Emotional aspects regarding DCE-MRI and mini-arthroscopy at baseline Group A* Mini-arthroscopy DCE-MRI P-value Preparation for procedure 85 (52-91) 82 (60-94) Fear of procedure 25 (6-46) 5 (0-51) Being reluctant to undergo procedure 18 (6-31) 4 (0-43) Group B* Mini-arthroscopy DCE-MRI P-value Preparation for procedure 90 (59-96) 89 (62-96) Fear of procedure 21 (7-57) 3 (0-7) Being reluctant to undergo procedure 16 (4-47) 1 (0-5) DCE-MRI: dynamic contrast enhanced MRI; All items measured on a visual analogue scale of mm; Results depicted as median (IQR). *Group A represents early arthritis patients, group B represents autoantibody-positive individuals without arthritis at risk for developing RA. 55

57 Table 3. A comparison of emotional aspects regarding DCE-MRI and mini-arthroscopy at baseline between group A and group B Group A* Group B* P-value Preparation for MRI 82 (60-94) 89 (62-96) Preparation for mini-arthroscopy 85 (52-91) 90 (59-96) Fear of MRI 5 (0-51) 3 (0-7) Fear of mini-arthroscopy 25 (6-46) 21 (7-57) Being reluctant to undergo MRI 4 (0-43) 1 (0-5) Being reluctant to undergo mini-arthroscopy 18 (6-31) 16 (4-47) DCE-MRI: dynamic contrast enhanced MRI; All items measured on a visual analogue scale of mm; Results depicted as median (IQR). *Group A represents early arthritis patients, group B represents autoantibody-positive individuals without arthritis at risk for developing RA. When focusing on the subgroups, within group A preference was as follows: at baseline 47% of the subjects did not have preference for either procedure, 35% preferred MRI and 18% preferred mini-arthroscopy. After both procedures 29% did not have preference, 24% preferred MRI and 47% preferred mini-arthroscopy (no difference was observed between preference after both procedures and before, p=0.755). Within group B, at baseline, 38% did not have preference for either procedure, 57% preferred MRI and 5% preferred mini-arthroscopy. After both procedures, these percentages were, 29%, 52% and 19%, respectively (no difference was observed between preference after both procedures and before (p=0.715). In addition, comparing study groups, there was no difference in preference between group A and B at baseline (p=0.271) or after both procedures (p=0.115). Of importance, after both procedures, 6 individuals of group A who did not have preference (n=2) or preferred MRI (n=4) at baseline changed to preference for miniarthroscopy. Four of the individuals of group B changed towards preference for miniarthroscopy, of which 3 individuals preferred MRI at baseline. In both groups, none of the subjects who preferred mini-arthroscopy at baseline changed to MRI and, of those, only 1 individual changed to no preference. See Figure 1 for preference at baseline, after both procedures and change in preference. Of subjects who underwent MRI in the open scanner, nobody changed to preference for MRI afterwards. Main remarks with regard to DCE-MRI were complaints about the noise coming from the MRI machine and being immobile for a long period of time, the latter in particular in patients with arthritis. Some patients indicated that mini-arthroscopy was better tolerated than expected, but two subjects complained about having more joint complaints until a few days after mini-arthroscopy. All study subjects were contacted by telephone one week after the procedures or consulted their rheumatologist within 3 weeks time and otherwise the procedures were well tolerated; no complications were reported. 56

58 PATIENT TOLERABILITY OF MINI-ARTHROSCOPY COMPARED TO MRI 4 Figure 1. Preference of mini-arthroscopy or dynamic contrast enhanced MRI Preference at baseline (a), change in preference (b) and preference after both procedures (c). Results are depicted as percentage within study group A or B. Group A represents early arthritis patients; group B represents autoantibody-positive individuals without arthritis at risk for developing RA. DISCUSSION In this small study we show that synovial biopsy sampling by means of mini-arthroscopy is well experienced when compared to DCE-MRI for studying synovial inflammation, both in early arthritis patients and in individuals without arthritis at risk for developing RA. Interestingly, we observed an increase, although not statistically significant, in the percentage of early arthritis patients preferring mini-arthroscopy after both procedures. Although at baseline the group of autoantibody-positive individuals without arthritis 57

59 had higher levels of fear of and reluctance to undergo mini-arthroscopy than MRI, in this group the percentage of individuals preferring mini-arthroscopy increased after procedures as well. Overall, these results refute assumptions that mini-arthroscopy would be a procedure not well experienced by study subjects. A factor that could be in favour of mini-arthroscopy may be that during miniarthroscopy patients have direct contact with physicians and nurses, whereas during MRI they are completely on their own in a distinct room. After having undergone both procedures, most arthritis patients preferred mini-arthroscopy, which may be explained in part by the more stringent need for immobilisation during MRI. In contrast to the arthritis group, most individuals without arthritis still favoured MRI, which might be due to a short period of relative rest necessary after mini-arthroscopy whereas after MRI no restrictions are imposed. Still, 19% of the subjects without arthritis favoured mini-arthroscopy after both procedures and none of these individuals preferring mini-arthroscopy at baseline changed to a preference for DCE-MRI. Of importance, the results of our study cannot be extrapolated to studies using conventional MRI, because scanning duration is generally longer for DCE-MRI and requires venipuncture in all cases, but do support the notion that mini-arthroscopy is generally well experienced, even in individuals without arthritis. In summary, our results show the important observation that mini-arthroscopy, compared to DCE-MRI, is well experienced in patients with early arthritis as well as in autoantibody-positive individuals without arthritis who are at risk for developing RA. These results support the use of mini-arthroscopy in a research setting from a patient perspective, which, together with the low complication rates 2-4 should help to start using mini-arthroscopy in additional research centres. ACKNOWLEDGEMENTS We thank our study subjects for participation in the study and the AMC mini-arthroscopy team for synovial biopsy sampling. We thank the Dutch Arthritis Association (grant ) and the European Community s FP6 funding (Autocure) for financial support. 58

60 PATIENT TOLERABILITY OF MINI-ARTHROSCOPY COMPARED TO MRI REFERENCE LIST 1. Tak PP, Bresnihan B. The pathogenesis and prevention of joint damage in rheumatoid arthritis: advances from synovial biopsy and tissue analysis. Arthritis Rheum 2000;43(12): Gerlag DM, Tak PP. How to perform and analyse synovial biopsies. Best Pract Res Clin Rheumatol 2009;23(2): Kane D, Veale DJ, Fitzgerald O, Reece R. Survey of arthroscopy performed by rheumatologists. Rheumatology (Oxford) 2002;41(2): Vordenbaumen S, Joosten LA, Friemann J, Schneider M, Ostendorf B. Utility of synovial biopsy. Arthritis Res Ther 2009;11(6): Axelsen MB, Stoltenberg M, Poggenborg RP et al. Dynamic gadolinium-enhanced magnetic resonance imaging allows accurate assessment of the synovial inflammatory activity in rheumatoid arthritis knee joints: a comparison with synovial histology. Scand J Rheumatol 2012;41(2): van der Leij C, van de Sande MG, Lavini C, Tak PP, Maas M. Rheumatoid synovial inflammation: pixel-by-pixel dynamic contrast-enhanced MR imaging timeintensity curve shape analysis--a feasibility study. Radiology 2009;253(1): Navalho M, Resende C, Rodrigues AM et al. Dynamic contrast-enhanced 3-T magnetic resonance imaging: a method for quantifying disease activity in early polyarthritis. Skeletal Radiol 2012;41(1): Ejbjerg B, Narvestad E, Rostrup E et al. Magnetic resonance imaging of wrist and finger joints in healthy subjects occasionally shows changes resembling erosions and synovitis as seen in rheumatoid arthritis. Arthritis Rheum 2004;50(4): Ostergaard M, Lorenzen I, Henriksen O. Dynamic gadolinium-enhanced MR imaging in active and inactive immunoinflammatory gonarthritis. Acta Radiol 1994;35(3): Ostergaard M, Stoltenberg M, Lovgreen- Nielsen P, Volck B, Sonne-Holm S, Lorenzen I. Quantification of synovistis by MRI: correlation between dynamic and static gadolinium-enhanced magnetic resonance imaging and microscopic and macroscopic signs of synovial inflammation. Magn Reson Imaging 1998;16(7): Tan AL, Tanner SF, Conaghan PG et al. Role of metacarpophalangeal joint anatomic factors in the distribution of synovitis and bone erosion in early rheumatoid arthritis. Arthritis Rheum 2003;48(5): de Hair MJ, Harty LC, Gerlag DM, Pitzalis C, Veale DJ, Tak PP. Synovial tissue analysis for the discovery of diagnostic and prognostic biomarkers in patients with early arthritis. J Rheumatol 2011;38(9): Gerlag DM, Raza K, van Baarsen LG et al. EULAR recommendations for terminology and research in individuals at risk of rheumatoid arthritis: report from the Study Group for Risk Factors for Rheumatoid Arthritis. Ann Rheum Dis 2012;71(5): van de Sande MG, de Hair MJ, van der Leij C et al. Different stages of rheumatoid arthritis: features of the synovium in the preclinical phase. Ann Rheum Dis 2011;70(5): van de Sande MG. Evaluating antirheumatic treatments using synovial biopsy: a recommendation for standardisation to be used in clinical trials. Ann Rheum Dis 2011;70(3):

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62 FEATURES OF THE SYNOVIUM OF INDIVIDUALS AT RISK OF DEVELOPING RHEUMATOID ARTHRITIS: IMPLICATIONS FOR UNDERSTANDING PRECLINICAL RHEUMATOID ARTHRITIS M.J.H. de Hair M.D. 1, M.G.H. van de Sande M.D. Ph.D. 1, T.H.Ramwadhdoebe M.Sc. 1 2, R. Landewé M.D. Ph.D. 1,3, C. van der Leij M.D. 2, M. Maas M.D. Ph.D. 2, D. van Schaardenburg M.D. Ph.D. 5, D.M. Gerlag M.D. Ph.D. 1, L.G.M. van Baarsen Ph.D. 1 2, P.P. Tak M.D. Ph.D. 1* 1 Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 3 Atrium Medical Center, Heerlen, The Netherlands, 4 Department of Radiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 5 Department of Rheumatology, Jan van Breemen Research Institute/Reade, Amsterdam, The Netherlands. *Current address also: GlaxoSmithKline, Stevenage, U.K. 5 Submitted for publication

63 ABSTRACT Objective Previous work has suggested that subclinical inflammation of the synovium does not coincide with the appearance of rheumatoid arthritis (RA) specific autoantibodies. The objective of this study was to examine the relationship between changes in the synovium and development of arthritis over time in a markedly larger, prospective study. Methods Fifty-five IgM rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positive individuals, without any evidence of arthritis upon physical examination, were included in the study. All individuals underwent MRI and mini-arthroscopic synovial biopsy sampling of a knee joint at inclusion and were prospectively followed. Proportional hazard regression analysis was performed to investigate whether changes in the synovium assessed by MRI and immunohistochemistry (IHC) of synovial biopsies were associated with onset of arthritis. Results After a median follow-up time of 13 (IQR 6-27, range 1-47) months, 15 individuals (27%) developed arthritis. We did not observe overt synovial inflammation during the preclinical stage. However, there was non-significant association between T cell numbers and subsequent development of clinically manifest arthritis (hazard ratio: 2.8; 95% confidence interval: (0.9 to 9.1; p=0.088)). Combined with ACPA-positivity, the presence of T cells in the synovium was associated with arthritis development (double-positive vs singlepositive or double-negative (HR (95%CI): 4.0 (1.4 to 11.4); p=0.010)). Conclusion These findings confirm and extend previous results showing the absence of clear cut synovial inflammation in individuals having systemic autoimmunity associated with RA. However, subtle infiltration by synovial T cells might precede signs and symptoms of arthritis in preclinical RA. 62

64 FEATURES OF THE SYNOVIUM IN THE PRECLINICAL PHASE OF RA Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of synovial tissue. Certain genes, such as MHC class II genes 1 and PTPN22 2, increase the susceptibility of RA. In subjects with genetic susceptibility environmental factors, including smoking and perhaps periodontitis, may lead to the development of autoantibodies like rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) 3, 4. These autoantibodies define individuals with systemic auto-immunity associated with RA 5. Whilst RA-specific autoantibodies can be found more than years before joint inflammation becomes clinically manifest 6-8, only a minority of individuals with RA-specific autoantibodies actually develops clinically manifest RA. We have previously proposed that, whereas the initial immune response leading to the production of autoantibodies may take place at sites other than the synovium, a second hit due to for instance a minor trauma or a viral infection may lead to citrullination of synovial proteins and subsequent epitope spreading 9. Consistent with the hypothesis that the initial changes may take place at sites other than the synovium, like the lung 10, 11, we found no evidence of overt synovial inflammation in the joints of 13 subjects at risk of developing RA 9. Because of the small sample size of that study, and in light of the importance of the implications for our understanding of the etiology of RA, we decided to validate and extend the results in a larger, prospective study. 5 MATERIALS AND METHODS Study subjects Individuals with arthralgia and/or a positive family history for RA, without any evidence of arthritis upon thorough physical examination, and who were positive for IgM-RF and/or ACPA were included in the study between June 2005 and August These individuals are at risk of developing RA and are characterized by the presence of systemic autoimmunity associated with RA (phase c according to 5 ) with or without environmental risk factors (phase b according to 5 ) and with or without symptoms without clinical arthritis (phase d according to 5 ). IgM-RF was measured using IgM-RF ELISA from Sanquin, Amsterdam, the Netherlands (upper limit of normal (ULN) 12.5 IU/mL) until December 2009 and thereafter using IgM-RF ELISA from Hycor Biomedical, Indianapolis, IN (ULN 49 IU/mL). IgM-RF levels were categorized into negative, <ULN, low positive, 3 times ULN, and high positive, >3 times ULN 12, 13. ACPA was measured using anti-ccp2 ELISA CCPlus from Eurodiagnostica, Nijmegen, the Netherlands; ULN 25 kau/l). These study subjects were recruited either via the outpatient clinic of the department of Clinical Immunology and Rheumatology at AMC, Amsterdam, via referral from the rheumatology outpatient clinic of Reade, Amsterdam, or via testing family members of RA patients in the outpatient clinic or at public fairs across the Netherlands. The study was performed according to the principles of the Declaration of Helsinki, approved by the institutional review board of the AMC, and all study subjects have given written informed consent. Study design At baseline demographic data were obtained. An MRI of an arbitrary knee joint was performed within one week before mini-arthroscopic synovial biopsy sampling of this 63

65 joint was performed 9. Study subjects were followed over time until arthritis onset or until January 1, 2012 (censored). There were yearly study visits. The development of arthritis, defined as a swollen joint, was the endpoint of this study. Individuals who developed arthritis underwent an interim visit, in order to confirm clinically apparent arthritis by two independent investigators (MS and DG or MH and DG)). Clinical parameters At every study visit the following clinical and disease activity parameters were obtained: 68-joint tender joint count (68TJC) and 66-joint swollen joint count (66SJC), duration of morning stiffness (minutes), IgM-RF- (IU/mL) and ACPA-levels (kau/l), erythrocyte sedimentation rate (ESR)(mm/hr), and serum levels of C-reactive protein (CRP)(mg/L). MRI Images were acquired on either a closed 1.5 Tesla (GE Signa Horizon Echospeed, LX9.0, General Electric Medical Systems, Milwaukee, WI) or open 1 Tesla (Panorama Open, Philips, Best, the Netherlands) MRI scanner, due to replacement of the former scanner. The minimum imaging protocol consisted of a sagittal STIR, an axial T2 with fat suppression and a sagittal T1 before and after contrast injection. A musculoskeletal radiologist with 15 years of experience and a research fellow with 4 years of experience in musculoskeletal radiology scored all images. Both were blind for the outcome of the study (arthritis). The presence of synovitis and hydrops were scored in 4 compartments of the knee joint (medial, lateral, central and suprapatellar; minimum and maximum score of 0 and 3, respectively, for each compartment), as well as the presence (1) or absence (0) of bone marrow edema, erosions and cartilage damage (each in 6 locations: the patella-femoral joint (2) the medial (2) and lateral (2) knee compartments). Mini-arthroscopic synovial biopsy sampling All study subjects underwent mini-arthroscopic synovial biopsy sampling of a knee joint at baseline 14. Six to 8 samples were collected for immunohistochemistry (IHC) to correct for sampling error, as described previously The synovial biopsy samples were snap-frozen en bloc in Tissue-Tek OCT (Miles, Elkhart, IN) immediately after collection. Sections (5 μm each) were cut and mounted on Star Frost adhesive glass slides (Knittelgläser, Braunschweig, Germany). Sealed slides were stored at -80 C until further use. Immunohistochemistry Synovial tissue sections were stained using mouse monoclonal antibodies for T cells (anti- CD3: clone SK7, Becton Dickinson, San Jose, CA; anti-cd4: clone SK3, Becton Dickinson; and anti-cd8: clone C8/144B; Dako, Glostrup, Denmark), B cells (anti-cd22; clone RFB4; Millipore, Billerica, MA), fibroblast-like synoviocytes (FLS) (anti-cd55; clone 67; AbD Serotec, Oxford, UK), macrophages (anti-cd68; clone EBM11; Dako), plasma cells (anti- CD138; clone B-B4; Immunotech, Marseille, France), blood vessels (anti-von Willebrand factor (vwf); clone F8/86; Dako) and citrullinated fibrinogen (anti-citrullinated fibrinogen; clone 20B2; ModiQuest Research, Nijmegen, the Netherlands). 64

66 FEATURES OF THE SYNOVIUM IN THE PRECLINICAL PHASE OF RA Staining was performed using a three-step immunoperoxidase method to detect bound anti-cd55, anti-vwf, anti-cd68 and anti-cd138 antibodies, as described previously 18. A two-step immunoperoxidase method was used to detect bound anti-cd4, anti-cd8 and anti-citrullinated fibrinogen antibodies. For anti-cd3 and anti-cd22 we used a 2-step immunoperoxidase method with a secondary polymer-horseradish peroxidase anti-mouse antibody (Envision+ System; Dako, Glostrup, Denmark). As negative control irrelevant isotype-matched immunoglobulins were applied to the sections instead of the primary antibody. The primary antibodies were incubated for 60 minutes (or overnight for CD4, CD8, CD55, vwf and citrullinated fibrinogen). AEC was used as chromogen (Vector Laboratories; Burlingame, CA). Slides were counterstained with Gill s hematoxylin and mounted in Kaiser s glycerol gelatin (Merck, Darmstadt, Germany). The intensity of the staining was scored using semi-quantitative analysis by two independent observers (MH and BS or MH and NS) on a five-point scale (range 0-4), including 5 RA patients as positive control, where 0 represented no expression and 4 represented the maximum expression of all tissue sections analyzed 19. Expression of CD68+ cells was performed separately for the intimal lining layer and synovial sublining. Since CD4 is not only expressed by T cells but also by macrophages (CD4dim) only bright staining was scored as positive for CD4+ T cells. Scoring of citrullinated fibrinogen was done in a dichotomous manner (presence vs absence of staining). When scores between the two observers did not match, a definite score was obtained upon mutual agreement. 5 Statistical analysis Continuous, normally distributed data were presented as means (SD), and differences between study groups were analyzed using t-test for unpaired samples. Not normally distributed data were presented as medians (IQR), and differences between study groups were analyzed using Mann-Whitney U-test. Categorical data were presented as numbers (percentages), and differences between groups were analyzed using Chi-square test. To investigate associations of synovial markers with arthritis onset, proportional hazard regression analysis (Cox) was performed. Follow-up duration was defined as the time between inclusion in the cohort and the onset of clinically manifest arthritis, or between inclusion and January 1, 2012 (censored). First, variables were tested variable-by-variable. Variables with a p-value <0.2 were arbitrarily selected for a multivariable analysis (forward and backward selection procedures). Meaningful statistical interactions were excluded upfront. Statistical analysis was performed using PASW Statistics 18 (SPSS Inc, Chicago, Il). A p-value of <0.05 was considered statistically significant. RESULTS Cohort description Of the 55 individuals included in the study, 41 individuals were single positive for RF or ACPA (19 were IgM-RF-positive only, 22 were ACPA-positive only) and 14 individuals were double positive. They were followed for a median duration of 27 (IQR 14-47; range 1-75) months. Fifteen of the 55 individuals (27%) developed arthritis over time, after a median 65

67 follow-up of 13 (IQR 6-27, range 1-47) months. Clinical characteristics and fulfillment of RA criteria at the moment of arthritis onset were described previously 20. Individuals who did not develop arthritis were followed for a median duration of 37 (IQR 19-52; range 3-75) months. Figure 1 shows the cumulative hazard of arthritis development in this study. Baseline clinical characteristics of individuals who developed arthritis during follow-up were generally comparable to those of individuals who did not develop arthritis during follow-up (see Table 1). Figure 1. Cumulative hazard of arthritis development. x-axis: follow-up time in months; y-axis: cumulative hazard of arthritis development. No overt synovial inflammation, but subtle synovial infiltration by T cells might precede the development of arthritis Complete synovial tissue samples of 6 individuals had to be excluded for IHC because of quality standards. The synovial tissue of a range of 35 to 49 individuals could be included in the analysis for expression of the various markers by IHC. In 6 individuals MRI was not performed due to logistic reasons. Semiquantitative scores for expression of inflammatory markers in the synovium were low compared to the 5 RA patients who were used as positive control, consistent with our previous study 9 (data not shown). In the autoantibody-positive individuals the scores for the expression of CD3 and citrullinated fibrinogen were either 0 or 1. For proportional hazard regression analysis the expression levels of the other markers were dichotomized. For the expression of CD4, CD8 and CD68 scores were 0 to 3 and categorized as follows: 0: negative; and 1: positive. CD55 expression was scored as 1 (low positive) or as 2 (high positive). Expression of vwf was scored in a range of 0 to 3 and categorized as 1 (low) for scores 1 and 2 (high) for scores 2. Expression of CD22 and CD138 was observed in only 1 individual (2 different persons) and therefore not included in statistical analyzes. 66

68 FEATURES OF THE SYNOVIUM IN THE PRECLINICAL PHASE OF RA Table 1. Baseline demographic and clinical characteristics Clinical characteristic No arthritis developed Arthritis developed N=40 * N=15 P-value Sex, female (n (%)) 28 (70) 9 (60) Age (years) (mean (SD)) 44 (12) 47 (8) Arthralgia (n (%)) 37 (93) 14 (93) Morning stiffness (min) (median (IQR)) 5 (0-30) 10 (5-30) IgM-RF positive (n (%)) 24 (57) 9 (60) IgM-RF level low positive (n (%)) ** 15 (62) 4 (44) IgM-RF level high positive (n (%)) ** 9 (38) 5 (56) ACPA positive (n (%)) 24 (60) 12 (80) ACPA level ** (kau/l) (median (IQR)) 466( ) 452( ) IgM-RF and ACPA both pos. (n (%)) 8 (20) 6 (40) ESR (mm/hr) (median (IQR)) 9 (2-20) 7 (5-15) CRP (mg/l) (median (IQR)) 2.1 ( ) 4.0 ( ) VAS pain (mm) (median (IQR)) 32 (5-66) 48 (11-76) VAS global disease activity (mm) 38 (10-64) 53 (9-76) (median (IQR)) 68TJC (n) (median (IQR)) 2 (0-8) 2 (0-8) SJC (n) (median (IQR)) *7 Individuals have been at risk for arthritis development for a shorter period of time: two patients have started with hydroxychloroquine for arthralgias, and two with anti-tnf therapy because of a diagnosis of ankylosing spondylitis (n=1) or Crohn s disease (n=1). Three patients were lost to follow-up after some time. IgM-RF: IgM-rheumatoid factor; ** only in positive individuals: low positive: 3 times upper limit of normal (ULN), high positive: >3 times ULN; ACPA: anti-citrullinated protein antibodies; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; VAS: visual analogue scale (range 0-100mm); 68TJC: tender joint count of 68 joints; 66SJC: swollen joint count of 66 joints. Proportional hazard regression analysis was performed to evaluate whether clinical and MRI parameters, as well as expression of inflammatory markers in the synovium, were associated with the onset of arthritis. First, all variables were analyzed variable-byvariable, see Table 2. ACPA-positivity showed a non-significant trend towards an association with arthritis development (hazard ratio (HR) (95%CI): 2.7 (0.8 to 9.7); p=0.119). Of note, subjects were selected based on being positive for RF and/or ACPA. Other clinical parameters were not associated with arthritis development (data not shown) as was the case for MRI parameters. With respect to synovial tissue analysis by IHC, there was no overt synovial inflammation during preclinical RA. However, the presence of CD3+ T cells at baseline showed a trend towards an association with arthritis development after follow up (HR (95%CI): 2.8 (0.9 to 9.1); p=0.088)). There was no association between expression of CD3 and presence of arthralgia in the biopsied knee joint (p=0.210). A similar trend was seen for the expression of CD8 in the synovium (HR (95%CI): 2.8 (0.7 to10.5; p=0.133)). It appeared that in particular expression of both CD3 and CD8 (CD3+CD8+) explained the trend towards an association with 67

69 Table 2. Univariate proportional hazard regression analysis for the development of arthritis Hazard ratio (95% confidence interval) P-value MRI factors Synovitis (per unit) 0.9 (0.6 to 1.4) Hydrops (per unit) 1.1 (0.9 to 1.5) Cartilage degeneration (per unit) 0.1 (0.0 to 9.4) Bone marrow edema (per unit) 0.6 (0.2 to 2.2) Erosions (per unit) 1.9 (0.2 to 15.2) Synovial tissue factors CD3 (pos (n=21) vs neg (n=28)) 2.8 (0.9 to 9.1) CD4 (pos (n=23) vs neg (n=12)) 2.9 (0.4 to 23.6) CD8 (pos (n=19) vs neg (n=17)) 2.8 (0.7 to 10.5) CD55 (low (n=15) vs high (n=31)) 2.2 (0.5 to 10.5) CD68 Sublining (pos (n=1) vs neg (n=38)) 0.0 (0.0 to infinite) CD68 Lining (pos (n=5) vs neg (n=34)) 0.5 (0.1 to 4.1) von Willebrand Factor (low (n=17) vs high (n=21)) 1.7 (0.5 to 5.7) Citrullinated fibrinogen (pos (n=28) vs neg (n=14)) 1.4 (0.4 to 4.3) pos: positive; neg: negative; CD3, CD4, CD8: markers for T cells; CD55: marker for fibroblast-like synoviocytes; CD68: marker for macrophages. Variables with p-values < 0.2 (in bold) included in multivariate analysis. arthritis development (double-positive vs single-positive or double-negative: HR (95%CI): 2.9 (0.9 to 9.4; p=0.086; double positive vs. double negative: HR (95%CI): 6.3 (0.8 to 53); p=0.088). This may suggest that synovial CD8+ T cells are involved in the earliest stages of RA. Expression of other synovial tissue markers for inflammatory cells and blood vessels was not associated with arthritis development, confirming and extending our previous results. Synovial T-cell infiltration combined with ACPA-positivity is associated with arthritis development After exclusion of potentially meaningful interactions between variables (data not shown), variables with p-value <0.2 in the univariate proportional hazard regression analysis were tested in a multivariable analysis. See Table 3 for the results of the proportional hazard regression analysis. Combining ACPA-status with CD3 expression in the synovium in one model resulted in an increased association for CD3 with arthritis development (HR (95%CI): 3.3 (1.0 to 11.0)) compared to a univariate model with only CD3. Combining CD8 with either ACPA and CD3 or both resulted in the absence of a significant association with development of arthritis (data not shown), which is most probably due to lack of power as a result of over stratification in this relatively small sample size. Of importance, individuals expressing CD3 in the synovial tissue were not only individuals who were ACPA positive (57% of these individuals), suggesting no direct association between CD3-positivity and ACPA-positivity. Within the subgroup of 68

70 FEATURES OF THE SYNOVIUM IN THE PRECLINICAL PHASE OF RA Table 3. Multivariate proportional hazard regression analysis for the development of arthritis Variables in model Hazard ratio (95% confidence interval) P-value Model 1: ACPA (pos vs neg) 2.7 (0.8 to 9.7) Model 2: CD3 (pos vs neg) 2.8 (0.9 to 9.1) Model 3: CD8 (pos vs neg) 2.8 (0.7 to 10.5) Model 4: ACPA (pos vs neg) CD3 (pos vs neg) 2.8 (0.8 to 10.5) 3.3 (1.0 to 11.0) ACPA: anti-citrullinated protein antibodies; pos: positive; neg: negative; CD3, CD8: markers for T cells. Table 4. Frequency table for development of arthritis within subgroups of ACPA-status and CD3- expression in the synovium ACPA+CD3+ * (N=12) ACPA+CD3- (N=20) ACPA-CD3+ (N=9) ACPA-CD3- (N=8) Follow up time in months, median (IQR) 22 (13-37) 25 (6-53) 40 (27-71) 36 (20-44) Arthritis developed, n (%) 7 (58%) 3 (15%) 2 (22%) 1 (12%) ACPA: anti-citrullinated protein antibodies; CD3: marker for T cells; * CD3-expression missing from 2 ACPA-positive individuals who developed arthritis, 2 ACPA-positive individuals who did not develop arthritis and 2 ACPA-negative individuals who did not develop arthritis. ACPA+CD3+ individuals, however, significantly more individuals developed arthritis than in the other subgroups (p=0.037) (Table 4). To investigate whether there is an association for subgroups of ACPA- and CD3- positivity with onset of arthritis we performed proportional hazard regression analysis for these subgroups as well. Positivity for both ACPA and CD3 was associated with arthritis development (double-positive vs single-positive or double-negative (HR (95%CI): 4.0 (1.4 to 11.4); p=0.010)), as depicted in Figure 2. Synovial expression of citrullinated fibrinogen in individuals at risk of developing arthritis The role of synovial citrullinated peptides in the pathogenesis of RA is unknown and has never been studied in individuals at risk of developing the disease 21. Therefore, we examined the synovial expression of citrullinated fibrinogen, one of the major candidate autoantigens in RA. Expression of citrullinated fibrinogen in the synovium was not only observed in individuals who developed arthritis after follow-up (42%), but also in those who did not (30%) (p=0.462), and was not predictive of arthritis onset. Moreover, expression of citrullinated fibrinogen was not dependent on ACPA status, being observed in 39% of ACPA positive and 21% of ACPA negative individuals (p=0.247). 69

71 Figure 2. Cumulative hazard for development of arthritis in subgroups of autoantibody positive individuals based on ACPA-status and expression of CD3 in the synovium. x-axis: follow-up time in months; y-axis: cumulative hazard for arthritis development. ACPA+/-: positive or negative for anticitrullinated protein antibodies (ACPA) in the serum; CD3+/-: presence/absence of expression of CD3 in the synovial tissue. Taken together, we confirm that there is no evident synovitis in individuals at risk of developing RA during phases c and d (according to 5 ). However, we found an indication for subtle T-cell infiltration preceding the development of clinically manifest arthritis. DISCUSSION We evaluated the synovium by MRI and IHC before the onset of clinical signs and symptoms of arthritis in autoantibody-positive individuals who are at risk of developing RA. After adjustment for differences in follow-up duration we observed that the presence of inflammatory cells or blood vessels in the synovial tissue was not associated with the development of arthritis. Consistent with these findings, MRI showed no indication of synovitis, confirming and extending our previous work 9. However, there was a trend towards increased synovial T cell numbers in subjects who subsequently developed arthritis compared to those who did not (yet); this effect was stronger when combined with ACPA-status. Thus, there is generally no overt subclinical synovitis present more than 1 month before the onset of arthritis in individuals who are at risk of developing RA. This suggests that infiltration of the synovial tissue by inflammatory cells would be a process relatively late in RA pathogenesis, which occurs close to the onset of clinically manifest disease. In contrast, systemic changes may be observed years before the onset of arthritis, as shown by the detection of RA-specific auto-antibodies (IgM-RF and ACPA) 6-8, increasing CRP levels towards arthritis onset 22, and increased monocyte chemotactic protein-1 (MCP-1) 23 in individuals who developed RA later on. To gain new insights into the earliest phase of RA pathogenesis it will therefore be important to study compartments of the immune system other than the synovium as well. For this reason, the first studies analyzing lymph node tissue in different phases of RA are underway 24. However, our results suggest that subtle infiltration of the synovium by T cells might precede the onset of clinically manifest arthritis. The exact role of T cells in the 70

72 FEATURES OF THE SYNOVIUM IN THE PRECLINICAL PHASE OF RA pathogenesis of RA is still not completely clear 25, 26. The abundant presence of synovial T cells in established RA and the association with certain MHC class II genes clearly support the involvement of T cells in the disease pathogenesis. We recently found highly expanded T-cell clones in the synovium of early RA patients compared to longstanding RA patients 27, suggesting a role for T-cell involvement in the early phase of disease. In the current study, it appeared that in particular expression of both CD3 and CD8 was associated with arthritis development. Activated CD8+ T cells may have cytotoxic activity by the production of granzymes and perforin, leading to cell death of (pathogenic) cells, and can produce proinflammatory cytokines like interferon (IFN)γ and TNF. We previously found that soluble granzyme B levels are an independent predictor of erosive disease in RF positive RA 28. The possible role of synovial CD8+ T cells during the preclinical stage of RA pathogenesis is however still unclear and our findings will first need to be confirmed in an independent cohort. Interestingly, our observations were done in the synovial tissue of knee joints which were not amongst the joints that initially showed clinical signs of arthritis in any of the individuals who developed arthritis after follow-up. This suggests a time lag between what is observed in the synovial tissue and clinical signs of arthritis. Another interesting observation is that expression of citrullinated fibrinogen in the synovial tissue was not associated with development of arthritis or ACPA status. Previous work has shown that the presence of citrullinated proteins is not specific for RA synovial tissue 29, 30, since citrullinated peptides can also be found in inflamed or cancer tissue outside the synovium 29, 31. Our data suggest that initial ACPA formation is not necessarily directed against joint specific peptides, but rather against citrullinated peptides in other compartments of the body. Citrullination of peptides in the lung, possibly as a result of smoking, suggests that the lung may be an early site of RA-related auto-immunity 3. Interestingly, in a comparable cohort of autoantibody-positive individuals at risk for RA, airway abnormalities were observed comparable to those found in RA patients, but significantly more than in autoantibody-negative controls 10. A possible limitation of our study is that synovial inflammation was examined in knee joints only whereas the disease usually presents in the small joints of the hands or feet. Obviously, it is difficult to obtain sufficient synovial tissue from non-arthritic small joints to allow reliable analysis. However, in 44% of the cases synovial biopsy sampling was performed in a symptomatic, painful knee joint (without swelling) and it can therefore be expected that if the pain was due to synovial inflammation in that joint, we would have detected this by MRI and synovial biopsy. Moreover, 25% of the individuals in the cohort underwent ultrasonography or MRI of the hands as well in the context of regular patient care, and there was also no sign of synovitis in the small joints in these subjects. In conclusion, in this prospective cohort study of autoantibody-positive individuals at risk of developing RA, we confirm in a relatively large study that there is no clear cut synovial inflammation before the development of clinically apparent arthritis. It is possible however that subtle infiltration of the synovium by T cells might precede the onset of arthritis, but this needs further validation. We propose a model where systemic autoimmunity may exist years before the onset of RA. Apparently, a second hit (for 5 71

73 instance a trauma or an infection leading to expression of citrullinated antigens) in the synovium is needed for arthritis development. This could subsequently lead to expansion of the ACPA repertoire 32 and progression towards synovial inflammation. ACKNOWLEDGEMENTS We thank our study subjects for participating in the study, the AMC arthroscopy team for synovial biopsy sampling and processing, and Noortje Smits and Britt Sotthewes for their laboratory assistance. The study was sponsored by the Dutch Arthritis Foundation grant and , the Netherlands Organisation for Health Research and Development (ZonMw) grant and the IMI EU funded project BeTheCure n

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75 onset of inflammatory response and development of overt rheumatoid arthritis. Ann Rheum Dis 2007;66(1): de Hair MJ, Zijlstra IA, Boumans MJ et al. Hunting for the pathogenesis of rheumatoid arthritis: core-needle biopsy of inguinal lymph nodes as a new research tool. Ann Rheum Dis 2012;71(11): Cope AP. T cells in rheumatoid arthritis. Arthritis Res Ther 2008;10 Suppl 1:S Fox DA. The role of T cells in the immunopathogenesis of rheumatoid arthritis: new perspectives. Arthritis Rheum 1997;40(4): Klarenbeek PL, de Hair MJ, Doorenspleet ME et al. Inflamed target tissue provides a specific niche for highly expanded T-cell clones in early human autoimmune disease. Ann Rheum Dis 2012;71(6): Goldbach-Mansky R, Suson S, Wesley R, Hack CE, El-Gabalawy HS, Tak PP. Raised granzyme B levels are associated with erosions in patients with early rheumatoid factor positive rheumatoid arthritis. Ann Rheum Dis 2005;64(5): Vossenaar ER, Smeets TJ, Kraan MC, Raats JM, van Venrooij WJ, Tak PP. The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum 2004;50(11): Makrygiannakis D, af KE, Lundberg IE et al. Citrullination is an inflammation-dependent process. Ann Rheum Dis 2006;65(9): Baka Z, Barta P, Losonczy G et al. Specific expression of PAD4 and citrullinated proteins in lung cancer is not associated with anti-ccp antibody production. Int Immunol 2011;23(6): van de Stadt LA, van der Horst AR, de Koning MH et al. The extent of the anti-citrullinated protein antibody repertoire is associated with arthritis development in patients with seropositive arthralgia. Ann Rheum Dis 2011;70(1):

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78 EXPRESSION OF PROSTAGLANDIN E 2 ENZYMES IN THE SYNOVIUM OF ARTHRALGIA PATIENTS AT RISK OF RHEUMATOID ARTHRITIS AND IN EARLY ARTHRITIS PATIENTS M.J.H. de Hair 1 *, P. Leclerc 2 *, E.C. Newsum 1, K.I. Maijer 1, M.G.H. van de Sande 1, T.H.Ramwadhdoebe 1 3, D. van Schaardenburg 4, L.G.M. van Baarsen 1 3, M. Korotkova 2, D.M. Gerlag 1, P.P. Tak 1 $ #, P.J. Jakobsson 2 # 1 Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, the Netherlands, 2 Rheumatology research Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden, 3 Department of Experimental Immunology, Academic Medical Center/University of Amsterdam, Amsterdam, the Netherlands, 4 Department of Rheumatology, Jan van Breemen Research Institute/Reade, Amsterdam, The Netherlands, $ Currently also: GlaxoSmithKline, Stevenage, U.K., * # Both authors contributed equally 6 Submitted for publication

79 ABSTRACT Introduction Arthralgia may precede the development of synovial inflammation in autoantibody positive individuals at risk of developing rheumatoid arthritis (RA). A major pathway involved in pain is the prostaglandin (PG) E 2 pathway. We investigated this pathway in the synovium of individuals with RA-specific autoantibodies and in early arthritis patients. Methods Nineteen autoantibody-positive individuals (IgM-rheumatoid factor and/or anti-cyclic citrullinated peptide antibodies) with arthralgia (n=15) and/or a positive family history of RA (n=8) but otherwise healthy, who had been prospectively followed for at least 2 years, were included. In addition, we selected disease-modifying antirheumatic drug naïve patients with early arthritis (disease duration < 1 year). After 2 years follow up they fulfilled classification criteria for RA (n=63), spondyloarthritis (SpA; n=14), or had unclassified arthritis (UA; n=27). At baseline, we assessed pain and disease activity, and performed synovial biopsy sampling by mini-arthroscopy. Tissue sections were examined by immunohistochemistry to detect PGE 2 pathway enzymes (mpges-1; COX-1 and -2; 15-PGDH); stained sections were evaluated by digital image analysis. Results Synovial expression of PGE 2 enzymes was not clearly related to pain sensation in autoantibody-positive individuals at risk of RA or in early arthritis patients. Six out of 19 autoantibody-positive individuals developed arthritis after follow up; the expression levels at baseline were not associated with the development of arthritis. However, as expected there was a marked expression of PGE2 enzymes in patients with various forms of arthritis and, interestingly, in early SpA patients the expression levels of mpges-1 and COX-1 were significantly increased compared to RA and UA patients. Conclusions Arthralgia in autoantibody-positive individuals without synovial inflammation who are at risk of developing RA and in early arthritis patients cannot be clearly explained by altered synovial expression of enzymes of the PGE 2 pathway. Pain in these patients may therefore be regulated by other pathways or originate at sites other than the synovium. 78

80 SYNOVIAL PGE 2 ENZYMES IN ARTHRALGIA AND EARLY ARTHRITIS PATIENTS Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovial inflammation with clinically apparent arthritis, which may lead to joint destruction. Joint pain is a major burden for RA patients and it is important to characterize the underlying molecular mechanisms. The RA-specific autoantibodies rheumatoid factor (RF) and anticitrullinated peptide antibodies (ACPA) can be present years before onset of clinical disease 1-3, defining individuals having an increased risk of developing RA 4, 5 and enabling us to study the preclinical phase of RA. The main target tissue in established RA is the synovium, which is characterized by hyperplasia of the intimal lining layer and accumulation of inflammatory cells in the synovial sublining 6. The changes in the synovium occur relatively late in the disease process, most likely not more than a few weeks to months before the development of clinically manifest arthritis 7. During the preclinical phase, patients may have arthralgia, even before the onset of synovial inflammation 7. It is currently not clear what the underlying mechanisms are explaining pain in the absence of synovial inflammation in subjects at risk of developing RA. We hypothesized that prostaglandin E 2 (PGE 2 ) might be involved in arthralgia in the absence of synovial inflammation. PGE 2 is a powerful lipid mediator not only of inflammation, but it can also act both locally and centrally to mediate hyperalgesia. It is part of the prostaglandin family of molecules, derived from the metabolism of arachidonic acid. To produce PGE 2, arachidonic acid is metabolized into prostaglandin H 2 (PGH 2 ) by the enzymes cyclooxygenase-1 (COX-1) or COX-2. COX-1 is constitutively expressed by a variety of cells and tissues and contributes to homeostasis. COX-2 expression, on the other hand, is almost absent under normal conditions but increases by proinflammatory stimuli. PGH 2 yield from cyclooxygenase activity is subsequently transformed into PGE 2 by one of three prostaglandin E synthases (PGES). Under inflammatory conditions, microsomal PGES-1 (mpges-1) plays, together with COX-2, a predominant role in the synthesis of PGE 2 as its expression is also upregulated by pro-inflammatory stimuli 8, 9. PGE 2 levels are further regulated at the catabolic end by the enzyme 15-hydroxy prostaglandin dehydrogenase (15-PGDH). PGE 2 pathway enzymes are expressed both at the mrna and protein level in the inflamed synovium of RA, spondyloarthritis (SpA) and osteoarthritis (OA) patients The prostaglandin pathway has a predominant role in arthritic pain, which is illustrated by the beneficial effects of nonsteroidal anti-inflammatory drugs (NSAIDs) and COX inhibitors (COXibs). Moreover, genetic deletion studies have highlighted the importance of COX and mpges-1 in different mouse models of arthritis, reducing incidence, severity and pain in knock-out mice under the relevant experimental settings Therefore, we investigated whether synovial expression of enzymes of the PGE 2 pathway was positively related to arthralgia and contributed to the development of arthritis in autoantibody-positive arthralgia patients at risk of developing RA. In addition, we studied the expression of these enzymes in early arthritis patients. 6 METHODS Study subjects Two groups were included. First, individuals with arthralgia and/or a positive family history of RA, but without any evidence of arthritis after thorough physical examination, have been recruited since June and followed over time to assess arthritis onset. 79

81 They are all positive for IgM-RF and/or ACPA. For this study, we selected 19 individuals who had been followed for at least 2 years: 15 individuals with arthralgia and 4 individuals without arthralgia, defined by absence of pain on joint examination and a score on a visual analogue scale (VAS) for pain (scale mm) of 0. These individuals are collectively referred to as autoantibody-positive individuals. The second group consisted of early arthritis patients (arthritis duration < one year, disease-modifying antirheumatic drug (DMARD) naïve) with an inflamed wrist, knee or ankle joint who were included in the early arthritis cohort of the Academic Medical Center (AMC) in Amsterdam 16, since August To analyse PGE 2 pathway enzymes expression exclusively in patients having a definite diagnostic classification, we selected all patients from this cohort who, after 2 years of follow-up, fulfilled either the 2010 ACR/ EULAR criteria for RA (n=63), or the European Spondyloarthritis Study Group criteria 17 for SpA (n=14) or who did not meet classification criteria for any established rheumatic disease and were classified as unclassified arthritis (UA, n=27). Only patients for whom synovial tissue slides were available for immunohistochemistry, based on our quality control system, were included. The study was performed according to the principles of the Declaration of Helsinki, approved by the institutional review board of the AMC, and all study subjects gave written informed consent. Study design At baseline and yearly study visits, the following clinical and laboratory parameters were obtained: patient s visual analogue scale (VAS) for global disease activity (scale mm); patient s VAS for pain in general; patient s VAS for pain in the biopsied joint; 68 tender joint count (68TJC) and 66 swollen joint count (66SJC); morning stiffness in minutes; IgM-RF levels using IgM-RF ELISA (Sanquin, Amsterdam, the Netherlands (upper limit of normal (ULN) 12.5 IU/mL until December 2009 and thereafter using IgM-RF ELISA (Hycor Biomedical, Indianapolis, IN (ULN 49 IU/mL)); ACPA using anti-citrullinated cyclic peptide (CCP)2 ELISA CCPlus (Eurodiagnostica, Nijmegen, the Netherlands (ULN 25 kau/l)); erythrocyte sedimentation rate (ESR); serum levels of C-reactive protein (CRP); x-rays of hands and feet. In the group of autoantibody-positive individuals, yearly study visits were performed and, for individuals who developed arthritis, an additional visit was performed at which the presence of arthritis was independently assessed by two investigators (MS and DG or MH and DG). Early arthritis patients were followed for 2 years, after which they were classified for arthritis outcome: self-limiting disease, defined as no arthritis on examination and no use of DMARDs or steroids in the preceding three months, persistent non-erosive disease, defined as presence of arthritis in at least 1 joint and/or of DMARDs or steroids in the preceding three months, or persistent erosive disease, defined as presence of joint erosions on radiographs of the hands and/or feet 18. Synovial biopsy sampling At baseline, all study subjects underwent arthroscopic synovial biopsy sampling as previously described 19, 20. In the autoantibody-positive individuals synovial biopsy sampling was performed in a knee joint in all cases and in the early arthritis patients in an inflamed wrist, 80

82 SYNOVIAL PGE 2 ENZYMES IN ARTHRALGIA AND EARLY ARTHRITIS PATIENTS knee or ankle. Six to 8 synovial biopsy samples were collected for immunohistochemistry (IHC) to correct for sampling error, as described previously 21. The synovial biopsy samples were snap-frozen en bloc in Tissue-Tek OCT (Miles, Elkhart, IN) immediately after collection. Sections (5 μm each) were cut and mounted on Star Frost adhesive glass slides (Knittelgläser, Braunschweig, Germany). Sealed slides were stored at -80 C until further use. Immunohistochemistry and analysis Synovial tissue sections were fixed in 2% formaldehyde and immunohistochemical staining was performed using rabbit polyclonal antiserum raised towards mpges-1 12, rabbit polyclonal anti-cox-1 (Cayman Chemical, Ann Harbor, MI) and mouse monoclonal anti-cox-2 (CX229; Cayman Chemical). Synovial tissue sections of the autoantibodypositive individuals were additionally stained using rabbit polyclonal anti-15-pgdh (Novus Biologicals, Littleton, CO). Staining was performed using a 2 step immunoperoxidase method as previously described 22. As negative control, isotype-matched immunoglobulins were applied to the sections instead of the primary antibody. Staining of the synovial tissue sections of autoantibody-positive individuals was developed using diaminobenzidine (DAB; Vector laboratories; Burlingame, CA) and quantified by computer-based image analysis using a Leica DM RXA2 microscope and the Leica Qwin pro software. Results were expressed as percentage of positive stained area per total tissue area. Staining of the synovial tissue of early arthritis patients was developed using 3-amino-9-ethylcarbazole (AEC; Vector Laboratories) and expression of the markers was quantified using digital image analysis as described previously 23, 24. Expression levels are presented as integrated optical density (IOD)/mm 2, an arbitrary unit representing the intensity of staining per mm These experiments were performed in four separate sessions; diagnoses were randomized over the sessions. To correct for between-session variation, the factor correction program was used 26. Previously, in a subset of the early arthritis patients synovial tissue sections were stained using mouse monoclonal anti-cd68 to detect macrophages, anti-cd3 for T cells and anti-cd55 for fibroblast like synoviocites (FLS), as described before Statistical analysis Continuous, normally distributed variables were presented as mean (standard deviation, SD) and differences between study groups were analyzed using ANOVA. Not normally distributed variables were depicted as median (interquartile range, IQR) and differences between study groups were analyzed using Kruskal-Wallis and Mann Whitney tests where appropriate. Categorical data were depicted as number (%) and differences between study groups analyzed using chi2 test. Bivariate correlations of not normally distributed variables were analyzed using Spearman s rank correlation test. Statistical analysis was performed using PASW Statistics 18 (SPSS Inc, Chicago, IL). RESULTS Autoantibody-positive individuals Baseline characteristics of autoantibody-positive individuals are depicted in Table 1. 81

83 Table 1. Baseline characteristics of autoantibody-positive individuals N=19 Sex, female (n (%)) 10 (53) Positive family history of RA (n (%)) 8 (21) Age, years (median (IQR)) 48 (43-54) IgM-RF positive (n (%)) 12 (63) ACPA positive (n (%)) 14 (74) IgM-RF and ACPA double pos (n (%)) 7 (37) ESR, mm/hr (median (IQR) 8 (3-19) CRP, mg/l (median (IQR) 2.2 ( ) Morning stiffness, minutes (median (IQR) 5 (0-15) Arthralgia (n (%)) 15 (79) Arthralgia in the biopsied knee joint (n (%)) * 7 (47) VAS pain, mm (median (IQR)) * 29 (6-57) VAS disease activity, mm (median (IQR)) 12 (1-40) 68 TJC (n) * 1 (0-2) 66 SJC (n) 0 NSAID use (n (%)) 4 (21) IgM-RF= IgM rheumatoid factor; ACPA= anti-citrullinated protein antibodies; ESR= erythrocyte sedimentation rate; CRP= C-reactive protein; VAS= visual analogue scale (rang mm); 68 TJC= tender joint count of 68 joints; 66 SJC= swollen joint count of 66 joints; * Only in individuals with arthralgia; IQR: interquartile range; NSAID: non steroidal anti-inflammatory drug. Synovial expression of PGE 2 enzymes is not clearly related to arthralgia and development of arthritis in autoantibody-positive individuals Synovial expression of PGE 2 pathway enzymes was not lower in the group of individuals who used NSAIDs (n=4) compared to those who did not (n=15). In addition the VAS pain was comparable between individuals who did or did not use NSAIDs (data not shown). A trend towards higher expression of COX-1 was observed in the 15 individuals with arthralgia compared to the 4 individuals without arthralgia (p=0.078) as well as for COX-2 (p=0.470) and 15-PGDH (p=0.352) (Figure 1, panel A). Expression of PGE 2 pathway enzymes did not correlate with pain scores (data not shown). Within the group of individuals with arthralgia, expression of PGE 2 pathway enzymes was not higher in the individuals having arthralgia in the biopsied joint compared to the individuals having arthralgia in other joints only (Figure 1, panel B). Next, we compared synovial expression of PGE 2 pathway enzymes in individuals who developed arthritis (n=6) with those who did not (n=13), irrespective of their arthralgia status (one individual without arthralgia had developed arthritis after follow-up). We did not observe any differences in the expression levels of COX-1, COX-2, mpges-1 or 15-PGDH between those two groups (data not shown). Collectively, these results suggest that synovial expression of the PGE 2 pathway has no major role in either arthralgia or the onset of arthritis in autoantibody-positive individuals at risk of developing RA. 82

84 SYNOVIAL PGE 2 ENZYMES IN ARTHRALGIA AND EARLY ARTHRITIS PATIENTS 6 Figure 1. Synovial expression of mpges-1, COX-1, COX-2 and 15-PGDH in autoantibody-positive individuals at risk of developing RA. Panel (A): comparison of individuals with or without arthralgia in general. Panel (B): comparison of individuals with or without arthralgia in the biopsied joint, within the group of individuals with arthralgia only; values expressed as median (IQR). Early arthritis patients Baseline characteristics of early arthritis patients are depicted in Table 2. Fifty-five percent of the early arthritis patients in this study used NSAIDs at the moment of synovial biopsy sampling. Expression of mpges-1, COX-1 and COX-2 was comparable between individuals who did or did not use NSAIDs (data not shown). This suggests that NSAID treatment will not bias the results of our analyzes. In addition the VAS pain was comparable between individuals who did or did not use NSAIDs (data not shown). Pain sensation is not related to synovial expression of PGE 2 pathway enzymes in early arthritis patients We did not find a positive correlation between synovial expression of PGE 2 pathway enzymes and pain sensation in early arthritis patients (Table 3). Results were comparable when analyzing diagnostic subgroups of RA, SpA and UA (data not shown). Next, since PGE 2 is also a potent pro-inflammatory molecule modulating 83

85 Table 2. Baseline characteristics of early arthritis patients N= 104 Sex, female (n (%)) 64 (62) Age, years (mean (SD)) 48 (15) IgM-RF positive (n (%)) * 29 (29) Anti-CCP positive (n (%)) 27 (27) IgM-RF and anti-ccp both pos (n (%)) * 22 (22) ESR, mm/hr (median (IQR) 23 (11-45) CRP, mg/l (median (IQR) 8.5 ( ) VAS pain general, mm (median (IQR)) 61 (39-77) VAS pain biopsied joint, mm (median (IQR)) 59 (29-80) DAS28 (median (IQR)) 4.4 ( ) 68 TJC (n) 5 (1-12) 66 SJC (n) 3 (1-8) NSAID use (n (%)) ** 57 (55) IgM-RF= IgM rheumatoid factor; ACPA= anti-citrullinated protein antibodies; ESR= erythrocyte sedimentation rate; CRP= C-reactive protein; VAS= visual analogue scale (rang mm); 68 TJC= tender joint count of 68 joints; 66 SJC= swollen joint count of 66 joints; * missing for 3 patients; ** missing for 1 patient; SD: standard deviation; IQR: interquartile range; NSAID: non steroidal anti-inflammatory drug. Table 3. Correlation analysis of pain scores and expression of PGE 2 pathway enzymes in the synovium of early arthritis patients Enzyme VAS pain general 68TJC VAS pain biopsied joint mpges-1 COX-1 COX-2 r= p=0.244 r= p=0.323 r=0.042 p=0.698 r= p=0.143 r= p=0.046 r= p=0.072 r= p=0.187 r= p=0.095 r= p=0.949 VAS: visual analogue scale; 68TJC: tender joint count of 68 joints; mpges-1: microsomal prostaglandin E synthase-1; COX: cyclooxygenase. vasodilation and edema, we investigated whether expression of the enzymes of the PGE 2 pathway could be positively related to inflammation rather than to pain sensation. As expected, we observed a positive correlation between expression of COX-1 and CD3 (T cells) and between expression of mpges-1 and COX-1 and expression of CD68 (macrophages) both in the synovial sublining and lining layer (Table 4). None of the PGE 2 pathway enzymes correlated to the systemic inflammatory parameters ESR and CRP, or to expression of CD55 (FLS) in the synovium. The expression of mpges-1 and COX-1 is increased in SpA compared to RA patients Baseline characteristics of the subgroups of early arthritis patients with a definite diagnostic classification of RA, SpA or UA after 2 years of follow-up are depicted in Table 5. 84

86 SYNOVIAL PGE 2 ENZYMES IN ARTHRALGIA AND EARLY ARTHRITIS PATIENTS Table 4. Correlation analysis of inflammatory markers and expression of PGE 2 pathway enzymes in the synovium of early arthritis patients Enzyme ESR CRP mpges-1 COX-1 COX-2 r= p=0.570 r=0.058 p=0.566 r= p=0.702 r= p=0.658 r= p=0.789 r=0.009 p=0.932 CD55 N=49 r=0.277 p=0.054 r=0.191 p=0.188 r=0.117 p=0.435 CD3 N=47 r=0.258 p=0.080 r=0.398 p=0.006 r=0.272 p=0.074 CD68SL N=45 r=0.359 p=0.016 r=0.439 p=0.003 r=0.180 p=0.253 CD68L N=48 r=0.326 p=0.024 r=0.460 p=0.001 r=0.203 p=0.181 ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; CD55: marker for fibroblast like synoviocytes; CD3: marker for T cells; CD68: marker for macrophages; SL: sublining layer; L: intimal lining layer; mpges-1: microsomal prostaglandin E synthase-1; COX: cyclooxygenase. 6 As expected, the number of patients being positive for IgM-RF and/or anti-ccp antibodies as well as the tender and swollen joint count and the DAS28 were significantly higher in the RA compared to the SpA and UA patients. The VAS for pain was significantly lower in SpA compared to UA and RA patients. Expression of mpges-1 and COX-1 was significantly different between the three diagnostic groups (p=0.002 and p=0.008, respectively) (Figure 2). When using Mann-Whitney U test for comparing groups pairwise, it appeared that expression of mpges-1 and COX-1 was increased in SpA compared to RA patients (p=0.001 and p=0.003, respectively) and a trend was observed for increased expression of mpges-1 in SpA compared to UA patients (p=0.067); not for COX-1. In addition, a trend towards significantly different expression of COX-2 within RA, UA and SpA patients was observed (p=0.063). When comparing groups pairwise COX-2 expression was increased in SpA compared to RA patients (p=0.047). The expression of PGE 2 pathway enzymes is comparable between diagnostic outcome groups within UA and RA patients To evaluate the discriminative value of synovial tissue expression of PGE 2 pathway enzymes between UA patients who later fulfill classification criteria for RA and those who remain UA, we compared patients with UA at baseline who were classified as RA after 2 years of follow-up (UA>RA) with UA patients at baseline who were still classified as UA after 2 years of follow-up (UA>UA) and compared those with patients classified as RA already at baseline (RA>RA). No significant difference could be detected in baseline synovial expression of mpges-1, COX-1 or COX-2 between the three diagnostic outcome groups (Table 6). Synovial expression of PGE 2 pathway enzymes is not related to disease persistence Lastly, we compared baseline expression of PGE 2 enzymes between prognostic outcome groups after 2 years of follow-up to evaluate whether RA patients developing persistent (erosive) disease had higher baseline expression levels than patients with self-liming 85

87 Table 5. Baseline characteristics of early arthritis patients classified as RA, SpA or UA after 2 years of follow-up RA N= 63 SpA N= 14 UA N= 27 P-value Sex, female (n (%)) 43 (68) 5 (36) 16 (59) Age, years (mean (SD)) 51 (15) 44 (14) 44 (15) IgM-RF positive (n (%)) 28 (45) * 0 (0) 1 (4)** Anti-CCP positive (n (%)) 27 (44)* 0 (0) 0 (0)** IgM-RF and anti-ccp both pos (n (%)) 22 (36) 0 (0) 0 (0) ESR, mm/hr (median (IQR) 24 (11-47) 17 (11-39) 22 (11-55) CRP, mg/l (median (IQR) 7.2 ( ) 9.0 ( ) 11.0 ( ) VAS pain general, mm (median (IQR)) 67 (45-78) 29 (18-57) 53 (36-76) VAS pain biopsied joint, mm (median (IQR)) 63 (34-81) 30 (17-73) 59 (30-76) DAS28 (median (IQR)) 4.7 ( ) 3.8 ( ) 3.9 ( ) TJC (n) 11 (4-22) 3 (1-5) 1 (1-3) SJC (n) 6 (2-11) 1 (1-3) 1 (1-2) NSAID use (n (%)) * 36 (57) 16 (62) 5 (36) IgM-RF= IgM rheumatoid factor; anti-ccp=anti-cyclic citrullinated peptide antibodies; ESR= erythrocyte sedimentation rate; CRP= C-reactive protein; VAS= visual analogue scale (rang mm); 68 TJC= tender joint count of 68 joints; 66 SJC= swollen joint count of 66 joints; * missing for 1 patient; ** missing for 2 patients; SD: standard deviation; IQR: interquartile range; NSAID: non steroidal anti-inflammatory drug. Figure 2. Baseline synovial expression of PGE2 pathway enzymes of early arthritis patients classified as RA, UA and SpA at 2 years follow-up. Representative immunohistochemical staining (A), Expression of mpges-1 (B) and COX-1 (C) is increased in SpA patients. D) A trend towards increased expression of COX-2 is observed in SpA patients; values expressed as median (IQR); RA: rheumatoid arthritis; UA: unclassified arthritis; SpA: spondyloarthritis; mpges-1: microsomal prostaglandin E synthase-1; COX: cyclooxygenase. 86

88 SYNOVIAL PGE 2 ENZYMES IN ARTHRALGIA AND EARLY ARTHRITIS PATIENTS disease. Prognostic outcome data were missing for 9 patients. No statistically significant differences between the prognostic outcome groups were observed (Table 7). Table 6. Baseline expression of PGE 2 pathway enzymes in the synovium of early arthritis patients classified as UA>UA, UA>RA or RA>RA at baseline>after 2 years of follow-up Enzyme mpges-1 (median (IQR)) UA>UA N= ( ) UA>RA N= ( ) RA>RA N=49 P-value 1202 ( ) COX-1 (median (IQR)) ( ) 9766 ( ) 8620 ( ) COX-2 (median (IQR)) 254 ( ) 133 (42-763) 143 (32-636) UA: unclassified arthritis; RA: rheumatoid arthritis; UA>UA both at baseline and after 2 years of follow-up classified as UA; UA>RA: classified as UA at baseline and as RA after 2 years of follow-up; RA>RA: classified as RA both at baseline and after 2 years of follow-up. mpges-1: microsomal prostaglandine E synthase-1; COX: cyclooxygenase; Values expressed as integrated optical density (IOD)/mm2; median (IQR: interquartile range). Table 7. Baseline synovial expression of PGE 2 pathway enzymes in different prognostic outcome groups of patients classified as RA after 2 years of follow-up Enzyme Self-limiting N=13 Persistent non-erosive N=30 Persistent erosive N=11 P-value mpges-1 (median (IQR)) 560 ( ) 1661 ( ) 3330 ( ) COX-1 (median (IQR)) 8570 ( ) 5045 ( ) ( ) COX-2 (median (IQR)) 111 ( ) 201 (51-876) 73 (53-155) mpges-1: microsomal prostaglandin E synthase-1; COX: cyclooxygenase. Values expressed as integrated optical density (IOD)/mm2; median (IQR: interquartile range). DISCUSSION Another hypothesis for our findings may be that PGE 2 would be involved in regulating arthralgia centrally (via spinal release) but not locally in the synovium. Measuring urinary excretion of the PGE 2 metabolite may be an alternative way to give information on systemic involvement of the PGE 2 pathway 31 and needs to be addressed in the future. We also have to consider that other prostanoids which have been shown to mediate pain hypersensitivity could be involved. For example, studies in experimental models of arthritis demonstrate involvement of PGI 2 (prostacyclin) in arthritic pain 32. Secondly, synovial expression of enzymes of the PGE 2 pathway was not related to development of arthritis in subjects at risk of developing RA and within RA patients we 87

89 did not observe a relation with the systemic inflammatory parameters ESR and CRP or with disease persistence. However, expression of these enzymes was positively correlated to the number of macrophages present in the synovium, which has been shown to be one of the main cell types expressing these enzymes 12. Thirdly, in early arthritis patients, synovial expression of mpges-1 and COX-1 was increased in SpA patients when compared to RA and UA patients and a similar trend was observed for COX-2, suggesting involvement of the PGE 2 pathway in the pathogenesis of SpA, supporting previous studies 11, 33. Of interest, the SpA patients in our cohort displayed significantly lower pain scores than the RA and UA patients. Previously, enhanced expression of COX-2 was observed in the intimal lining layer of ankylosing spondylitis (AS) patients (axial SpA) compared to psoriatic arthritis (PsA) and RA patients, and in the sublining layer in AS and PsA patients compared to RA patients 11. In that study, however, it could not be excluded that lower expression of COX-2 in the RA patient group was the result of the use of corticosteroids. In the current study, no patients had used corticosteroids for joint complaints or for any other disease in the last 3 months before inclusion, hereby excluding a treatment effect. Moreover, a role for the PGE 2 pathway in the pathogenesis of SpA has been shown by the positive effects of the use of NSAIDs. NSAIDs are the first-line drug treatment for complaints of pain and stiffness in AS patients 34 and in SpA patients in general. In addition, the beneficial effect of NSAIDs on the ossification of the spine in this patient group has repeatedly been shown 35. This is most probably the result of interference with the stimulatory effects of PGE 2 on osteoblastogenesis by increasing the replication and differentiation of osteoblasts 33. Our results suggest that this might be linked to the presence of PGE 2 produced in the synovial tissue. CONCLUSIONS We did not find clear evidence that arthralgia in subjects at risk of developing RA or in early arthritis patients can be explained by altered synovial expression of enzymes of the PGE 2 pathway, which are known to be involved in pain sensation. Pain in these patients may therefore be regulated by other pathways or originate at sites other than the synovium, which needs to be addressed in future studies. Synovial expression of mpges-1 and COX-1 is increased in SpA patients compared to RA and UA patients, supporting involvement of the PGE 2 pathway in the pathogenesis of SpA. ACKNOWLEDGEMENTS We would like to thank our study subjects for participation in the study, the AMC arthroscopy team for obtaining and processing synovial biopsies, and Noortje Smits and Jan Visscher for laboratory assistance. 88

90 SYNOVIAL PGE 2 ENZYMES IN ARTHRALGIA AND EARLY ARTHRITIS PATIENTS REFERENCE LIST 1. Aho K, Heliovaara M, Maatela J, Tuomi T, Palosuo T. Rheumatoid factors antedating clinical rheumatoid arthritis. J Rheumatol 1991;18(9): Nielen MM, van SD, Reesink HW et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum 2004;50(2): Rantapaa-Dahlqvist S, de Jong BA, Berglin E et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003;48(10): Bos WH, Wolbink GJ, Boers M et al. Arthritis development in patients with arthralgia is strongly associated with anti-citrullinated protein antibody status: a prospective cohort study. Ann Rheum Dis 2010;69(3): Gerlag DM, Raza K, van Baarsen LG et al. EULAR recommendations for terminology and research in individuals at risk of rheumatoid arthritis: report from the Study Group for Risk Factors for Rheumatoid Arthritis. Ann Rheum Dis 2012;71(5): Tak PP, Smeets TJ, Daha MR et al. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum 1997;40(2): van de Sande MG, de Hair MJ, van der Leij C et al. Different stages of rheumatoid arthritis: features of the synovium in the preclinical phase. Ann Rheum Dis 2011;70(5): Stichtenoth DO, Thoren S, Bian H, Peters- Golden M, Jakobsson PJ, Crofford LJ. Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells. J Immunol 2001;167(1): Thoren S, Jakobsson PJ. Coordinate upand down-regulation of glutathionedependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by NS-398 and leukotriene C4. Eur J Biochem 2000;267(21): Gheorghe KR, Thurlings RM, Westman M et al. Prostaglandin E2 synthesizing enzymes in rheumatoid arthritis B cells and the effects of B cell depleting therapy on enzyme expression. PLoS One 2011;6(1):e Siegle I, Klein T, Backman JT, Saal JG, Nusing RM, Fritz P. Expression of cyclooxygenase 1 and cyclooxygenase 2 in human synovial tissue: differential elevation of cyclooxygenase 2 in inflammatory joint diseases. Arthritis Rheum 1998;41(1): Westman M, Korotkova M, af KE et al. Expression of microsomal prostaglandin E synthase 1 in rheumatoid arthritis synovium. Arthritis Rheum 2004;50(6): Chen M, Boilard E, Nigrovic PA et al. Predominance of cyclooxygenase 1 over cyclooxygenase 2 in the generation of proinflammatory prostaglandins in autoantibody-driven K/BxN serum-transfer arthritis. Arthritis Rheum 2008;58(5): Myers LK, Kang AH, Postlethwaite AE et al. The genetic ablation of cyclooxygenase 2 prevents the development of autoimmune arthritis. Arthritis Rheum 2000;43(12): Trebino CE, Stock JL, Gibbons CP et al. Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase. Proc Natl Acad Sci U S A 2003;100(15): de Hair MJ, Harty LC, Gerlag DM, Pitzalis C, Veale DJ, Tak PP. Synovial tissue analysis for the discovery of diagnostic and prognostic biomarkers in patients with early arthritis. J Rheumatol 2011;38(9): Dougados M, van der Linden S, Juhlin R et al. The European Spondylarthropathy Study Group preliminary criteria for the classification of spondylarthropathy. Arthritis Rheum 1991;34(10): Visser H, le CS, Vos K, Breedveld FC, Hazes JM. How to diagnose rheumatoid arthritis early: a prediction model for persistent (erosive) arthritis. Arthritis Rheum 2002;46(2): Gerlag DM, Tak PP. How to perform and analyse synovial biopsies. Best Pract Res Clin Rheumatol 2009;23(2): van de Sande MG, Gerlag DM, Lodde BM et al. Evaluating antirheumatic treatments using synovial biopsy: a recommendation for standardisation to be used in clinical trials. Ann Rheum Dis 2011;70(3): Dolhain RJ, Ter Haar NT, De KR et al. Distribution of T cells and signs of T-cell activation in the rheumatoid joint: implications for semiquantitative comparative histology. Br J Rheumatol 1998;37(3): Ulfgren AK, Lindblad S, Klareskog L, Andersson J, Andersson U. Detection of cytokine producing cells in the synovial membrane from patients with rheumatoid arthritis. Ann Rheum Dis 1995;54(8):

91 23. Haringman JJ, Gerlag DM, Zwinderman AH et al. Synovial tissue macrophages: a sensitive biomarker for response to treatment in patients with rheumatoid arthritis. Ann Rheum Dis 2005;64(6): Kraan MC, Haringman JJ, Ahern MJ, Breedveld FC, Smith MD, Tak PP. Quantification of the cell infiltrate in synovial tissue by digital image analysis. Rheumatology (Oxford) 2000;39(1): van der Hall PO, Kraan MC, Tak PP. Quantitative image analysis of synovial tissue. Methods Mol Med 2007;135: Ruijter JM, Thygesen HH, Schoneveld OJ, Das AT, Berkhout B, Lamers WH. Factor correction as a tool to eliminate betweensession variation in replicate experiments: application to molecular biology and retrovirology. Retrovirology 2006;3: van de Sande MG, de Hair MJ, Schuller Y et al. The features of the synovium in early rheumatoid arthritis according to the 2010 ACR/EULAR classification criteria. PLoS One 2012;7(5):e Ito S, Okuda-Ashitaka E, Minami T. Central and peripheral roles of prostaglandins in pain and their interactions with novel neuropeptides nociceptin and nocistatin. Neurosci Res 2001;41(4): Schaible HG, Richter F. Pathophysiology of pain. Langenbecks Arch Surg 2004;389(4): Schaible HG, von Banchet GS, Boettger MK et al. The role of proinflammatory cytokines in the generation and maintenance of joint pain. Ann N Y Acad Sci 2010;1193: Murphey LJ, Williams MK, Sanchez SC et al. Quantification of the major urinary metabolite of PGE2 by a liquid chromatographic/mass spectrometric assay: determination of cyclooxygenase-specific PGE2 synthesis in healthy humans and those with lung cancer. Anal Biochem 2004;334(2): Pulichino AM, Rowland S, Wu T et al. Prostacyclin antagonism reduces pain and inflammation in rodent models of hyperalgesia and chronic arthritis. J Pharmacol Exp Ther 2006;319(3): Schett G, Rudwaleit M. Can we stop progression of ankylosing spondylitis? Best Pract Res Clin Rheumatol 2010;24(3): Braun J, van den Berg R, Baraliakos X et al update of the ASAS/EULAR recommendations for the management of ankylosing spondylitis. Ann Rheum Dis 2011;70(6): Sieper J. Developments in therapies for spondyloarthritis. Nat Rev Rheumatol 2012;8(5):

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96 HUNTING FOR THE PATHOGENESIS OF RHEUMATOID ARTHRITIS: CORE- NEEDLE BIOPSY OF INGUINAL LYMPH NODES AS A NEW RESEARCH TOOL Maria J.H. de Hair 1, IJsbrand A. J. Zijlstra 2, Maria J.H. Boumans 1, Marleen G.H. van de Sande 1, Mario Maas 2, Danielle M. Gerlag 1, Paul P. Tak 1 1 Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands, 2 Department of Radiology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands 7 Ann Rheum Dis Nov;71(11):1911-2

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98 CORE-NEEDLE BIOPSY OF INGUINAL LYMPH NODES AS A NEW RESEARCH TOOL FOR RA Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting mainly the synovial joints. Processes involved in the initiation of inflammation in RA are largely unknown. Recent work has shown that systemic autoimmunity precedes synovial inflammation in autoantibody positive individuals developing RA1 and animal models have suggested that lymph node changes may precede inflammation in the synovial tissue2. Ultrasound guided lymph node biopsy under local anesthesia has previously been used as a diagnostic tool in hematology and oncology3. Therefore, we started to use this technique to provide insight into the pathogenic processes involved in the earliest phases of RA, by analyzing lymph node tissue obtained during different phases of the disease. Our experience includes use of this technique in autoantibody positive individuals without arthritis who are at risk of developing RA4, early arthritis patients and established RA patients. The studies have been approved by the institutional review board of the Academic Medical Center/ University of Amsterdam. Here we describe the technique and the initial experience, which may facilitate research in other centres. Study candidates are informed about the background and purpose of the study and the biopsy procedure and possible complications (in particular hematoma). Written informed consent is obtained. Anticoagulant therapy is stopped. The lymph node biopsy procedure is performed on an inguinal lymph node in an outpatient clinical setting by a radiologist and assistant, see Figure 1. Figure 1. Ultrasound guided core-needle biopsy procedure of an inguinal lymph node. A. Utensils are placed on a sterile table B. A bar gun is used to obtain biopsies under ultrasound guiding C. Ultrasound image of the biopsy needle within the lymph node D. The biopsy is placed on a wet gauze E. The biopsy is put in medium. 97 7