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Available onlinehttp://arthritis-research.com/supplements/6/S3 Arthritis Research & Therapy Volume 6 Supplement 3, 2004 Meeting abstracts Global Arthritis Research Network: 4th World Congress on Arthritis in Montreal Montreal, Quebec, Canada 20–22 September 2004 Published online: 13 September 2004 These abstracts are online at http://arthritis-research.com/supplements/6/S3 © 2004 BioMed Central Ltd Message from World Health Organization lection will vary in different socioeconomic and geographic areas. Guidance on sampling methods is also being developed. Health needs assessment of musculoskeletal ConclusionsThe comparability of data collected across the globe will conditions: an international bone and joint burden improve by the application of agreed upon indicators that consider key of disease study domains for the different musculoskeletal conditions and problems in epidemiological studies conducted in different populations. N Khaltaev1, B Pfleger1, AD Woolf2, K Åkesson3, JM Hazes4, References D Symmons5, and the Bone and Joint Monitor Group 1. The burden of musculoskeletal diseases at the start of the 1Management of Noncommunicable Diseases, World Health new millenium.Report of a WHO Scientific Group (WHO Tech- Organization, Geneva, Switzerland; 2Duke of Cornwall Rheumatology nical Report Series No 919). Geneva: World Health Organization; Department, Royal Cornwall Hospital, Truro, UK; 3Department of 2003. Orthopaedic Surgery, Malmö University Hospital, Malmö, Sweden; 2. International Classification of Functioning, Disability and 4Department of Rheumatology, University Hospital of Rotterdam, The Health 2004[http://www3.who.int/icf/icftemplate.cfm] Netherlands; 5Epidemiology Research Unit, University of Manchester, UK Arthritis Res Ther 2004, 6(Suppl 3):103 (DOI 10.1186/ar1334) Introduction The Bone and Joint Monitor Project was developed to ORAL PRESENTATIONS quantify the global burden of musculoskeletal conditions and develop Session I — Genomic/genetic/proteomic: the strategies for their prevention. Experts within the Monitor Project have worked previously with officers at the World Health Organization added value to arthritis research and (WHO) to estimate morbidity and mortality associated with rheumatic treatment conditions. The present collaboration seeks means of providing additional and more current burden data. 1 Objective To develop recommendations for performing epidemiologi- Osteoarthritis: degeneration of matrix or cells? cal studies in sample populations with musculoskeletal conditions and problems, accounting for determinants and consequences to the indi- T Aigner vidual and society. Osteoarticular and Arthritis Research, Department of Pathology, MethodsRecommendations have been developed identifying the most University of Erlangen–Nürnberg, Germany relevant domains for measuring and monitoring the various muscu- Arthritis Res Ther 2004, 6(Suppl 3):1 (DOI 10.1186/ar1335) loskeletal conditions by review of epidemiological data on occurrence, determinants and outcomes, and by expert opinion. Instruments that Osteoarthritis is the most common disabling condition of man in the measure these domains were reviewed. Western world. Biologically, osteoarthritis implies the (symptomatic) ResultsThe domains recommended follow the principles of the WHO degeneration of the whole joint with many structures involved such as International Classification of Functioning, Disability and Health [1,2], subchondral bone and the periarticular soft tissues. Still, the degenera- and consider: health condition; body function and structure; activity lim- tion of the articular cartilage appears to be the focus of the structural itation; participation restriction; personal and environmental contextual changes taking place during the osteoarthritic disease process. factors; and, in addition, the resource utilisation and social conse- Cartilage degeneration implies mostly a failure of the extracellular quences. The musculoskeletal conditions and problems considered matrix of the articular cartilage. The matrix represents the functional were osteoarthitis, inflammatory arthritis, osteoporosis, spinal prob- component responsible for the biomechanical integrity of the tissue. lems, musculoskeletal trauma and injuries, and musculoskeletal pain Thus, many studies over decades in osteoarthritis and cartilage with restricted activity. The selection of indicators for each domain con- research have focused on the understanding of degenerative or sidered the feasibility of their use in a health interview survey (HIS), a degradative processes taking place within the matrix. This has led to health examination survey (HES), a register or a clinical study. Consen- significant continuous advances in our understanding of the biochem- sus on case definition was reached depending on the study methodol- istry and pathobiochemistry of molecules and their assembly within the ogy. For example, osteoporosis defined by bone densitometry cannot cartilage matrix. be ascertained in an HIS, whereas the outcome of osteoporosis (i.e. The cells of the tissue, which do not exert per se structural functions fragility fracture) can be. Osteoarthitis can be identified as joint pain in within the tissue, were considered largely responsible for the proper an HIS but the preferred definition is pain with X-ray changes and can matrix turnover. They were thought to react to external mediators and only be ascertained in an HES. Previously validated generic and biomechanical stimuli or assaults and to be more or less able to com- disease-specific instruments have been identified that include indica- pensate for continuous (bio)mechanical tissue destruction. More tors for all or most of the recommended domains for the consequences recently, however, besides matrix (patho)biochemistry, the cellular phe- of the different conditions and problems. The indicators of the domains notype during the disease process has come more and more into the for resource utilisation and social consequences and feasibility for col- focus of interest. This was induced on the one hand by upcoming (mol- S1 Arthritis Research & Therapy Vol 6 Suppl 3 Abstracts of the 4th World Congress of the Global Arthritis Research Network ecular) technology, but also by increasing evidence that also the cells dependent on many polymorphic genes operating in concert. So far degenerate per se. Osteoarthritic chondrocytes are not only no longer two genes in this concert have been identified; the MHC class II Ab able to compensate for matrix degeneration, but themselves are (co)ini- gene in the mouse [1] and the Ncf1 gene in the rat [2]. The Ncf1 tiators and (co)promoters of the disease process. protein is a part of the NADPH oxidase complex involved in generation of the inducible oxidative burst. The discovery of the Ncf1 polymor- 2 phism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive T cells. Genetics of osteoarthritis This hypothesis has now been further documented by the identification J Loughlin, A Ferreira, B Dowling, L Southam, Z Mustafa, of an Ncf1 mutation in the mouse that reproduces the effects earlier T Forster, K Chapman observed in the rat. Mice with the deficient Ncf1 allele, and expressing Institute of Musculoskeletal Sciences, Botnar Research Centre, the MHC class II allele Aq, binding CII peptides, could be shown to be University of Oxford, UK dramatically more susceptible to collagen-induced arthritis, and also Arthritis Res Ther 2004, 6(Suppl 3):2 (DOI 10.1186/ar1336) developed a chronic form of arthritis. Interestingly, the immune response to CII was enhanced by the Ncf1 deficiency linking the Ncf1 Epidemiological studies have demonstrated a major genetic compo- pathway to the adaptive immune response. nent to osteoarthritis (OA), with heritability estimates of over 50% for References most joint sites. These studies have also highlighted differences in the 1. Brunsberg U, Gustafsson K, Jansson L, Michaëlsson E, Ährlund- degree of OA heritability between joint sites and between the sexes, Richter L, Pettersson S, Mattsson R, Holmdahl R: Expression of implying a high level of heterogeneity. a transgenic class II Ab gene confers susceptibility to colla- We published a genome-wide scan in 1999. We had focused on fami- gen-induced arthritis.Eur J Immunol1994, 24:1698-1702. lies containing siblings with severe large-joint OA ascertained by joint- 2. Olofsson P, Holmberg J, Tordsson J, Lu S, Åkerström B, Holmdahl replacement surgery. Our data showed that OA genetic susceptibility R: Positional identification of Ncf1 as a gene that regulates did exhibit joint specificity and that this susceptibility had a greater role arthritis severity in rats. Nat Genet2003, 33:25-32. in female disease. During the past 5 years we have been investigating our linkage regions and we have so far identified FRZB(chromosome 4 2q32.1), COL9A1 (6q12-q13), BMP5 (6p12.1) and IL4R (16p12.1- Genome-wide single nucleotide polymorphism p11.2) as encoding for OA susceptibility. Common variants at these analyses on rheumatoid arthritis genes affect either the structural properties of the protein (FRZBand IL4R) or the transcription of the gene (BMP5and COL9A1). The vari- K Yamamoto1,2, A Suzuki2, S Tokuhiro2,Y Kochi1,2, R Yamada2 ants are particularly relevant to the development of hip OA in females. 1Department of Allergy and Rheumatology, The University of Tokyo, What is particularly interesting about our recent discoveries is that the Japan; 2Laboratory of Rheumatic Diseases, SNP Research Center, proteins encoded for by IL4R, BMP5and FRZBare involved in chon- RIKEN, Yokohama, Japan drocyte cell signalling and signal transduction pathways. It appears Arthritis Res Ther 2004, 6(Suppl 3):4 (DOI 10.1186/ar1338) probable, therefore, that OA genetic risk for the hip is principally accounted for by aberrant cell signalling. This was not anticipated. Although the etiology of rheumatoid arthritis (RA) is still unknown, it is In this presentation I will focus on our latest genetic findings. I will also generally believed that a combination of multiple polymorphic genetic discuss the results from other studies. A number of OA genome-wide factors and environmental contributions lead to the pathogenic auto- scans have been performed, some on large joints and others investigat- immune reactions characterized by destructive polyarthritis and various ing hand disease. Many loci appear unique to one particular study, with extra-articular inflammatory involvements. A genetic approach to iden- only some loci being positive in multiple studies. The reasons for this tify RA-associated genes from the whole genome is proposed to be will be discussed. one of the promising methodologies to elucidate the etiology of RA. A concern often expressed at orthopaedic and rheumatology meetings Although multiple genome-wide linkage studies and many association is that genetic linkages and associations are not consistently repro- studies to test an individual gene that is functionally relevant to RA duced. These are reasonable criticisms but it needs to be remembered have been performed to identify RA-associated nonhuman leukocyte that the genetic component of a complex trait such as OA will not be antigengenes, no region or gene has been conclusively shown to be mediated by fully penetrant risk alleles. Instead, any one allele will con- one of the RA-susceptible loci or genes. Therefore, a linkage disequilib- tribute only a fraction of the overall risk and this allele will, by chance, rium approach using single nucleotide polymorphisms (SNPs) as have varying frequencies in different cohorts. It is unreasonable, there- genetic markers to identify disease-associated genes was considered fore, to expect a linkage or an association in one cohort to be repli- a promising alternative method for RA, as well as other common dis- cated in all cohorts. eases with complex genetic predispositions. Acknowledgements Our research was funded by Research into Our RIKEN SNP Research Center has established a high-throughput Ageing, The Arthritis Research Campaign, The Nuffield Foundation and SNP-genotyping system and has been investigating multiple common The Wellcome Trust. diseases using a whole-genome gene-based linkage disequilibrium mapping approach. In 2003, we reported that functional variants of the 3 PADI4 gene were associated with RA. PADI4 is an enzyme that con- verts arginine residue in proteins to citrulline. The citrullinated proteins Genetic control of T-cell autoimmunity and arthritis are targets of the most specific autoantibodies in RA. We have identi- R Holmdahl fied this gene in a whole-genome, hypothesis-free approach. Therefore, Section of Medical Inflammation Research, Lund University, Lund, this made our methodology strongly reliable in that the RA-associated Sweden gene identified by our hypothesis-free approach was functionally Arthritis Res Ther 2004, 6(Suppl 3):3 (DOI 10.1186/ar1337) strongly relevant to the pathogenesis of RA. Recently we also found that SLC22A4 and RUNX1 are associated Inbred animals are useful for studies of the identification of genes asso- with RA. SLC22A4 is an organic cation transporter with unknown ciated with rheumatoid arthritis (RA) since they are a more efficient tool physiologic function, and RUNX1 is a hematologic transcriptional regu- for identification genes controlling complex diseases. There are several lator that has been shown to be responsible for acute myelogenic arthritis models, each of which may reflect various variants of the het- leukemia. It is suggested that the association of RUNX1 with RA is due erogeneity of RA in humans. Examples are collagen-induced arthritis to its regulation of expression of SLC22A4. Because the physiologic and pristane-induced arthritis, which both fulfil the clinical diagnostic function of SLC22A4 is still unclear, further investigation is needed into criteria for RA. how SLC22A4 affects RA susceptibility. Although the association of Type II collagen (CII) is immunogenic and contains peptides that can RUNX1 with RA was identified as a regulatory factor of SLC22A4, it is be bound to major histocompatibility complex (MHC) class II and pre- possible that RUNX1 is a key molecule in autoimmunity, as it has been sented to T cells, whereas pristane is not immunogenic by itself. Both reported to be associated with systemic lupus erythematosus and pso- S2 diseases are genetically complex and the susceptibility is, as RA, riasis, two other autoimmune diseases. Available onlinehttp://arthritis-research.com/supplements/6/S3 5 important for ‘ER-associated degradation (ERAD)’. Its overexpression Rheumatoid arthritis: a heterogeneous disease causes arthropathy, resembling RA in mice. Moreover, the heterozygote of Synoviolin (+/–) is remarkably resistant to collagen-induced arthritis. evaluated by DNA-microarray technology These ‘gain of function’ and ‘loss of function’ analyses clearly indicate PV Kasperkovitz1, T Timmer1, T Smeets2, PP Tak2, the important role of Synoviolin in arthropathy. TWJ Huizinga3, GS Firestein4, F Rustenburg1, Therefore, we present a new pathogenic mechanism for RA that is TCTM van der Pouw Kraan1, M Fero5, CL Verweij1 related with ERAD, an essential system for ER homeostasis by eliminat- 1Department of Molecular Cell Biology and Immunology, VUMC, ing unfolded proteins from the organelle. Recent studies have shown Amsterdam, The Netherlands; 2Department of Clinical Immunology that the accumulation of unfolded proteins mediated by impaired ERAD and Rheumatology, Academic Medical Center, Amsterdam, The system results in ER stress-induced apoptosis of cells and causes Netherlands; 3Department of Rheumatology, LUMC, Leiden, The various degenerative human diseases. Besides, the ERAD system is Netherlands; 4Division of Rheumatology, Allergy and Immunology, tightly linked with cell growth, apoptosis and differentiation. This evi- UCSD, La Jolla, California, USA; 5Stanford Functional Genomics, dence led us to a new hypothesis that ‘hyper function of ERAD system’ Stanford University, California, USA may cause synovial cell overgrowth through its anti-apoptotic effect in RA. Thus, we propose that inhibition of Synoviolin is one of the promis- Arthritis Res Ther 2004, 6(Suppl 3):5 (DOI 10.1186/ar1339) ing strategies for RA treatment by targeting synovial cells. The molecular pathogenesis of rheumatoid arthritis (RA) is still poorly understood and its clinical course can vary widely. We apply a systems Session II — Role of leptin and eicosanoids in biology approach to gain insight into the complex pathogenesis and arthritic diseases disease heterogeneity. Genomics studies revealed considerable heterogeneity in global gene expression signatures between synovial 7 tissue specimens from different patients with RA. Based on the molecular signatures, at least two distinct subsets of RA tissues could be A role for leptin in immune-mediated inflammatory identified. One class revealed abundant expression of gene clusters diseases? indicative of an ongoing activation of the adaptive immune response, C Gabay, G Palmer whereas the other class resembled the expression pattern of osteoarthritic tissues, which are characterized by a low inflammatory Division of Rheumatology, University Hospital of Geneva and gene expression signature and increased tissue remodeling. The mole- Department of Pathology and Immunology, University of Geneva cular heterogeneity is featured not only at the whole synovial tissue School of Medicine, Geneva, Switzerland level, but also at the level of fibroblast-like synoviocytes (FLS) cultured Arthritis Res Ther 2004, 6(Suppl 3):7 (DOI 10.1186/ar1341) from those tissues. One of the most impressive features of our gene expression profiling studies is the clear correlation of the FLS pheno- Leptin, the product of the Ob gene, is a polypeptide hormone that type with that of paired synovial tissue from which the cells were plays an important role in the regulation of body weight by inhibiting derived. One class of FLS is tightly related to the presence of lympho- food intake and stimulating energy expenditure. Moreover, leptin cytes in the lesions, whereas the other class of FLS suggests that syn- exhibits a variety of other effects including the regulation of endocrine oviocyte-mediated invasion appears to be less dependent on infiltrating function, reproduction and hematopoiesis. Consistently, leptin-deficient immune cells. These data support the notion that heterogeneity mice are not only obese, but display major hormonal disturbances, observed between synovial tissues is reflected in the FLS as a stable including hypercorticosteronemia, diabetes, and infertility. In addition, it trait. Clearly, the list of genes that are differentially expressed between has been known for many years that leptin-deficient (ob/ob) and leptin the tissue and FLS subgroups facilitates our understanding of the receptor-deficient (db/db) mice have an altered immune response. pathophysiology of the molecular forms of disease. Moreover, the dif- More recent studies have shown that T cells and B cells express the ferences in the gene expression profiles reflect important aspects of long signaling leptin receptor isoform (ObRb) and that leptin exerts biological variation within the clinically diagnosed disease that provide direct effects on T lymphocytes, including the stimulation of cell prolif- a molecular basis for the well recognized but as yet poorly understood eration, the promotion of Th1 responses, and the protection of thymo- heterogeneity in RA and may help to (sub)classify rheumatic diseases. cytes from corticosteroid-induced apoptosis. Consistently, ob/ob mice AcknowledgementsThe authors are grateful to Dr Pat Brown and Dr are protected from inflammation mediated by T cells and B cells in David Botstein, in whose laboratories part of the work described in this some models such as experimental colitis, experimental autoimmune report was performed. Supported in part by the Howard Hughes encephalomyelitis, and concanavalin A-induced hepatitis. Conversely, Medical Institute, a grant from the National Cancer Institute, a grant the administration of recombinant leptin increased the severity of exper- from the Netherlands Organization for Scientific Research, and the imental autoimmune encephalomyelitis. Using a model of antigen- Dutch Arthritis Foundation. induced arthritis, we demonstrated that ob/ob and db/db mice have a milder form of arthritis and that cellular and humoral immune responses 6 to the injected antigen are decreased as compared with wild-type mice. Furthermore, ex vivostimulated lymph node cells from ob/ob and Hyper-ERAD: a novel pathomechanism for db/db mice produced less interferon gamma and more IL-10 than cells rheumatoid arthritis obtained from control mice. T Nakajima1, N Yagishita1, S Yamasaki1, K Tsuchimochi1, Leptin production is regulated by various proinflammatory cytokines T Amano1, A Fukamizu2, I Maruyama3, K Nishioka4 during the acute-phase response, and leptin stimulates the production 1Department ofGenome Science and 4Department of Rheumatology, of both proinflammatory and antiinflammatory cytokines in vitro. ob/ob mice exhibit increased susceptibility to lipopolysaccharide-induced and Immunology and Genetics Program, Institute of Medical Science, St Marianna University, Kawasaki, Japan; 2Institute of Applied tumor necrosis factor alpha-induced lethality, suggesting that leptin is also involved in regulating the innate immune response. We recently Biochemistry and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan; 3Department of Dermatology examined the role of leptin in zymosan-induced arthritis (ZIA), where joint inflammation is dependent on innate immunity. Our results demon- and Laboratory of Molecular Medicine,Kagoshima University, Faculty strate that, as opposed to antigen-induced arthritis, ZIA is not impaired of Medicine, Kagoshima, Japan in ob/ob and db/db mice. On the contrary, these mice exhibit a delayed Arthritis Res Ther 2004, 6(Suppl 3):6 (DOI 10.1186/ar1340) resolution of the inflammatory process, with increased circulating levels Rheumatoid arthritis (RA) is a joint-affecting disease and is character- of IL-6 and serum amyloid A, and a tendency to develop more severe ized with overgrowth of articular synovial cells, so-called ‘pannus’. To joint damage. Leptin deficiency thus appears to interfere with adequate understand the pathomechanism of RA, we attempted to characterize control of the inflammatory response in ZIA. the rheumatoid synovial cell and found a novel protein, ‘Synoviolin (syn- Although data obtained in different models of inflammatory diseases ovial cell + protein)’. Structural analysis indicated that Synoviolin is an suggest that leptin plays a role in immune response, the analysis of endoplasmic reticulum (ER)-resident E3 ubiquitin ligase, which is direct effects of leptin on lymphocytes in vivo is precluded by the S3 Arthritis Research & Therapy Vol 6 Suppl 3 Abstracts of the 4th World Congress of the Global Arthritis Research Network important hormonal and metabolic alterations, which are linked to leptin 9 deficiency. To our knowledge, it has never been thoroughly investi- Regulation of eicosanoid synthesis by leptin gated to which extent immune defects in ob/ob or db/db animals are P Mancuso1, HH Mehta2, C Canetti2, M Peters-Golden2, linked to leptin deficiency or, respectively, leptin receptor deficiency per se, as opposed to confounding factors such as hypercorticos- BJ Roessler2, LJ Crofford2 teronemia. To examine the importance of direct effects of leptin on lym- 1Department of Environmental Health Sciences, University of phopoiesis and immune response, we generated bone marrow Michigan, Ann Arbor, Michigan, USA; 2Department of Internal chimeras by transplantation of db/db bone marrow cells into lethally Medicine, University of Michigan, Ann Arbor, Michigan, USA irradiated normal recipient mice, which provide a normal environment Arthritis Res Ther 2004, 6(Suppl 3):9 (DOI 10.1186/ar1343) for the grafted cells. Donor db/db mice display a marked atrophy of the thymus as compared with db/+ controls. In bone marrow chimeras, the Leptin is an adipocyte-derived hormone that is found in the peripheral size and cellularity of the thymus were not different between mice circulation in correlation with total body fat mass. While leptin is best grafted with db/db or db/+ bone marrow 12 weeks after the graft, sug- known for its ability to regulate energy homeostasis, it is a pleotropic gesting that the thymic atrophy observed in db/db donors is not due to hormone whose receptor is widely distributed in various tissues includ- direct effects of leptin on lymphocytes. Moreover, RT-PCR analyses ing cells of the immune system. We have previously reported that suggest that CD4/CD8 double positive thymocytes do not express leukotriene (LT) synthesis in macrophages from leptin-deficient mice is ObRb. Further studies are in progress in order to examine which cell attenuated and that the provision of exogenous leptin restores this types respond to leptin in the thymus and whether impaired leptin sig- defect. To explore the possibility that leptin regulates eicosanoid syn- naling in lymphocytes affects immune responses after antigen chal- thesis in cells from normal animals, we cultured rat alveolar and murine lenge in db/db bone marrow chimeras. peritoneal macrophages overnight with media alone or with increasing doses of leptin. Following stimulation with calcium ionophore (A23187) 8 or zymosan, we observed that leptin enhanced prostaglandin E and LT 2 synthesis in a dose-dependent manner. Since arachidonic acid (AA) is Is leptin the link between obesity and the metabolic precursor of the eicosanoids, we next asked whether osteoarthritis? leptin regulates AA release and phospholipase A (PLA ) activity. Rat 2 2 P Pottie, N Presle, H Dumond, B Terlain, D Mainard, D Leuille, alveolar macrophages (AMs) were prelabeled overnight with [3H]AA P Netter and cultured with increasing doses of leptin on the following day. Leptin enhanced A23187-stimulated [3H]AA release in a dose-depen- UMR 7561 CNRS-UHP, Faculty of Medicine, Nancy, France dent manner that peaked at 5 ng/ml (300% above control). Using rat Arthritis Res Ther 2004, 6(Suppl 3):8 (DOI 10.1186/ar1342) AMs pretreated overnight with leptin, we observed that leptin Leptin is a small, 16-kDa protein produced and secreted primarily by enhanced both calcium-dependent and calcium-independent PLA 2 adipocytes found in white adipose tissue. Initially discovered as a activity. To determine the time required for leptin to enhance AA central regulator of appetite and energy expenditure, leptin may also be release, AMs were prelabeled overnight with [3H]AA, cultured with involved in the regulation of metabolic activity in the growth plate and leptin (5 ng/ml) on the following day, and cell culture medium was col- bone. Since obese individuals have an increased incidence of lected following stimulation with A23187. Leptin enhanced AA release osteoarthritis (OA), we evaluated the contribution of leptin to cartilage at 30 min, and 1, 2, 4, and 16 hours following A23187 stimulation. To changes associated with this degenerative disease. explore the mechanism by which short-term leptin pretreatment Leptin levels in synovial fluid samples obtained from OA patients under- enhances AA release, we assessed cPLA αphosphorylation in rat AM 2 going joint replacement were measured by ELISA assay. In addition, lysates following leptin pretreatment. Western blot analysis of immuno- histologic sections of tibial plateau cartilage and osteophytes obtained precipitated cPLA α revealed that leptin pretreatment of rat AMs 2 from OA patients were graded using the Mankin score, and were increased cPLA α phosphorylation (ser505) in a dose-dependent 2 immunostained using antibodies to leptin, transforming growth factor manner that peaked at 15 min (sevenfold increase with 10 ng/ml) beta 1 (TGF-β1), and insulin-like growth factor 1 (IGF-1). For experi- before returning to baseline levels at 60 min. To determine the mecha- mental studies, various doses of leptin (10, 30,100, and 300 µg) were nism by which long-term leptin pretreatment enhances macrophage AA injected into the knee joints of rats. Tibial plateaus were collected and release and LT synthesis, we assessed the expression of 5-lipoxyge- processed for proteoglycan synthesis by radiolabeled sulfate incorpo- nase, 5-lipoxygenase activating protein, and cPLA α in murine peri- 2 ration, and for expression of leptin and growth factors by RT-PCR and toneal macrophages and of cPLA γ in rat AMs using western blot 2 immunohistochemical analysis. analysis. Overnight pretreatment with leptin increased the expression of Our results indicated that leptin was observed in synovial fluid from the 5-lipoxygenase (200%) in murine peritoneal macrophages and of human OA-affected knee (n = 14 men, 8.16 ± 5.5 µg/l; and n = 6 cPLA γ(180%) in rat AMs. Our data suggest that the increased inci- 2 women, 12.95 ± 8.92 µg/l). These leptin concentrations were corre- dence of inflammatory diseases, such as asthma and osteoarthritis, lated with the body mass index (r= 0.572, P < 0.01). Interestingly, a associated with obesity may be related to the ability of leptin to marked expression of leptin was observed in OA cartilage, especially in increase AA release and eicosanoid synthesis. fibrillated cartilage with clusters and in osteophytes, while few chondrocytes produced leptin in normal cartilage. Furthermore, the pattern 10 and level of leptin expression were related to the grade of cartilage Transcriptional regulation of the mPGES-1 gene in destruction, and paralleled those of growth factors (IGF-1 and TGF- β1). When injected into rat knee joints, leptin stimulated chondrocytes primary cultured articular chondrocytes anabolic functions and induced the synthesis of IGF-1 and TGF-β1 in P Bausero, C Salvat, V Meynier de Salinelles, A Pigenet, cartilage at both mRNA and protein levels. M Raymondjean, F Berenbaum Taken together, these findings provide a new peripheral function to UMR 7079 CNRS, University Paris 6 Pierre & Marie Curie, Paris, leptin as a key regulator of chondrocyte metabolism, and indicate that France leptin may play an important role in the pathophysiology of OA. Arthritis Res Ther 2004, 6(Suppl 3):10 (DOI 10.1186/ar1344) Acknowledgement This study was supported by grants from the Contrat de Programme de Recherche Clinique, CHU Nancy, France. Healthycartilage is maintained in a state of dynamic equilibrium by matrixsynthesis and matrix degradation by the chondrocytes. Any dys- regulation with increased degradation and/or inadequate synthesis leads to hteloss of tissue structure and function, as in rheumatoid arthritis and osteoarthritis. The proinflammatory cytokine IL-1 plays a major role inthis phenomenon. IL-1 acts on chondrocytes in part by stimulating the release of prostaglandin E (PGE ) at the sites of 2 2 inflammation. Recently, a human membrane-associated prostaglandin S4 E synthase-1 (mPGES-1) was cloned. This enzyme catalyzes the con- 2 Available onlinehttp://arthritis-research.com/supplements/6/S3 version of prostaglandin H to PGE in a highly specific manner. We 12 2 2 previously demonstrated that mPGES-1 mRNA is induced by IL-1 in Regulation of neutrophil trafficking by the lipid chondrocytes in a dose-dependent and time-dependent manner. mediators of inflammation In order to study the transcriptional regulation of mPGES-1 in primary rabbit articular chondrocytes, we have cloned its promoter upstream of S Marleau1, J Lefebvre2, H El Iman1, C Bélanger1, P Borgeat2 the CAT ORF (vector pCAT3-basic; Promega, Charbonnières-les- 1Faculty of Pharmacy, Université de Montréal, Québec, Canada; Bains, France). We show by transient transfection experiments that the 2Centre de recherche en Rhumatologie et Immunologie, Centre de mPGES-1 promoter is stimulated by IL-1. A close examination of puta- recherche du CHUQ (CHUL), Sainte-Foy, Québec, Canada tive binding sites has revealed the presence of two CCAAT/Enhancer Arthritis Res Ther 2004, 6(Suppl 3):12 (DOI 10.1186/ar1346) Binding Protein (C/EBP) binding sequences (TTNNGNAAT) located between –548 to –558 base pairs and –610 to –619 base pairs. Co- Background The lipid mediators of inflammation include platelet-acti- transfections of expression vectors encoding the two different isoforms vating factor (PAF) and several classes of metabolites of arachidonic of C/EBP (βand δ) strongly stimulate the promoter activity. To further acid derived from the cyclooxygenase (prostaglandins and thrombox- study the role of C/EBP in mPGES-1 expression, we performed gel anes) and lipoxygenase pathways, such as leukotrienes (LTs) and lipox- shift experiments on wild-type and mutated oligonucleotides derived ins. Of these lipid mediators, LTs and PAF are potent agonists of from the mPGES-1 sequence. These experiments confirm the specific leukocytes and endothelial cells and show important proinflammatory binding of C/EBP on the mPGES-1 promoter. Taken together, our properties. Several elegant studies have recently provided strong results suggest that C/EBP factors indeed bind and regulate the support for a role of one of these mediators, LTB , in animal models of 4 mPGES promoter in articular chondrocytes. rheumatoid arthritis [1–3]. Interestingly, LTB , LTC /LTD and PAF are 4 4 4 concomitantly generated at the blood–endothelium interface when neu- 11 trophils bind to activated endothelial cells at inflammatory sites in vivo and are exposed to a chemoattractant. The hypothesis explored in this ALXR, a common GPCR gateway for project is that the three classes of lipid mediators collectively and syn- antiinflammatory and/or proinflammatory cell ergistically act to promote and facilitate neutrophil extravasation and activation: relevance of selective kinase and accumulation at inflammatory sites. The hypothesis also implicates that, phospholipase signaling by uteroglobin, lipoxin A given the high level of redundancy in the abilities of the three lipid medi- 4 and serum amyloid A in ALXR-mediated ators to activate neutrophils and endothelial cells, the blockade of one of these mediators can only have a modest and incomplete inhibitory chemotaxis effect on neutrophil extravasation. G Antico1, S Sodin-Semrl2, J Varga2, L Miele1, S Fiore2 Objective The objective of these studies is therefore to compare the 1Biopharmaceutical Sciences, University of Illinois, COM, Chicago, role of each individual mediator and the collective effect of the three Illinois, USA; 2Section of Rheumatology, University of Illinois, COM, mediators in regulating neutrophil trafficking. Another objective is to Chicago, Illinois, USA assess whether or not the combined use of antagonists to the three Arthritis Res Ther 2004, 6(Suppl 3):11 (DOI 10.1186/ar1345) classes of mediators could result in a stronger inhibitory effect on neutrophil trafficking. The lipoxin A receptor (ALXR) is expressed in fibroblast-like synovio- MethodsThe studies are performed using potent and selective LTB , 4 4 cytes and is targeted by both antiinflammatory and proinflammatory LTD and PAF antagonists in both in vitroand in vivomodels. The sep- 4 endogenous ligands. Recently, annexin-1 (lipocortin) has been identi- arate and collective role of the three classes of lipid mediators will be fied to be an ALXR ligand. Uteroglobin (Ug) is secretoglobin that investigated in the migration of human neutrophils through a monolayer shares with annexin-1 properties such as potent inhibition of phospholi- of human endothelial cells grown on a gel of extracellular matrix pro- pase A (PLA ) as well as sequence similarities in the ‘antiflammin’ teins. The studies also involve the use of models of dermal inflammation 2 2 consensus region. Potential receptor interactions of Ug with ALXR in rats and rabbits and the mouse air pouch model. were therefore investigated and PLA activity assays were performed in ResultsThe results show that, in the various models tested, neutrophil 2 CHO and HL-60 cell lines stably overexpressing ALXR. Serum amyloid migration elicited by proinflammatory agents such as IL-1, tumor necro- A (SAA), an ALXR ligand displaying proinflammatory activities, was sis factor alpha and lipopolysaccharide is generally decreased by LTB 4 compared with two antiinflammatory ligands, Ug and lipoxin A (LXA ). or PAF antagonists, and that the combination of both antagonists 4 4 While Ug and LXA did not alter baseline PLA activity (Ug) or pro- results in a greater inhibitory effect, supporting the concept that several 4 2 voked a pulsed peak of activity (LXA ), SAA did induce a delayed and lipid mediators generated locally (at the blood–endothelium interface 4 prolonged activation of PLA . SAA-induced PLA activation was and/or at inflammatory sites) regulate neutrophil trafficking. Additional 2 2 sharply inhibited in the presence of Ug. Ug inhibition of SAA-depen- experiments involving antagonists to the three classes of lipid media- dent phospholipase activity occurred at submillimolar Ca2+concentra- tors, as well as LTB and PAF receptor knockout mice, are in progress. 4 tions, suggesting an effect on cytosolic PLA (rather than secretory References 2 PLA , a known target inhibited by Ug and annexin-1 but requiring 1. Griffiths RJ, Pettipher ER, Koch K, Farrell CA, Breslow R, Conklyn 2 millimolar Ca2+). Studies of Erk, p38 and AKT kinase phosphorylation MJ, Smith MA, Hackman BC, Wimberly DJ, Milici AJ, et al.: confirmed that SAA and Ug interact with ALXR to oppositely regulate Leukotriene B plays a critical role in the progression of colla- 4 GPCR-coupled downstream signaling events. Overall the analysis of gen-induced arthritis. Proc Natl Acad Sci USA 1995, 92:517- phospholipase D activity, kinase phosphorylation and cAMP levels also 521. suggested that proinflammatory and antiinflammatory ligands of ALXR 2. Griffiths RJ, Smith MA, Roach ML, Stock JL, Stam EJ, Milici AJ, engage differently coupled pathways, leading to activation of these Scampoli DN, Eskra JD, Byrum RS, Koller BH, Mcneish JD: Colla- processes (SAA) or to their inhibition (LXA and Ug). These signaling gen-induced arthritis is reduced in 5-lipoxygenase-activating 4 events are functionally matched by the ability of SAA to stimulate NF- protein-deficient mice.J Exp Med1997, 185:1123-1129. κB activity, IL-8 release and cell chemotaxis, and that of LXA and Ug 3. Tsuji F, Oki K, Fujisawa K, Okahara A, Horiuchi M, Mita S: 4 to strongly inhibit them. Expression of Ug and SAA mRNAs was also Involvement of leukotriene B in arthritis models. Life Sci 4 detected in human fibroblast-like synoviocytes, suggesting that ALXR 1999, 64:PL51-PL56. may play a pivotal role in the pathophysiology of arthritis. In addition, annexin-1 and Ug inhibition of PLA offers enticing new venues to 2 control inflammatory arthritides by limiting, for example, cascade signaling amplification via synthesis of proinflammatory eicosanoids, along with a redirection of ALXR signaling toward antiinflammatory feedback mechanisms. Acknowledgements This work was supported by NIH-NIAMS (AR- 45931 to SF) and the Arthritis Foundation (National Postdoctoral fel- lowship to SSS). S5 Arthritis Research & Therapy Vol 6 Suppl 3 Abstracts of the 4th World Congress of the Global Arthritis Research Network Session III — Autoimmunity and inflammation: 15 from toll to reg Post-transcriptional regulation of tumor necrosis factor alpha expression 13 S Brooks, W Rigby The nonsense allele obliviousreveals a sensor of Department of Medicine, Section of Rheumatology, Dartmouth Medical di-acylglycerides acting in conjunction with TLR2 School, Lebanon, New Hampshire, USA and TLR6 Arthritis Res Ther 2004, 6(Suppl 3):15 (DOI 10.1186/ar1349) K Hoebe1, K Tabeta1, P Georgel1, X Du1, S Mudd1, S Sovath1, The success of tumor necrosis factor (TNF) antagonists in the therapy L Shamel1, T Hartung2, Ul Zähringer3, B Beutler1 of inflammatory arthritides has established the central role of this 1The Scripps Research Institute, La Jolla, California, USA; cytokine in the pathogenesis of these disorders. TNF is notable in that 2Department of Biochemical Pharmacology, University of Konstanz, it is predominantly controlled at the post-transcriptional level in Konstanz, Germany; 3Research Center Borstel, Leipniz-center for macrophages. TNF biosynthesis is highly regulated by the AU-rich Medicine and Bioscience, Borstel, Germany element (AURE) found in its 3′ untranslated region. This AURE regu- Arthritis Res Ther 2004, 6(Suppl 3):13 (DOI 10.1186/ar1347) lates both the stability and rate of translation of TNF mRNA. In the The mammalian Toll-like receptors (TLRs) activate cells of the innate macrophage, the ERK, JNK, and p38 SAPK signaling pathways con- immune system when stimulated by diverse ligands of microbial origin. verge on the AURE in regulating the nuclear export, stability and trans- In some instances, these ligands are directly engaged by the TLRs; lation of TNF mRNA. Second, the C3H zinc finger protein, however, this is not necessarily true in all cases. TLR2 recognizes mul- tristetraprolin (TTP), appears to play a major role in the post-transcrip- tiple, structurally disparate microbial ligands, consistent with a require- tional regulation of TNF by binding the AURE. Recent studies have ment for co-receptors in ligand binding. Using N-ethyl-N-nitrosourea, identified that TTP is a target of the p38 SAPK/MAPKAP K2 kinase we generated the recessive immunodeficiency phenotype oblivious, in (MK2) pathway. Phosphorylation of TTP by MK2 has been reported in which macrophages show diminished awareness of the S-enantiomer vitroand in vivo; data suggest that MK2 activation inactivates the func- of the di-acylated bacterial lipopeptide MALP-2 and lipoteichoic acid, tion of TTP as a destabilizing protein. together with spontaneous ocular colonization by Gram-positive organ- Intriguingly, TTP binding to the AURE does not appear to be regulated isms and hypersusceptibility to Staphylococcus aureus infection. by this phosphorylation. Rather, the function of TTP seems to be modu- Oblivious macrophages readily detect the tri-acylated bacterial lated through interactions with specific proteins that alter its subcellular lipopeptide PAM CSK as well as zymosan, revealing that some TLR2 localization. This creates a model where TTP provides specificity in 3 4 ligands are activated via an Oblivious-independent pathway. The gene binding cytokine-type AURE but the consequences of this interaction responsible for the oblivious phenotype has been positionally cloned. are determined by protein–protein interactions. Second, we have iden- In its ability to carry the lipoteichoic acid and MALP-2 signal to the tified that TTP does not bind all AURE, but rather exhibits specificity for transmembrane signaling receptors TLR2 and TLR6, Obliviousserves nUAUUUAUn sequences. Third, we have identified that TTP regulates a function analogous to CD14, which concentrates the lipopolysaccha- its own mRNA stability. Fourth, we have demonstrated that TTP local- rdide signal for transduction by TLR4. Besides microbial molecules, izes to the polysomes in the context of macrophage activation by obliviousalso serves as a receptor for endogenous molecules and may lipopolysaccharide. Fifth, we have demonstrated that TTP is expressed mediate (some) of the inflammatory events involved in the development in many different hematopoietic cells and appears to function as an of atherosclerosis. AURE binding protein. Thus, although the role of TTP in TNF biology is best understood in the macrophage, it appears that some, if not all, of 14 these concepts may be relevant to other cells involved in the immune and inflammatory response. An apoptotic signaling pathway activated by Nod1 Acknowledgements Supported by funding from the Veteran’s Admin- J da Silva Correia, Y Miranda, N Austin-Brown, J Mathison, J istration and National Institutes of Health Han, RJ Ulevitch Department of Immunology, The Scripps Research Institute, La Jolla, 16 California, USA A proinflammatory chemokine, CCL3, sensitizes Arthritis Res Ther 2004, 6(Suppl 3):14 (DOI 10.1186/ar1348) the heat-gated and capsaicin-gated ion channel, Nod1 and Nod2 are two cytosolic proteins thought to play a role in TRPV1 innate immunity. Both detect the presence of microbes through recog- N Zhang1, S Inan2, A Cowan2, AD Guler3, R Sun1, J Wang1, nition of peptidoglycan fragments but also may initiate apoptosis. The TJ Rogers2, M Caterina3, JJ Oppenheim1 nod2gene has been strongly associated with several autoimmune dis- 1Laboratory of Molecular Immunoregulation, Intramural Research eases and particularly Crohn’s disease; in contrast, nod1 polymor- Support Program, Frederick, Maryland, USA; 2Department of phisms have not been linked to any genetic disorders. Here we provide Pharmacology, Fels Institute for Cancer Research and Molecular multiple lines of evidence showing that Nod1 participates in apoptosis. Biology, Center for Substance Abuse Research, Temple University Nod1-deficient breast cancer cells (MCF-7) were more resistant to School of Medicine, Philadelphia, Pennsylvania, USA; 3Department of tumor necrosis factor-induced cytotoxicity, and this was accompanied by a reduction in caspase signaling. Further, γTriDAP, a naturally occur- Biological Chemistry and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA ring tripeptide product from peptidoglycan and a ligand for Nod1, induced cell death consistent with apoptosis in wild-type MCF-7 cells Arthritis Res Ther 2004, 6(Suppl 3):16 (DOI 10.1186/ar1350) but not in Nod1-deficient cells. γTriDAP triggered processing of PARP Pain, a critical component of host defense, is one of the hallmarks of and many caspases, including caspase 6, caspase 7, caspase 8 and the inflammatory response. We therefore hypothesized that pain might caspase 9. Only caspase 9 inhibitor totally abrogated γTriDAP cytotoxi- be exacerbated by proinflammatory chemokines. To test this hypothe- city. RIP2/RICK, a downstream protein kinase of Nod1, appears to be sis, CCR1 was co-transfected into HEK293 cells together with the an essential component of the Nod1 proapoptotic pathway since Vanilloid Receptor 1 (TRPV1), a cation channel required for certain expression of a dominant negative form of RIP2 abolished γTriDAP- types of thermal hyperalgesia. Capsaicin induced calcium influx by induced cell death. This is a newly defined activity for Nod1 and sug- TRPV1. When CCR1:TRPV1/HEK293 cells were pretreated with gests that Nod1-induced apoptosis may be responsible for cell injury in CCL3, the sensitivity of TRPV1-mediated calcium flux was increased a variety of disease states. about fivefold. Pertusis toxin inhibited CCL3-elicited sensitization of TRPV1, indicating the involvement of G-protein signaling. RT-PCR analysis data showed that a spectrum of chemokine and cytokine receptors are expressed in rat dorsal root ganglia (DRG). Immunohisto- S6 chemical staining of the DRG showed that CCR1 coexpressed with Available onlinehttp://arthritis-research.com/supplements/6/S3 TRPV1 on over 85% of small diameter neurons. CCR1 on neuronal reacted against HC Gp39 with the production of IL-10, but not inter- cells was functional, as demonstrated by CCL3-induced calcium flux feron gamma. Ex vivo assays indicated that the natural occurring HC and protein kinase Cactivation. Pretreatment with CCL3 enhanced the Gp39-directed immune response in bulk is capable of suppressing response of DRG neurons to capsaicin, and this sensitization was cytotoxic T-cell and recall responses, indicating that, rather than being inhibited by pertussis toxin, U73122, or staurosporine. Futhermore, unresponsive, the HC Gp39-directed immune response in healthy indi- injection of CCL3 into mice spine cords enhances the sensitivity of the viduals is biased towards a regulatory phenotype. Moreover, CD4+T- mice tails toward the hot water, indicative of chemokine-induced sensi- cell lines directed against HC Gp39 expressed CD25, GITR, CTLA-4 tization effects in vivo. The fact that a proinflammatory chemokine, by and Foxp3 molecules and were capable of suppressing other immune interacting with its receptor on small-diameter neurons, sensitizes responses. Cell–cell contact was required for this suppression. In con- TRPV1 reveals a novel mechanism of receptor cross-sensitization that trast, the quality of the HC Gp39-directed immune response in patients may contribute to hyperalgesia during inflammation. with RA exhibits polarization toward a proinflammatory Th1 phenotype. Together these findings indicate that the presence of HC Gp39-spe- 17 cific immune responses in healthy individuals may have a profound inhibitory effect on inflammatory responses in areas were HC Gp39 is A genetic analysis of lupus present, and imply that the balance of autoreactive proinflammatory and AN Theofilopoulos antiinflammatory HC Gp39-directed immune responsiveness is dis- Department of Immunology, The Scripps Research Institute, La Jolla, rupted in RA patients. California, USA Arthritis Res Ther 2004, 6(Suppl 3):17 (DOI 10.1186/ar1351) Plenary lecture – The use of tissue engineering for joint structure repair: a future? Systemic lupus erythematosus is a complex multigenic inherited disease with susceptibility determined by a combination of genetic, Current tissue engineering approaches to cartilage environmental and stochastic factors. Although not yet defined, recent repair technical advances have provided the means to dissect the component genetic contributions of polygenic traits. We have applied such RS Tuan approaches to mouse models of spontaneous systemic lupus erythe- Cartilage Biology and Orthopaedics Branch, National Institute of matosus, and in this presentation I will summarize our genome-wide Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of mapping studies that identified loci predisposing to several major Health, Bethesda, Maryland, USA lupus-related traits. Through the generation and study of interval con- Arthritis Res Ther 2004, 6(Suppl 3):104 (DOI 10.1186/ar1353) genic lines, precise mapping, and screening of candidate genes, identification of the specific genes and mechanisms associated with some of Chondral defects, generated as a result of trauma and injury or degen- the major loci is currently being pursued. erative joint diseases, such as osteoarthritis, represent some of the Acknowledgement The work of the authors reported herein was sup- most challenging orthopedic conditions in terms of natural tissue ported by National Institutes of Health grants AR39555 and AR30103. healing and repair. Damages to the articular cartilage fail to elicit significant reparative activity, due to the acellularity of the tissue. Clonal pro- 18 liferation of articular chondrocytes is often seen, and results in the production of mechanically inferior fibrocartilage. Extensive degenera- Cellular-based immune modulation in arthritis tion of the articular surface eventually necessitates total joint arthro- REM Toes1, J van Bilsen1, H van Dongen1, L Lard1, plasty. There is, therefore, a timely need for the development of EIH van der Voort1, D Elferink2, A Bakker1, A Miltenburg3, biological approaches to cartilage repair. The emerging research disci- FC Breedveld1, TH Huizinga1, RRP de Vries2 pline of tissue engineering aims to develop functional tissue substitutes 1Department of Rheumatology, Leiden University Medical Center, The by combining experimental approaches in biology and engineering, and Netherlands; 2Department of Immunohematology and represents a particularly attractive technology for the treatment of Bloodtransfusion, Leiden University Medical Center, The Netherlands; skeletal diseases, most of which involve tissue degeneration or failure 3Organon, Oss, The Netherlands to heal. Functionally, it is instructional to approach cartilage regenera- tion in the context of developmental chondrogenesis; that is, the forma- Arthritis Res Ther 2004, 6(Suppl 3):18 (DOI 10.1186/ar1352) tion of cartilage from a progenitor mesenchymal cell population during While the symptoms associated with rheumatoid arthritis (RA) are well embryonic development. In particular, adult tissue-derived mesenchy- described, the factors that are crucially involved in induction and/or mal stem cells (MSCs), which display multilineage differentiation poten- progression of RA are poorly defined. Nonetheless, it is generally tial, are currently considered a highly promising source of progenitor accepted that cells belonging to the adaptive immune system (i.e. B cells for tissue engineering. Chondrogenesis in the developing verte- cells and T cells) are intimately engaged in the processes responsible brate limb consists of a highly coordinated and orchestrated series of for RA induction and/or progression. As these cells require triggering events involving the commitment and differentiation of mesenchymal of their antigen receptors before they can exert their functions, it is cells to mature chondrocytes. This process is regulated by the sequen- likely that antigen recognition is important in the pathological tial and coordinated expression of genes that encode for specific cell processes driving RA. Unfortunately, no antigens casually related to the adhesion molecules, growth factors, and extracellular matrix molecules induction/perpetuation of RA are known. Nonetheless, studies that carry out morphogenetic and signaling activities. Recently, addressing the question of how immunity against autoantigens is regu- members of the Wnt family of signaling molecules have been function- lated, combined with studies aiming at defining the molecular identity ally implicated in limb development. The Wnts comprise a large family of antigens involved in disease, will be important to gain a better under- of cysteine-rich glycoproteins that perform a number of inductive and standing of the pathways involved in the induction/progression of RA. regulatory functions in both normal development and oncogenic trans- Recently, we have found that CD4+CD25+ regulatory T cells play a formations. The expression of various Wnts has been identified in the pivotal role in the control of arthritis in mice. More importantly, treat- developing limb, and our recent studies showed that Wnts play an ment of established arthritis by adoptive transfer of these cells stopped important role in mesenchymal chondrogenesis. Interestingly, our further progression of disease. These data indicate the feasibility to recent work with human MSCs indicates the functional involvement of inhibit ongoing disease by specific manipulation of the immune system. Wnts and the cell adhesion molecular, N-cadherin, as well as members The data described also indicate that the class of immune response of the transforming growth factor beta superfamily in MSC chondroge- against autoantigens could profoundly influence the susceptibility for, nesis, suggesting that regenerative chondrogenesis and developmental and/or outcome of, autoimmune responses. However, until now, there chondrogenesis are likely to share common regulatory pathways. By has been only limited evidence for autoantigen-specific regulatory combining MSCs, biodegradable polymeric scaffolds, and the applica- responses in humans. Here we analyzed the natural immune response tion of growth and differentiation factors, we are currently developing against Human Cartilage gp39 (HC-Gp39), a candidate autoantigen in cartilage constructs in vitrothat are potentially applicable for cartilage RA. Peripheral blood mononuclear cells from healthy individuals repair in vivo. Specifically, we recently fabricated a nanofibrous scaf- S7 Arthritis Research & Therapy Vol 6 Suppl 3 Abstracts of the 4th World Congress of the Global Arthritis Research Network fold using a synthetic biodegradable polymer, poly(ε-caprolactone), 20 and demonstrated its ability to support in vitro chondrogenesis of Galectin-3 in osteoarthritis: from a protective to a MSCs. The electrospun porous scaffold consisted of uniform, randomly destructive role oriented nanofibers (700 nm diameter); MSCs seeded into the scaffold and cultured in the presence of transforming growth factor beta 1 dif- C Boileau1, J Martel-Pelletier1, M Guévremont1, J-P Pelletier1, ferentiated to chondrocytes, indicated by gene expression and histo- F Poirier2, P Reboul1 logical profiles, at a level similar to that observed in high-density pellet 1Osteoarthritis Research Unit, Centre hospitalier de l’Université de cultures. The physical nature and improved mechanical properties of Montréal, Hôpital Notre-Dame, Montréal, Québec, Canada; such scaffolds, particularly in comparison with cell pellets, suggest that 2Laboratoire de Génétique et Dévelopepment des Mammifères, Institut these constructs may serve as a practical carrier for MSC transplanta- Jacques Monod, Paris, France tion in cell-based tissue engineering approaches to cartilage repair, Arthritis Res Ther 2004, 6(Suppl 3):20 (DOI 10.1186/ar1355) supported by our preliminary animal model study. The future success of cartilage tissue engineering is dependent on a number of requirements, BackgroundOsteoarthritic (OA) chondrocytes are able to re-express including expansion of progenitor cells, optimization of biomaterial scaf- numerous genes normally activated in the growth plate, and more par- fold design, and molecular enhancement of cell differentiation and ticularly in the hypertrophic zone. Among several genes, we are inter- growth using biologics and gene therapeutic means. We believe that ested in studying galectin-3 (gal-3) since we have recently the understanding of developmental chondrogenesis serves as a ratio- demonstrated that its expression was increased in OA cartilage [1]. nal and powerful paradigm for cartilage tissue engineering. gal-3 is a mammalian lectin, which interacts with β-galactoside residues and is involved in numerous functions such as adhesion, splic- Session IV — Novel therapeutic strategies: ing activity, cell cycle regulation, as well as a receptor for advanced gly- cation end products (AGE receptor). These functions are related to the don’t miss the target gal-3 cellular localization. Indeed, this protein may be found in the plasma membrane, in cytoplasm and in the nucleus. 19 Objective In the present study, we investigated the role(s) of gal-3 Regulating innate immune response cytokines in using both the mono-iodoacetate-induced OA model and in vitroexper- synovitis iments. Methods OA was induced by a single injection of iodoacetate (5 IB McInnes, D Xu, JA Gracie, X Wei, FY Liew mg/ml, 2 µl) into each knee joint of 4-month-old mice (WT) or gal-3 null Division of Immunology, Infection and Inflammation, University of mice (KO). Mice were sacrified 7, 14 and 21 days after the single injec- Glasgow, UK tion. Histologic evaluation was performed on sagittal sections of mouse Arthritis Res Ther 2004, 6(Suppl 3):19 (DOI 10.1186/ar1354) knee joint. The severity of the OA lesions was graded on a scale of 0–14 in a blinded fashion, by two independent observers, using the Several studies have provided powerful proof of concept for the notion histologic/histochemical scale of Mankin. Intracellular and extracellular that single cytokine targeting can manifest effective immune suppres- roles of gal-3 were investigated in both human chondrocytes and in sion even in the context of complex networks of cytokine activities. We chondrogenic ATDC5 cells, a mouse cell line derived from the 129 have studied the biology and functional expression of the innate strain. response cytokine IL-15 and, more recently, members of the IL-1/IL-1 Results Intra-articular injection of mono-iodoacetate, which induced receptor superfamily in inflammatory synovitis. These cytokines are osteoarthritis, upregulated the expression of gal-3 in WT mice 7 days characterised by early upregulation during inflammatory responses and post injection, reaching a statistical significance 14 days post injection characteristic functional synergy and cross-regulation in the intact (P < 0.05). The histologic grading score indicated that KO mice immune response. IL-15 is an IL-2-like cytokine of 15 kDa that pos- (control group) had a poorer quality of cartilage compared with WT sesses broad proinflammatory activities via its heterotrimeric receptor mice (control group). Moreover, the induction of OA in KO mice (IL-15Rα/IL-15/2Rβ/common γ), including promoting T-cell maturation, showed a marked decreased of bone area, noticeable 7 days post activation, and cytokine production, natural killer cell maturation and injection (P < 0.05). effector function, neutrophil activation, dendritic cell activation, According to the results obtained, it seemed that gal-3 was important endothelial cell rescue from apoptosis, macrophage activation and B- for the cartilage homeostasis. Colnot and colleagues have suggested cell activation and isotype switching. IL-15 is expressed at mRNA and that gal-3 could be implicated in chondrocyte survival [2]. Therefore, protein levels in several inflammatory arthritides including rheumatoid we treated OA chondrocytes with sodium nitroprusside (SNP), which arthritis. When targeted in rodent models of inflammation using either is known to generate chondrocyte cell death. Our results showed that soluble IL-15Rαor modified IL-15 mutant fusion proteins, amelioration gal-3 was much further decreased than was Bcl2 in experiments per- of onset and of existing arthritis is observed. A recent phase I study in formed under the same conditions [3]. Moreover, SNP decreased the which a fully human IgG monoclonal antibody (HuMax-IL15; Genmab, gal-3 phosphorylation, which is a key process in the capacity of gal-3 1 Copenhagen, Denmark) was administered to 30 patients with active to prevent cell death. Finally, ATDC5 cells transfected with a gal-3- rheumatoid arthritis indicated that IL-15 blockade was well tolerated up expressing vector were more resistant to SNP-induced cell death com- to 8 weeks, with early indications of efficacy detected. We have now pared with those transfected with the empty vector. On the other hand, observed close interactions between IL-15 and IL-1 receptor superfam- Ohshima and colleagues found gal-3 in synovial fluid, particularly ily signalling in promoting T-cell activation. Specifically, IL-15 syner- during inflammation [4]. Therefore, we investigated the potential role of gises with the TLR2 ligand BLP to promote memory T-cell activation exogenous gal-3 in chondrocyte cultures. Surprisingly, we found that (co-stimulatory) and also via a cell membrane-dependent pathway to exogenous gal-3 induced chondrocyte death. promote adjacent macrophage activation and tumour necrosis factor One of the most fascinating phenomena is the regulation of gal-3 release. We have also observed that a further member of the IL-1 secretion. Indeed, several cells produced gal-3 but not all are able to receptor superfamily, namely ST2, is expressed in synovial fibroblasts. secrete a great amount of it, chondrocytes belonging to the latter cate- Membrane-bound ST2 negatively regulates type I IL-1 receptor and gory. Conversely, gal-3 is secreted in a much greater quantity by inflam- TLR4 signalling by sequestrating the adaptors MyD88 and Mal. Intrigu- matory cells that could affect — at least locally (i.e. at the pannus level) ingly, soluble ST2 suppresses development of collagen-induced arthri- — chondrocyte survival. tis in DBA/1 mice if given prophylactically, and also established References collagen-induced arthritis if administered after disease onset. Together 1. Guévremont M, Martel-Pelletier J, Boileau C, Liu FT, Richard M, these data suggest that complex interactions between the common γ- Fernandes JC, Pelletier JP, Reboul P: Human adult chondro- receptor chain and IL-1 receptor superfamily signalling cytokines are of cytes express galectin-3 to their surface: a potential substrate importance in developing synovial inflammatory responses and that elu- for collagenase-3. Annals Rheum Dis2004, 63:636-643. cidation of these pathways offers therapeutic utility. 2. Colnot C, Sidhu SS, Balmain N, Poirier F: Uncoupling of chondrocyte death and vascular invasion in mouse galectin 3 null S8 mutant bones.Dev Biol2001, 229:203-214. Available onlinehttp://arthritis-research.com/supplements/6/S3 3. Notoya K, Jovanovic DV, Reboul P, Martel-Pelletier J, Mineau F, 22 Pelletier JP: The induction of cell death in human osteoarthritis Mapping out the mitogen activated protein kinase chondrocytes by nitric oxide is related to the production of pathway prostaglandin E2 via the induction of cyclooxygenase-2. J Immunol2000, 165:3402-3410. GS Firestein 4. Ohshima S, Kuchen S, Seemayer CA, Kyburz D, Hirt A, Klinzing S, UCSD School of Medicine, La Jolla, California, USA Michel BA, Gay RE, Liu FT, Gay S, Neidhart M: Galectin3 and its Arthritis Res Ther 2004, 6(Suppl 3):22 (DOI 10.1186/ar1357) binding protein in rheumatoid arthritis. Arthritis Rheum 2003, 48:2788-2795. The mitogen activated protein (MAP) kinases represent another attrac- Acknowledgements This work was supported by the Canadian Insti- tive target for rheumatoid arthritis (RA) because they can regulate cell tutes of Health Research (CIHR) and the Canadian Arthritis Society. proliferation, apoptosis, cytokine expression, and metalloproteinase CB is recipient of a PostDoctoral award from the CIHR/R&D. PR is a production. They represent a complex, inter-related signal transduction recipient of a New Investigator Award from the Canadian Arthritis mechanism that integrates extracellular stresses and induces an appro- Society. priate cellular response. The three major MAP kinase families, c-Jun-N- terminal kinase (JNK), extracellular regulating kinase (ERK) and p38 21 kinase, differ in their substrate specificity and subsequent responses to stress depending on the cell type and the environmental influences. Angiogenesis and arthritis: a potential therapeutic The MAP kinases regulate various genes via both transcriptional and target? post-transcriptional mechanisms. The upstream MAP kinase kinases J Grosjean, B Sivakumar, E Paleolog (MAPKK) serve as regulators of MAP kinase activity by phosphorylating Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial specific threonine and tyrosine residues. MAPKKs are, in turn, regu- College, London, UK lated, by MAPKK kinases (MAPKKK or MAP3K). The p38 MAP kinase is of particular interest and several inhibitors have Arthritis Res Ther 2004, 6(Suppl 3):21(DOI 10.1186/ar1356) progressed into clinical trials. There are at least four isoforms of p38 Musculoskeletal disorders such as rheumatoid arthritis (RA) are a (α, β, γand δ), although the αform is probably the most important in common cause of pain and disability. Over the past decade, advances macrophages for cytokine regulation. Many of the cytokine regulatory in understanding RA pathogenesis, based on studies of human cells effects appear to be mediated through the downstream p38 substrate and animal models of disease, have led to the identification of new MAPKAP-2. Some evidence suggests that tumor necrosis factor pro- targets for therapeutic intervention. Nevertheless, despite the clinical duction, in particular, is regulated by this kinase. Preclinical models success of anticytokine biologicals, further initiatives in understanding demonstrate that p38 inhibitors are effective in a number of animal RA pathogenesis to aid further drug discovery are highly desirable. models of arthritis, including murine collagen-induced arthritis. Of note, An early event in RA is an alteration in synovial blood vessel density. p38 inhibitors also suppress joint destruction in these models, perhaps The hyperplasia of the synovium necessitates a compensatory increase due to a combination of indirect effects on cytokine expression and a in the number of blood vessels to nourish and oxygenate the tissue. A direct effect on metalloproteinase production. key response to increased requirement for nutrients and/or reduced In vitrostudies previously identified the MAPKKs MKK3 and MKK6 as oxygen is to form new blood vessels (angiogenesis). In RA, the synovial the primary regulators of p38 phosphorylation and activation. To inves- blood vessel number has been found to correlate with hyperplasia, tigate a potential role for MKK3 and MKK6 in RA, we evaluated their mononuclear cell infiltration and indices of joint tenderness. Endothelial expression and regulation in RA synovium and cultured fibroblast-like cells lining blood vessels within RA synovium have been shown to synoviocytes (FLS). Immunohistochemistry demonstrated that MKK3 express cell-cycle antigens. A variety of angiogenic mediators, includ- and MKK6 are expressed in RA and osteoarthritis (OA) synovium. ing cytokines and growth factors, have been identified in rheumatoid Digital image analysis showed no significant differences between OA joints. We and other workers have shown that expression of the angio- and RA with regard to expression or distribution. However, phosphory- genic factor vascular endothelial growth factor (VEGF) is increased in lated MKK3/MKK6 expression was significantly higher in RA synovium RA. Importantly, circulating levels of VEGF are elevated in patients with and was localized to the sublining mononuclear cells and the intimal established RA and are reduced by anti-tumour necrosis factor alpha lining. Western blot analysis of synovial tissue lysates confirm the antibody treatment, suggesting that part of the beneficial effect of anti- increased expression of phosphorylated MKK3/MKK6 in RA. Western tumour necrosis factor alpha in RA may be reduced angiogenesis. blot analysis demonstrated constitutive expression of MKK3 and MKK6 However, new blood vessel formation may not keep pace with synovial in RA and OA FLS. Phospho-MKK3 levels were low in medium-treated proliferation leading to regions of hypoperfusion. Intra-articular oxygen FLS, but were rapidly increased by IL-1 and tumor necrosis factor tension is significantly lower in RA patients when compared with con- alpha, although phospho-MKK6 levels only modestly increased. p38 trols, and hypoxia-inducible transcription factors such as HIF-1α and co-immunoprecipitated with MKK3 and MKK6 from cytokine-stimulated HIF-2αare significantly upregulated. Of relevance in the context of RA, FLS, and the complex phosphorylated ATF2 in an in vitrokinase assay. VEGF is significantly upregulated by hypoxia, which would promote Studies using dominant negative constructs in cultured synoviocytes further blood vessel formation. As well as being a key angiogenic suggest that both are required for full activation of p38. These data are factor, VEGF also promotes vascular endothelial cell survival, by the first documentation of MKK3 and MKK6 activation in human inflam- increasing expression of anti-apoptotic proteins such as Bcl-2, and matory disease. By forming a complex with p38 in synovial tissue and members of the inhibitor of apoptosis family (namely cIAP-1, XIAP and FLS, these kinases can potentially be targeted to regulate the produc- survivin), which directly bind to and inhibit caspases. Our data suggest tion of proinflammatory cytokine production in inflamed synovium. that induction of these apoptosis inhibitors is mediated via the NF-κB pathway. We are currently further investigating pathways activated by VEGF, as well as dissecting out the response of synovial cells to hypoxia, both in terms of induction of angiogenic molecules such as VEGF and upregulation of inflammatory cytokines such as tumour necrosis factor alpha. There have been a number of studies showing that angiogenesis blockade is effective in animal models of arthritis. Since VEGF is elevated early during the course of RA, and since RA is most active in the initial stages of disease, early treatment might reduce subsequent joint erosion and damage. RA may be a potential target for anti-angiogenic therapy, and targeting VEGF may prove to be especially beneficial. Acknowledgements The Kennedy Institute of Rheumatology receives a core grant from arc (Registered Charity No. 207711). BS is supported by the Restoration of Appearance and Function Trust. S9 Arthritis Research & Therapy Vol 6 Suppl 3 Abstracts of the 4th World Congress of the Global Arthritis Research Network 23 Session V — Development of DMOADS: a long The ααvββ3 integrin as a therapeutic target for and risky business inhibition of bone resorption 24 SJ Dixon1, J Bashutski1, M-L Rütz1, HA Goldberg1, SM Sims1, Issues in structure-modifying osteoarthritis drug A Gramoun2, S Shorey2, M Manolson2, JN Heersche2, H Zhang3, development: new insights regarding radiographic J Suzich3, S-Y Mao3 clinical trial methods 1CIHR Group in Skeletal Development and Remodeling, University of Western Ontario, London, Canada; 2Dental Research Institute, Faculty JF Beary, GA Cline of Dentistry, University of Toronto, Canada; 3MedImmune Inc., Procter & Gamble Pharmaceuticals. Mason, Ohio, USA Gaithersburg, Maryland, USA Arthritis Res Ther 2004, 6(Suppl 3):24 (DOI 10.1186/ar1359) Arthritis Res Ther 2004, 6(Suppl 3):23 (DOI 10.1186/ar1358) Background Over the past several decades, the osteoarthritis (OA) An early feature of inflammatory arthritis is an increase in the number, field has relied on radiographs in clinical trials, in order to assess longi- size and activity of osteoclasts leading to the destruction of periarticu- tudinal structural changes in weight-bearing joints such as the knee. lar and subchondral bone and mineralized cartilage [1]. Osteoclastic Because alternative imaging modalities such as magnetic resonance resorption is regulated by a number of processes, including the prolifer- imaging are beginning to mature, it is timely to compare and contrast ation, differentiation and recruitment of osteoclast precursors, the the utility of these imaging methods for the purpose of conducting migration and activation of mature osteoclasts, and programmed cell future structure-modifying OA drug clinical trials death. Regulatory mechanisms involve soluble mediators, cell–cell and Objective Insights from a large, multicenter knee OA study will be cell–matrix interactions. The latter involve integrins — heterodimeric shared in the context of considering the next generation of longitudinal receptors that link the cytoskeleton to the extracellular matrix and imaging methods to study OA. mediate outside-in and inside-out signaling. Mature osteoclasts Methods Patients in the study all had radiographic and symptomatic express three integrins (αvβ3, αvβ1 and α2β1), with the αvβ3 vit- knee OA. A total of 2400 patients were randomized to the study, with ronectin receptor predominating. an 85% study completion rate. The radiographic data for the study We used a single cell assay to demonstrate that macrophage-colony were collected at baseline, month 12 and month 24, using a highly stimulating factor (M-CSF) and transforming growth factor beta are standardized radiographic method directed at the medial compartment chemotaxins for mammalian osteoclasts. Interestingly, the RGD- of the signal knee. Fluoroscopic confirmation of proper knee position containing disintegrin echistatin, which binds αvβ3 integrin, inhibited was achieved at each study visit. M-CSF-induced chemotaxis at concentrations as low as 0.1 nM [2]; ResultsA 2-year, 2400-patient study of knee OA has been completed whereas it did not inhibit migration induced by transforming growth and is being analyzed. Insights will be shared at the GARN Conference. factor beta [3]. We have recently examined the effects of Vitaxin®, a humanized mono- Session VI — The use of imaging and clonal antibody that blocks human and rabbit αvβ3 integrins [4]. Vitaxin®caused a concentration-dependent decrease in the number of biomarkers in the assessment and follow-up rabbit osteoclasts attached to plastic. Moreover, Vitaxin® inhibited of arthritis resorption by rabbit osteoclasts cultured on slices of bovine bone (Vitaxin® at 0.7 nM reduced resorption by ~50%). Vitaxin® also sup- 25 pressed bone resorption by in vitro-derived human osteoclasts, with a Overview: imaging, biomarkers and arthritis maximum of 63% inhibition observed at concentrations greater than 2 nM. Vitaxin® does not react with rodent αvβ3 integrin; therefore, we C Peterfy used a blocking anti-rat αvβ3 integrin to assess effects on rat osteo- Synarc, Inc., San Francisco, California, USA clast motility. This antibody induced rapid retraction of M-CSF-treated Arthritis Res Ther 2004, 6(Suppl 3):25 (DOI 10.1186/ar1360) rat osteoclasts but, unlike echistatin, did not inhibit chemotaxis induced by M-CSF. The recent introduction of effective structure-modifying therapies for Evidence from our laboratories and others, using disintegrins, antibod- rheumatoid arthritis (RA) has changed the way that rheumatologists ies, RGD-containing peptides and small molecules that bind the αvβ3 manage patients with RA, and this has created new demands on imaging integrin, point to its potential as a target for the development of antire- and biochemical markers both in clinical practice and in clinical research. sorptive therapies. The role of αvβ3 integrins in angiogenesis may Among other things, it has shifted therapeutic strategy towards early, make such therapeutics especially useful for the treatment of rheuma- aggressive treatment before the onset of erosive joint damage in order to toid arthritis and metastatic bone disease. Differences in responses to prevent irreversible functional disability. Additionally, it has made it unethi- various agents and to genetic ablation of αvβ3 integrins point to multi- cal to withhold active therapy and therefore to perform true placebo-con- ple underlying mechanisms and underscore the need for further investi- trolled clinical trials. This has necessitated using active comparator study gations. designs instead, which require more patients, more clinical sites and References longer studies to test the efficacy of putative new therapies. This adds 1. Goldring SR: Pathogenesis of bone and cartilage destruction time and cost to drug development, which slows progress and potentially in rheumatoid arthritis. Rheumatology (Oxford) 2003, 42 raises the cost of new therapies that do get approved. Enriching study (Suppl 2):ii11-ii16. populations with rapidly progressing patients may offset some of this 2. Nakamura I, Pilkington MF, Lakkakorpi PT, Lipfert L, Sims SM, effect, but this necessitates the availability of prognostic markers that can Dixon SJ, Rodan GA, Duong LT: Role of ααvββ3 integrin in osteo- accurately identify which patients are most likely to develop erosive clast migration and formation of the sealing zone. J Cell Sci damage and functional disability. This can be challenging in early RA, as 1999, 112:3985-3993. 30% or more of the patients in early RA cohorts do not progress. Early 3. Pilkington MF, Sims SM, Dixon SJ: Transforming growth factor- prognosticators are also needed by clinical practitioners to determine ββinduces osteoclast ruffling and chemotaxis: potential role in which patients need aggressive treatment before the narrow window of osteoclast recruitment.J BoneMiner Res2001, 16:1237-1247. opportunity for containing erosive disease closes. 4. Wilder RL: Integrin alpha V beta 3 as a target for treatment of Magnetic resonance imaging (MRI) provides greater sensitivity for bone rheumatoid arthritis and related rheumatic diseases. Ann erosions than radiography does, especially in early RA, when radiogra- Rheum Dis2002, 61(Suppl II):ii96-ii99. phy is relatively uninformative. This, along with its ability to detect pre- Acknowledgements These studies were supported in part by the erosive features, such as synovitis and bone edema, allows MRI to Canadian Institutes of Health Research and by the Canadian Arthritis identify patients with the aggressive phenotype of RA very early in the Network. course of their disease. Supplementing MRI with specialized molecular markers, such as CTX-I, CTX-II, glucosyl-galactosyl pyridinoline, COMP S10 and others, may provide additional predictive power. These MRI and

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health examination survey (HES), a register or a clinical study. Consen- sus on case .. Healthy cartilage is maintained in a state of dynamic equilibrium by . 1Faculty of Pharmacy, Université de Montréal, Québec, Canada; . oblivious also serves as a receptor for endogenous molecules and may.

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