Animal models of rheumatoid arthritis and their relevance to human disease

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1 Pathophysiology 12 (2005) Animal models of rheumatoid arthritis and their relevance to human disease Krishnaswamy Kannan, Robert A. Ortmann, Donald Kimpel Department of Internal Medicine, Division of Rheumatology and Immunology, University of Virginia Health System, P.O. Box , Charlottesville, VA 22908, USA Abstract Rodent models of rheumatoid arthritis (RA) are useful tools to study the pathogenic process of RA. Among the most widely used models of RA are the streptococcal cell wall (SCW) arthritis model and the collagen-induced arthritis (CIA). Both innate and adaptive immune mechanisms are involved in these rodent models. While no models perfectly duplicate the condition of human RA, they are easily reproducible, well defined and have proven useful for development of new therapies for arthritis, as exemplified by cytokine blockade therapies. Besides SCW and CIA models, there are numerous others including transgenic models such as K/BxN, induced models such as adjuvant-induced and pristane models, and spontaneous models in certain mouse strains, that have been used to help understand some of the underlying mechanisms. This review provides an update and analysis of RA models in mice and rats. The array of models has provided rheumatologists and immunologists a means to understand the multifactorial disease in humans, to identify new drug targets, and to test new therapies Published by Elsevier Ireland Ltd. Keywords: Animal models; Rheumatoid arthritis; Rodents It is often difficult to discern the nature of an edifice from a study of its ashes. 1. Introduction Rheumatoid arthritis (RA) is a systemic inflammatory disorder that affects approximately 1% of the population worldwide. Even after decades of research, our understanding of the pathogenesis of the disease and the underlying mechanisms remains rudimentary. Clues to the etiology of RA reside in the preclinical stage where the initial triggering and induction events leading to disease take place. Unfortunately, this phase of human RA is not readily accessible to investigation and therefore remains speculative. The spectrum and disease progression of RA is governed by multiple factors including immune, genetic and environmental factors [1,2]. In patients with clinically evident disease, RA is readily diagnosed by joint involvement and indices of immune activation Corresponding author. Tel.: ; fax: address: dlk9t@virginia.edu (D. Kimpel). and inflammation, however it remains a clinical challenge for Rheumatologists to effectively treat and manage the disease. Multiple components of immunity and inflammation play a role in established disease including T and B lymphocytes, neutrophils and monocytes, and vascular endothelium. Treatment thus typically requires multi-drug therapy. Therefore, a better understanding of the immune and genetic mechanisms during the early phases of RA is critical to the development of novel therapies that may one day lead to a cure of the disease. The significance of understanding the mechanism of RA is exemplified by the recently developed anti-tnf blockade therapy, which has raised the bar for therapeutic effectiveness in the management of patients. Yet, the relative resistance of RA to various immunotherapeutic strategies remains an enigma and therefore a challenge to both basic scientists and Rheumatologists [2 4]. Rodent models of RA serve as valuable tools to investigate the underlying mechanisms at early, intermediate and late stages of RA [5 7]. With the recent advances in molecular biology, immunology, bioinformatics, and drug designing techniques, the possibility of developing novel therapies for RA and other inflammatory diseases is all the more promising /$ see front matter 2005 Published by Elsevier Ireland Ltd. doi: /j.pathophys

2 168 K. Kannan et al. / Pathophysiology 12 (2005) The concern that no two arthritis models are alike and none is exactly like human RA, makes it all the more important to understand these models given the clinical observation that no two RA patients are exactly alike, and the etiologic stimulus (or stimuli) is unknown. This review encompasses various rodent models of RA that are used to understand the pathogenesis and pathophysiology of RA, and for the evaluation of therapies. Special emphasis has been placed on streptococcal cell wall (SCW) arthritis in rats and collagen-induced arthritis (CIA) in mice, two widely used animal models. The numerous models of RA available to-date have contributed to our understanding of the role of MHC and non-mhc genes, cell lineages involved, cell surface receptors, cytokines and chemokines, and vascular adhesion molecules that are involved in immunologic and inflammatory process leading to arthritis. The contributions and limitations of animal models are discussed in relation to human RA. 2. Animal models of rheumatoid arthritis Rodent models of RA have been developed in both rats and mice. Other species have also been used over the years, however rodent models are most common, due to cost, homogeneity of the genetic background, and in mice, the capacity to use genetically modified strains. Most RA in animals is produced by treatment with an inducing agent, and even spontaneous models can be considered induced since they develop by the introduction or deletion of specific genes in animals with the proper immunologic milieu for susceptibility. Examples of genetic mouse models include the double transgenic K/BxN model of Mathis and coworkers [8,9], IL- 1 receptor antagonist knockout mice [10] and human tumor necrosis factor transgenic (hutnf Tg) mice [11]. Many of the commonly used arthritis models could fill a chapter if not a book with the nuanced understanding we have of each, however we will provide an overview of RA models by reviewing several rat models with a special emphasis on the streptococcal cell wall model as an example, and review some of the commonly used mouse models with particular attention to the collagen-induced arthritis model and the KBxN transgenic model. Then we will summarize the mechanistic features of inflammatory arthritis with reference to these models Rat models of rheumatoid arthritis The first model of polyarthritis was developed in rats when Stoerk et al. [12] and Pearson and Wood [13] found that injection of rats with complete adjuvant induced polyarthritis, possibly by a mechanism involving heat shock proteins (HSP). This model is termed the adjuvant arthritis (AA) model, and has been used to test new drugs for inflammatory arthritis. Interestingly HSPs have been implicated in the pathogenesis of human RA as well [14]. Many early studies of this model addressed the role of lymphocytes, suppressor cells, and the type of antigen (or lack thereof) administered with the adjuvant for induction of arthritis [15]. In fact it was recently found that AA could be induced by incomplete Freund s adjuvant (IFA) alone in the DA strain of rats. More recently this model has also been reproduced in mice [16]. Adjuvant arthritis has been used in the evaluation of nonsteroidal inflammatory drugs (NSAIDs) such as phenulbutazone and aspirin during the early 1960s [17,18], and later COX-2 inhibitors such as celecoxib [19]. AA in rats shares many features with human RA including genetic linkage, synovial CD4 + cells and T cell dependence [20]. One of the major differences between the AA model and human RA is simply that the inciting agent is known in the model, though the need for any specific antigen is controversial. Other useful rat models of arthritis are streptococcal cell wall-induced arthritis and collagen induced arthritis (CIA). There are clear genetic differences among rat and mouse strains in susceptibility to inflammatory arthritis. In some cases, the responses vary depending on the target antigen, induction protocol, and environmental factors [18,21]. Most models of RA concentrate on the late, chronic, autoreactive stage of arthritis. The SCW model, in contrast, offers investigators an opportunity to examine both early and late phases of arthritis in a predictable manner starting within days of SCW injection [1,22] Streptococcal cell wall arthritis in rats An array of bacterial cell wall components have been shown to induce arthritis in susceptible strains of rodents. The streptococcal cell wall model of arthritis in female Lewis rats is one of the most reliable and best characterized experimental models of RA [23]. Schwab and colleagues described SCW arthritis in the 1970s, and subsequently Schwab and Wilder separately characterized important features of this model (reviewed in [1]. A single intraperitoneal injection of the SCW component peptidoglycan-polysaccharide (PGPS), suspended in an aqueous phase will induce a chronic, severe, erosive arthritis in female Lewis rats [1,24], while males are less sensitive to induction of arthritis. Besides female Lewis rats, other susceptible inbred rat strains include LOU/MN, LA/N, and NSD/N. On the other hand Fisher F344/N rats, WKY, BUF/N, and other strains are resistant to PGPS induced arthritis [25]. The mechanism of susceptibility remains unknown. A dominant role for MHC in arthritis susceptibility is discounted because Lewis and Fisher share the same rat MHC, but differ in their susceptibility [1]. As in human RA, female rats develop arthritis more readily than male rats. The high female susceptibility in SCW arthritis was initially thought to be due to high estrogen levels in the blood and its secondary effects on the reticuloendothelial system. Clearly, other factors play a role in light of the evidence that female Fisher F344/N rats have comparable levels of serum estrogen to female Lewis rats but do not develop arthritis following PGPS injections [26,27]. An association between the susceptibility of Lewis rats to the development of SCW-induced arthritis with the inability of their hypothalamic pituitary adrenal (HPA) axis to

3 adequately respond to inflammatory stimuli was shown by Calogero and colleagues [26]. As compared to F344/N rats, Lewis rats had significantly blunted or no detectable levels of plasma ACTH and corticosterone. Furthermore, LEW/N hypothalami released less immunoreactive CRH (icrh) in response to acetylcholine, nor-epinephrine, serotonin, and quipazine (a selective 5-HT 3 agonist) than F344/N hypothalami suggesting an HPA axis defect. The above data make this an attractive hypothesis; however other factors clearly play a role, as our data show that ACTH receptor knockout mice on the C57BL/6 background are not susceptible to PGPS arthritis (unpublished observations). The differences between Lewis and Fisher HPA axis responses may be secondary to multiple factors. K. Kannan et al. / Pathophysiology 12 (2005) Induction of streptococcal cell wall arthritis: practical considerations. SCW arthritis is induced by a single intraperitoneal injection of PGPS prepared from Group A streptococcal cell walls. The inflammation inducing property of PGPS has been attributed to the peptidoglycan (PG) moiety, whereas protection from degradation in vivo has been attributed to the polysaccharide component [28]. Immunofluorescent studies have demonstrated that cell wall fragments are relatively resistant to enzyme degradation and can persist for weeks and sometimes months depending on the preparation of PGPS used for RA induction [24]. The severity of arthritis and persistence of fragments depends on the source strain of SCW and can vary with the batch of PGPS and specific preparative procedure. The incidence of acute and chronic arthritis is approximately 95% with a proper preparation of SCW 10S PGPS fragments (25 g rhamnose/g body wt) and healthy female Lewis rats ( g). PGPS can be obtained commercially and used for the induction of RA in animals (Becton Dickinson laboratories, San Diego, CA) or prepared according to standard protocols [29]. During the first 24 h, a T cell independent, possibly complement-dependent acute phase develops followed by visible joint swelling by day 3 [24]. The joint inflammation progresses from an initial acute phase (1 5 days) through a remission phase (day 10), followed by a spontaneous reactivation phase as shown in Fig. 1 [22,30,31]. The reactivation phase starts around day 14 and the joint swelling progressively increases and becomes chronic by 4 weeks and persists for months [22]. The chronic phase is T cell dependent as shown by several methods including thymectomy, T cell depletion, and lack of occurrence in athymic nude rats. Assessment of arthritis can be carried out by various means, both objective and subjective. Ankle swelling by any of these methods shows essentially the same biphasic pattern in appropriately susceptible strains. Measurement of ankle swelling by volume displacement in a plethysmograph (Buxco Electronics, Sharon, CT, USA; Kent Scientific, Torrington, CT, USA) is one such method. Ankle diameter measurement using calipers is an alternative approach, which is less costly, but is also dependent on an experienced Fig. 1. Time kinetics of arthritis development in female Lewis rats injected with SCW antigen. Female Lewis rats were injected with 10 s fraction of PGPS by a single i.p. injection and the day of PGPS injection designated as 0 day. Rats were scored for arthritis (arthritis index) daily for the first 10 days, then thrice weekly by a set visual criterion as previously described [30]. A score of 0 4 was assigned to each paw with a theoretical maximum of 16 per mouse as follows: (1) hyperemia and redness with little or no swelling; (2) swelling was confined predominantly to the ankle region; (3) joint swelling was extended to ankle and metatarsal regions and when the swelling area was extensive and more obvious; and (4) maximal paw swelling in both ankle and metatarsal regions and or involving disability in mobility typically manifested by rat dragging the hind paws. The data included in this figure is obtained from two individual rats during a 4 week period. observer to be reproducible. Visual scoring of ankle swelling is most commonly used, and is reliable when the observer is blinded to treatment. The scoring index is based on hyperemia and paw swelling on a scale of 0 4 for each paw [30,31]. An example of the ankle scoring during the course of SCW arthritis are is shown in Fig. 1. A variation on the chronic SCW model is the monoarticular reactivation model, which utilizes a slightly different preparation of PGPS injected intra-articularly to induce a transient joint inflammation. The contra-lateral ankle is injected with saline and serves as a control. Though the arthritis is acute, the joint is apparently primed for arthritis development, because systemic injection of various agents including lipo-polysaccharide (LPS) (gram negative bacteria product which stimulates the innate response via Toll-like receptors, or TLRs), superantigen, or PGPS can reactivate the arthritis [32 34]. Superantigens stimulate a T cell subset based on the particular V region used by the T cell receptor, supporting a role for T cells in the reactivation models as well. Interestingly, the arthritis can also be re-activated by bacterial overgrowth in a blind loop of bowel, suggesting a role for endogenous flora in stimulation of at least some forms of arthritis [35]. The reactivation model has been used for studies of gene expression in joint tissues [36], and for gene therapy experiments [37]. Perhaps one of the most interesting features is that cell wall preparations from other bacteria can also reactivate arthritis, suggesting clinical and perhaps biological parallels to the waxing and waning course of RA flares in humans [1].

4 170 K. Kannan et al. / Pathophysiology 12 (2005) Mouse models of rheumatoid arthritis Numerous approaches have been developed to induce arthritis in mice by immunization or injection. Genetically modified mouse strains have provided other, usually spontaneous, models of arthritis and are reviewed below. Collageninduced arthritis is the best known induced model and is perhaps the easiest to appreciate conceptually, as it involves immunization with a known cartilage component. This model is discussed in more detail below. Antigen-induced arthritis (AIA) is another model, induced with various antigens by intra-articular injection after previous sensitization of the animal to the protein. Ovalbumin and bovine serum albumin (BSA) are commonly used in mice, as well as other species such as rat and rabbit, and demonstrate that a joint-specific antigen is not required. It is important to note that many of these models are transient and modification of the antigen (e.g. methylation) is necessary to induce a chronic arthritis in mice. Besides Freund s adjuvant, other immune stimuli induce arthritis such as pristane, which is an immune stimulant and adjuvant. The pristane arthritis model was originally developed in rats, but has since been found to induce arthritis and lupus-related autoantibodies in certain mouse strains [38]. Proteoglycan-induced arthritis (PGIA) is induced by systemic (intra-peritoneal) injection of BALB/c mice with proteoglycan aggrecan emulsified in dimethyl-dioctadecylammonium bromide (DDA) adjuvant [39]. The PG is isolated from human cartilage obtained during joint replacement surgeries. This is not a commonly used model, but has been used for adoptive transfer of disease with lymphocytes and to investigate gene expression profiles in acute and early chronic arthritis [39] Induced models of arthritis Collagen induced arthritis Collagen-induced arthritis is an extensively studied animal model of RA because it shares both immunological and pathological features of human RA. CIA is primarily an autoimmune disease of joints, requiring both T and B cell immunity to autologous type II collagen (CII) for disease manifestation. This model is reproducible in genetically susceptible strains of mice with MHC haplotypes H-2 q or H-2 r by immunization with heterologous type II collagen in complete Freund s adjuvant (CFA). Susceptible strains are DBA/1, B10.Q, and B10.RIII. Collagen from a variety of sources has been used including bovine, porcine, chick, and human, and response varies with strain and injection conditions. Not surprisingly, mouse collagen gives a poor response. Highly purified collagen prepared under a defined protocol should be used, for the presence of minor contaminants or deglycosylated protein preparation yield either false positive results or may be less arthritogenic [40]. Rats injected with native type II collagen also develop polyarthritis [41], but the mouse model is most commonly used for its applicability in genetically modified strains. Specific T cell and antibody epitopes of CII have been defined in CIA [42,43] and a restricted use of TCR V genes is seen in arthritic joints and lymph nodes of mice with this T cell-dependent arthritis [44]. On the other hand, genomic deletion of certain TCR V genes alone is not in itself sufficient to confer resistance to CIA [45]. Similarly, expression of a TCR transgene in H-2 matched but CIA resistant mouse strain SWR/J was not sufficient to overcome disease resistance [46]. Results of these studies suggest that resistance/susceptibility to arthritis development in CIA models require a complex set of factors including MHC class II, TCR, and non-mhc susceptibility genes. In recent work, Campbell et al. [47] have shown that C57BL/6 (B6; H-2b) can develop CIA with a high incidence (60 70%) and sustained severity. Both clinically and histologically, B6 disease resembles that of DBA/1 (H-2q) mice and is dependent on B and CD4 + T cells. In contrast, 129/Sv mice, which share H-2b, are resistant to CIA. Similarly, Biozzi HI and HII mice (H-2q) both of which are complement sufficient and have no TCR V deletions, are high antibody responders to CII but show, respectively, susceptibility and resistance to CIA [48]. Collectively, this reflects that susceptibility to CIA requires more than MHC class II H-2 q or H-2 r haplotypes. Important factors in disease development include immunization conditions, the tendency of individual strains to preferentially produce certain cytokines, and the involvement of non-mhc genes. Taken together, these results suggest that CIA is governed by a complex set of genes in the induction phase involving interaction of both B and CD4+ T cells, cytokines, and non-mhc genes for the development of disease. CIA is an important rodent model for the analysis of non-mhc genes and their role in RA development. To approach the question of MHC and CIA association David and colleagues have demonstrated CIA induction is more pronounced in the presence of the human MHC susceptibility alleles for RA (DR4 and DQ8), however, this model is produced on a mixed genetic mouse background and other genetic elements may play a role [48]. The requirement for T cells in the development of CIA is clear, whereas the underlying mechanisms are not. Contrary to expectations, passive transfer of collagen-ii specific T cells induces only minor changes in the synovium, while the transfer of collagen-ii specific antibody results in severe inflammation, and co-transfer of antibody and T cells induces chronic disease. Interestingly, despite the fact that Th1 phenotype (preferential production of IFN and IL-12 and related inflammatory mediators) is implicated in the pathogenesis of CIA, interferon-gamma (IFN ) is not required for the development of autoimmune response in the disease process. In fact, IFN KO mice exhibit enhanced susceptibility to CIA [49,50],soIFN may play a more important role in immune regulation, perhaps in development of regulatory cells. Although T cells play a prominent role in the regulation and development of CIA, autoantibody to murine CII appears

5 K. Kannan et al. / Pathophysiology 12 (2005) to be the primary mechanism of immunopathogenesis in this model. During the early stages of disease development, anticollagen antibodies bind to the joint cartilage and activate the complement cascade [46]. Consistent with autoantibody being a major pathogenic factor is the fact that passive transfer of anti-collagen II sera produces an inflammatory arthritis not only in strains considered genetically susceptible to CIA, but also in CIA-non-susceptible strains [51] Passive antibody transfer of collagen-induced arthritis A rapid onset model of CIA can be induced by the use of a commercially available monoclonal antibody cocktail to type II collagen arthritogenic epitopes (Arthrogen, Chemicon International, Temecula, CA) [41,52]. In this model RA can be induced in mice, which do not possess CIA-susceptible MHC haplotypes (i.e. H-2 q, and H-2 r ). Severe and persistent arthritis can be induced by a combination of monoclonal antibody cocktail and LPS (mab-lps-induced CIA) [52]. In the mab-lps induced CIA model, nearly 100% of mice, such as BALB/c, DBA/1, B10.RIII and C.B.17 SCID/SCID mice develop arthritis. Arthritis progresses rapidly within hours, instead of weeks as in the classic CIA model. Within h, arthritis can be seen, and the inflammation reaches a maximum score by day 5 7 and persists for 2 weeks. Both acute and chronic phases of arthritis can be observed, though the inflammatory arthritis will decline after 3 weeks, unless exacerbated by an additional injection of LPS (50 ug) 1 week later. The mechanism of this transient, antibody-induced arthritis may differ from those models requiring a complete immune response, and may have similarities to the K/BxN model described below Immune-complex induced arthritis It is instructive that an immune-complex arthritis was elicited in naive mice by van Lent using a non-self-antigen [53], demonstrating that the development of arthritis does not require a joint-specific response. The fact that the immune complexes (IC) do not include a self-antigen, provides some insights which may help us to understand other models, and the pathogenesis of disease in humans. Mice were injected intravenously with heat-inactivated polyclonal rabbit anti-lysozyme serum, followed by an injection with poly-llysine-coupled lysozyme in the joint. This leads to inflammatory arthritis, which may have similar pathogenesis to the immune-complex-mediated disease produced by K/BxN serum transfer, described below. Using the K/BxN serum transfer model Wipke et al. showed that immune complexes which were not joint-specific accumulated in the synovial tissue, possibly due to lack of decay activating factor (DAF) in this tissue [54] Streptococcal cell wall arthritis in mice In addition to various strains of rats used for the induction of SCW arthritis, BALB/c and other mouse strains develop an acute arthritis following IP injection of PGPS [55]. Muramyl di-peptide (MDP), the minimal immune stimulatory component of PGPS has also been used by IP injection to induce arthritis [56]. In both of these cases the arthritis was apparently acute, as it was not reported past 7 days, and no pannus or erosions were seen. In our hands, BALB/c is the most responsive mouse strain to arthritis induction by PGPS and the C57Bl/6 strain is virtually resistant to this arthritis. Some groups have succeeded in inducing acute arthritis using intraarticular injection as is used for the rat, however it may be dependent on the specific PGPS preparation [57]. Compared to Lewis rats, the SCW arthritis in mice is less robust and the chronic phase is absent. At present, there is no SCW model of RA in mice that is comparable to the rat model in chronicity Transgenic and knockout mice as models of rheumatoid arthritis Genetically modified mice have a dual role in studies of arthritis. Deletion or introduction of genes for particular receptors, signaling molecules, cytokines, or other factors help test the role of these genes in immunologic mechanisms. Sometimes however, spontaneous inflammation occurs, resulting in arthritis or another inflammatory disorder. Outside of providing another model for the study of arthritis, such serendipitous findings also provide valuable insights into immune (dys) regulation and mechanisms of autoimmunity Spontaneous arthritis due to recognition of a ubiquitous self-antigen: the K/BxN model Several mouse strains develop arthritis without the administration of any external antigen, adjuvant, or antibody. For example, the K/BxN mouse (KRN T cell receptor transgenic mouse on the C57BL/6 x NOD background) spontaneously develops chronic, progressive inflammation [58]. Clinically visible joint inflammation is observed as early as 3 weeks of age and it progressively evolves to a severe chronic inflammatory arthritis. In addition to T cell involvement, B cells secrete autoantibodies that promote joint destruction. Similar to the CIA model, arthritis can be induced by transfer of serum and is induced in most strains regardless of MHC, but arthritis is transient, consistent with the need for persistence of antibody as immune complex to drive the arthritis, and the finding that complement and Fc receptor (FcR) play a role [59]. The K/BxN model is thus an important tool to study the role of antibodies in the development of RA [46]. This model also demonstrates that a joint-specific antibody is not required for induction of arthritis. The KRN T cell receptor recognizes glucose-6-phosphate isomerase (G6PI) in the context of the NOD-derived I-A g7 class II MHC molecule [60]). This immune recognition of G6PI gives rise to autoantibodies to the isomerase which, when purified, can transfer disease. The relevance of this reactivity to the etiology of RA is unclear (reviewed in [18]. Interestingly, the transgenic T cell receptor of the krn strain was

6 172 K. Kannan et al. / Pathophysiology 12 (2005) originally selected for its recognition of an unrelated bovine antigen, and discovery of the model was entirely serendipitous. Benoist and Mathis have performed a series of elegant studies to define the underlying mechanisms of the initiation and effector phases, and have implicated mast cells, complement, FcR, and other elements of innate immunity (reviewed in [60]. In yet another variation on this model, immunization with G6PI induces a T cell dependent inflammatory arthritis in normal mice, providing an opportunity for additional insights into this interesting model [61] Other genetic mouse arthritis models NZB/NZW mice, the F1 generation of a cross between NZB and NZW mice develop spontaneous inflammatory arthritis. This strain produces IgM and IgG rheumatoid factors similar to human disease [46]. As with some other arthritis models, such as the pristane model, production of other autoantibodies has lead to the use of this strain as a model of systemic lupus erythematosus (SLE). Identification of the genes involved in arthritis development and autoantibody production will help us understand varying manifestations of autoimmunity. The proliferation of genetically modified mice to alter immune function has often unexpectedly lead to new models of inflammatory arthritis and other autoimmune diseases Table 1 Rheumatoid arthritis models in rodents Rat models Streptococcal cell wall (SCW) Antigen induced arthritis (AIA) Adjuvant arthritis (AA) Pristane-induced arthritis (PIA) Mouse models Induced Collagen-induced arthritis (CIA) Pristane-induced Arthritis Proteoglycan-induced arthritis Zymosan-induced arthritis Immune complex arthritis Serum transfer models Genetic K/BxN NZB/NZW HuTNF Tg TNF gene mutation in AUUUA motif Tristetraprolin / IL-1RA / along with enhanced appreciation for the nuances and complexity of the immune system. Several of the transgenic and knockout mice are also discussed below in the section on Mechanisms. A sampling of the genetically modified mice Fig. 2. Genetic models of arthritis: IL-1 receptor antagonist gene deletion and K/BxN mice. IL-1ra gene knockout mice on the BALB/c background developa mild arthritis in the hindpaws (A); compared to littermate controls (B). The arthritis is evident in the mild thickening of the midfoot, and on the medial aspect of the ankle with loss of distinction of the achilles tendon. The IL-1 / mice also exhibit a distinct scruffy appearance to their coat (C); compared to littermate controls (D). When krn mice, which carry the transgene for a specific TCR, are crossed to NOD mice or mice transgenic for the H2g7 of NOD an inflammatory arthritis develops spontaneously (E); which is not present in the littermate controls which lack the TCR transgene (F).

7 K. Kannan et al. / Pathophysiology 12 (2005) with arthritis is listed in Table 1, and a few are described below. TNF transgenic mice with a human TNF- transgene modified in the 3 region express high levels of both soluble and membrane bound TNF-. Using this model, the pathogenic role of excess TNF- and the interdependency of TNF and IL-1 in the pathogenesis of inflammatory arthritis were confirmed. When these mice were injected with a monoclonal antibody directed to IL-1 receptor thrice weekly from birth, the onset of RA was completely prevented. The TNF transgene was backcrossed onto DBA/I mice resulting was accelerated onset of RA, paralleling the susceptibility of DBA/I mice to CIA. Similar to other models of RA, the levels of TNF-, IL-1, and IL-6 were found elevated [46]. Other mice which exhibit arthritis as a component of their altered immune function include IL-1 receptor antagonist (IL-1ra) knockout (see Fig. 2C E), and two gene mutations which result in TNF overexpression, an AUUUA motif deletion and a Tristetraprolin deletion, discussed in more detail below. The IL-1ra / mice on the BALB/c background do not have as robust an arthritis as that seen in CIA or K/BxN, but do have an interesting scruffy appearance to their coat, which makes it easy to identify the knockout mice since this is not observed in littermate controls (Fig. 2C and D). While useful for understanding mechanisms of immunity, genetic modifications of mice often have unpredictable consequences to the immune system and to other phenotypic features. 4. Understanding mechanisms of arthritis from animal models The immune mechanisms in the pathogenesis of RA have been difficult to understand given the inability to identify an initiating event. The putative mechanisms have evolved as our immunologic knowledge has expanded, and have varied with the trends in immunology. Throughout these developments in understanding human RA, animal models have played a key role in defining mechanisms and establishing some common features. They have also often challenged our cherished beliefs in what factors are required for development of arthritis. Following is an overview of some of the mechanisms identified using animal models of RA, and how they may apply to human RA Role of MHC RA is closely associated with the shared epitope (SE) QKRAA, QRRAA, or RRRAA found in DR4 and related HLA-DRB1 alleles, a finding which has supported the T cell-centered hypothesis of the etiology of RA. However it is unclear if the critical element is the SE or another gene in the shared haplotype. Candidate linked genes include the DQ gene such as DQ8 [38], or non-mhc genes such as TNF [62]. The conclusions are no more clear from animal models of RA. For instance the SCW model is not purely MHC associated, as F334 rats, which share the same MHC as Lewis, are resistant, while other strains with different MHC are susceptible [63,64]. The clearest association is the K/BxN model in which the unusual H-2g7 haplotype of the NOD strain is necessary for disease. This was shown by susceptibility of the H-2g7 transgenic mouse, and identification of the specific antigen glucose 6 phosphate isomerase (G6PI) bound to H-2g7 molecules [60]. CIA seems associated with H-2 q or H-2 r, and recognized T and B cell epitopes have been identified, however the relation to RA has been questioned [65]. Additionally, induction of CIA by David in HLA-DR and DQ transgenic mice with deleted H-2 raises questions about the role of H-2 q or H-2 r [66]. If either human or mouse MHC can play a role, then it speaks to the multi-component susceptibility which leads to RA and RA-like disease in animal models T cell dependent mechanisms The presence of large numbers of T cells in rheumatoid synovium, the MHC class II association, and the ability of T cells to interact with many cell types relevant to RA (e.g. fibroblast-like synoviocytes; Reviewed in a separate article in this issue) has sustained the theory that the original immunologic event of RA may be T cell mediated [22,67]. Our understanding is complicated by the evidence in multiple models for involvement of some of the innate immune mechanisms in both initiation phase and effector phase, such as Toll-like receptors, NK cell activation, and the activation, migration and differentiation of mononuclear phagocytes [68]. It appears that T cells function at the crux of converting an acute arthritis to a chronic inflammatory disease, so whether a model shows T cell dependency may be related to its chronicity. In SCW arthritis an array of evidence demonstrates that the chronic arthritis, but not the acute, is dependent on CD4 + T lymphocytes [69]. This evidence includes: (a) histology and immunohistochemistry showing synovial proliferation with infiltration by CD4 + T lymphocytes [1]; (b) absence of disease in athymic rats [70]; (c) prevention of disease by treatment with T cell-targeted agents such as cyclosporin A [71]) or FK506 [72]; (d) ability to transfer disease with T cell clones [73]; (e) prevention of disease by treatment with monoclonal antibody (mab) to, -TCR [74]; and (f) prevention of disease by treatment with mab to CD4 [69]. Our time course data demonstrates an initial innate activation of monocytes preceding the T cell activation in SCW arthritis, with increases in the percentage of CD4 + T cells expressing activation markers in spleen, lymph nodes, and synovial fluid [22]. Nevertheless, similar to human RA, SCW arthritis in rats seems to be governed by multiple factors, and the role of T cells in the pathogenesis of RA remains controversial [75,76]. Numerous other models have been used to study inflammation and the monocyte and neutrophil infiltrate of arthritis, but many are relatively acute diseases, not expected to require T cells for a cognate response. CIA and K/BxN on the other

8 174 K. Kannan et al. / Pathophysiology 12 (2005) hand have clear-cut T cell involvement. In the K/BxN model T cells bearing the appropriate TCR are one critical element for initiating this antibody-dependent disease, whereas in the serum transfer variation no such requirement has been described. On the other hand when arthritis is induced by immunization with G6PI, depletion of CD4 + T cells could cure or prevent disease [77]. In CIA transfer of disease using T cell lines has been less clear, as disease was variable and mild in appearance. However, total splenocytes could transfer disease effectively [78]. Control of pathologic immune responses is thought to reside in the CD4 + CD25 + regulatory T cells (T regs ). Their role in arthritis is not clear, however synovial fluid T cells from RA patients have some of the features of T reg cells. In AIA, Frey demonstrated that depletion of CD25 + expressing cells using mab exacerbates disease while transfer of this subset decreased severity of arthritis [79]. Other features associated with the putative T reg subset include production of the anti-inflammatory cytokines IL-4, IL-10, and TGF ; expression of CTLA-4, CCR2, GITR, and FoxP3; and development in gut-associated lympoid tissue (GALT) when oral tolerance is induced [80 84]. Depletion or blockade of these cells in the CIA model has shown efficacy but timing was critical to effectiveness [80,81,84]. When antibody against CCR2 was used, arthritis could be suppressed or exacerbated, depending on the phase of arthritis during treatment [80]. The role of T regs in immune responses and the molecular markers, which identify the subset are evolving areas of research, and the potential for utilization of this subset to control arthritis is yet to be defined Autoantigen, antibodies, and B cells No specific autoantigen has been identified that is responsible for arthritis development in RA and in animal models, though rheumatoid factor immunoglobulin against the Fc portion of IgG and more recently anti-citrulinated cyclic peptide (anti-ccp) antibodies are used to help characterize patients with RA. The role of antibodies and B cells in RA are discussed in a separate section of this issue. Among the RA models, those dependent on antibody CIA and K/BxN clearly require B cells, and the antisera can be used to transfer disease, but the variety of models do not provide a consensus on antigen. The CIA model suggests that articular collagen is at least one possible target antigen. Numerous other models do not require a specific antigen. For example, in the adjuvant arthritis model an array of antigens have been used, and in some cases no antigen is used. Likewise the AIA model can use foreign antigens such as Ovalbumin or BSA injected intra-articularly, however induction of antibodies to the specific antigen are required. In SCW arthritis there does not appear to be any association between the arthritis and antibody response to PGPS, which is minimal [1]. Interestingly there are similarities between SCW and the rat AA model in which Kohashi et al. were able to induce arthritis with MDP emulsified in IFA [85]. MDP is the minimal adjuvant component of PGPS and streptococcal cell wall, is also found in mycobacteria, and is non-immunogenic [26]. Similar results were obtained with the non-antigenic adjuvant CP20961, a lipoidal amine from Pfizer [26]. Likewise Pristane-induced arthritis requires no specific antigen. Perhaps these apparent differences can be explained by data from the K/BxN model in which immune complexes are the key and tissue differences in clearing ICs from synovium leads to the development of arthritis. In the K/BxN model the presence of non-specific ICs along with anti-g6pi complexes was part of the arthritis process [54]. Interestingly arthritogenic mabs from K/BxN mice do not induce arthritis individually. Multiple mabs are required with the capacity to form multimers of MAb with antigen (G6PI) by recognition of different epitopes. This is consistent with the need for complement and FcRs in the K/BxN model [59]. Likewise, the mab-lps-induced CIA model utilizes 4 different mabs against CII to induce disease [52]. One intriguing explanation for a role of B cells is that B T interaction is a two-way street. T cells not only provide help to B cells, but B cells can present antigen to and provide activation signals to T cells, and at least two studies suggest that B cells in RA synovial tissue are uniquely able to activate T cells [86,87]. A B cell-centered model would require activated B cells to drive the well-described T cell changes observed in arthritis. Thus, it is not clear whether chronic inflammation in these models is due to an immunopathologic response to a particular autoantigen or to a cascade of events set in motion by a particular type of antigen antibody complex. The role of T cells and B cells and their use as therapeutic targets will continue to be areas of intense investigation Cytokines, chemokines, and humoral factors Cellular interactions are not all carried out by direct cellto-cell contact, thus animal models provide insights into the role of soluble factors in RA. Pro-inflammatory cytokines such as TNF-, IL-1, IL-6, IL-15 and IL-18 regulate inflammatory and immune responses in patients with RA. Similar pro-inflammatory cytokines play a pivotal role in driving the disease in models of arthritis. Chemokines also provide intercellular signals and direct cell trafficking, and are discussed in a separate article in this issue. Time course studies have consistently found IL-1, IL-6, and TNF and other key pro-inflammatory cytokines and chemokines expressed in a variety of models including CIA [88], AIA [89], SCW and Immune complex arthritis [89 92]. Other soluble mediators such as nitric oxide are also observed [31,93]. Methods have included serum and synovial fluid ELISA, ribonuclease protection assays, immunohistochemistry, and gene expression profiling. TNF- and IL-1 promote cartilage and bone destruction, whereas others, such as IL-4 and IL-10, provide antiinflammatory and immunoregulatory actions [94]. The evidence for a pivotal role of certain cytokines has been con-

9 K. Kannan et al. / Pathophysiology 12 (2005) vincing enough to lead to the development of anti-cytokine therapies, for which anti-tnf and anti-il-1 treatments are currently available. To follow up on these observations genetically modified mice have been used to confirm the role of cytokines. As noted earlier, such perturbation of the immune response also sometimes leads to surprises and new models of RA. The role of TNF has been exemplified in several knockout and transgenic mice. Mice with a deletion of the AUUUA motif in the TNF gene had elevated TNF levels and developed spontaneous arthritis and colitis [95]. Mice lacking tristetraprolin, a protein that binds to the AUUUA region, also had elevated TNF and developed arthritis. Mice transgenic for the human TNF gene overexpress this protein and develop spontaneous arthritis [46]. These models helped establish an understanding of the importance of TNF in the disease process. IL-6 is produced by a multitude of cell types, and can stimulate the inflammatory response which TNF and IL-1 are fairly deep in the inflammatory cascade. The role of IL-6 in CIA has been demonstrated using an IL-6 / mouse which had reduced arthritis development [96]. In antigen-induced arthritis and the non-immunologically mediated zymosaninduced arthritis similar attenuation of arthritis occurred in IL-6 / mice. These results have helped lead to current trials of anti-il-6 therapy in RA. Other cytokines are divided by their T cell source into Th1 (IL-2, IFN ) or Th2 (IL-4, IL-5, -9, -10, -13). Th1 responses are associated with organ-specific autoimmunity and inflammation, while Th2 responses are associated with humoral immunity and allergy, though specific Th1 or Th2 cytokines are difficult to detect in serum [97]. RA is generally considered a Th1 disease and IFN is found in most synovial fluid from patients with inflammatory arthritis. IL-2 is harder to detect [98] and a minority of the T cells in synovial tissue express IL-2 and IFNg by immunohistochemical analysis. This was nevertheless greater than expression in non-inflamed joints [98], Similarly, in CIA IL-2 production is observed in arthritic, but not in unaffected paws [88]. In the rat SCW model we were unable to detect Th1 or 2-specific cytokines in serum, or in mrna of lymphoid tissues [90]. To address the role of IL-2 in arthritis and in other immunologic processes mice have been genetically modified by gene deletion for expression of IL-2 and its receptor. Unexpectedly, the phenotype was spontaneous colitis, as has also been seen with gene deletion for IL-10, and certain TCR trangenics [99], pointing to the complex pathogenesis of disease. Similarly IFN KO mice have been generated, and are more susceptible to CIA than are normal C57BL/6 mice. The reason for this finding may be that although IFN is considered a marker of pathogenic T cells, it also regulates development of the immune system, again demonstrating the complex cytokine interactions involved in disease pathogenesis. In the ever-increasing world of cytokines there are always new possibilities to explore. For example, the IL-18 role has been evaluated in CIA and the investigators found that neutralization of IL-18 decreased the severity of arthritis [100]. The importance of the major pro-inflammatory cytokines seems well established by these studies, but the details are always interesting, and these models will continue to be used to identify cytokine, chemokine, and other messengers standing at the gateway between immunity and autoimmune disease Innate immune mechanisms The innate immune response is the first line of defense against foreign threats with recognizable danger signals, and should perhaps be the first mechanism of disease discussed. However most components of innate immunity also function in inducing tissue damage, and by their interactions with the cognate immune cells play an important role in perpetuating inflammation. Components of non-cognate immunity include monocytes, NK cells, platelets, and soluble mediators such as complement, fibrinogen, and the cytokines released by cellular components. The role of the non-cellular components in amplifying the immune system in arthritis is only generally appreciated at present. The need for innate stimulus is clearly demonstrated in various serum and antibody transfer models. Development of arthritis in animal models requires an innate stimulus in addition to any specific stimulus. For instance, the standard CIA model utilizes adjuvant, while the mab-lps-induced CIA model utilizes LPS to activate the immune response [52]. Similarly, the peptidoglycan componenet of PGPS is a functional LPS analogue with adjuvant activity [101,102]. Other components of the immune system, which help shape the innate immune response include complement and Fc receptors. Studies in CIA and in K/BxN arthritis have demonstrated the role of the complement cascade and of inhibitory and activating Fc receptors [ ]. Because the K/BxN experiments were performed using the serum transfer method, they demonstrated the important point that a pronounced activation of the innate response by cognate immunity could take place, instead of the converse (activation of cognate immunity by innate), and these components of innate immunity were important in the effector phase of joint damage. Besides their role in animal models, innate stimulus by moieties such as LPS and peptidoglycan may be responsible for the flare-ups seen in autoimmune diseases such as RA. Various studies have shown that products from gram positive cocci, such as PG and lipotichoic acid (LTA) also found in sterile human spleen, may stimulate cytokine production in CNS, blood cells, and Kuppfer cells [107] Toll-like receptor signaling The role of Toll-like receptors in the pathogenesis of inflammatory arthritis is not well understood, but it is possible that RA synovial fibroblasts activated via TLR pathways contribute to the pathogenesis of inflammatory arthritis in the initial stages and/or chronic phase of arthritis. There are at least 10 numbered TLRs described, the two of primary

10 176 K. Kannan et al. / Pathophysiology 12 (2005) interest for our discussion being TLR2 which binds gram positive cell wall products, and TLR4 which binds LPS of gram negative bacteria. Seibl et al. reported TLR2 expression and TLR-mediated signaling in RA synovial fibroblasts and showed that synovial cells upregulated TLR2 mrna expression, not TLR4, in cells treated with IL-1, TNF-, LPS, and synthetic bacterial lipopeptide [108]. Interestingly, there seem to exist at least two pathways of chemokine induction, one mediated by cytokines such as TNF-, IL-1 and IL- 18 and the other (alternative pathway) mediated by TLR2, independent of cytokines (reviewed in [109]). It is likely that inflammatory arthritis events are initiated by the activation of TLRs, either at the secondary lymphoid organs or at the RA synovium, since TLRs are known to mediate activation of NFkB and MAPKinases resulting in the production of pro-inflammatory cytokines. Such a hypothesis is further supported by the experimental observation that RAsynovial fibroblasts (RA-SF) respond to TLR2 ligands by a strong up-regulation of various CC and CXC chemokines that are of importance in the inflammatory process [109]. It is noteworthy to mention that bacterial DNA, and bacterial peptidoglycans are found in the joint tissues of patients with RA [110] suggesting a causal role of these microbial products in TLR activation and RA induction. In HEK293 cell line transfected with TLR2, PGPS directly binds to peptidoglycan derived from S.aureus and stimulates TLR2, possibly in a CD14-dependent manner [111,112]. The lipoarabinomannan moiety of the Mycobacterium cell wall, a component of CFA also binds to TLR 2 [111]. On the other hand DNA in the preparation likely plays a role in stimulating via TLR-9, as DNAse treatment of CFA inhibits both adjuvant induced arthritis in rats [113] and collagen induced arthritis in mice (Ortmann, unpublished data). Additionally, a synthetic TLR-9 ligand ODN 1826 can substitute for mycobacterium to induce CIA. Clearly TLR stimulation is used to initiate the inflammatory response, and it is likely that exacerbations or perpetuation of arthritis can occur via TLR stimulation as with TLR-4 stimulation by LPS in the mab-lps CIA model described previously. In many of these models the stimulus in some cases incomplete adjuvant without antigen is given IP, and it is possible that this inflammation enhances permeability to intestinal antigens, providing potential TLR ligands and/or antigenic stimulus from gut flora. A critical mediator of intracellular TLR signaling is transcription factor NF- B, which regulates the production of various cytokines, adhesion molecules and antiapoptotic factors [ ]. A more recently described family of intracellular pathogen recognition molecules, Nod1 and Nod2, also signal through NFkB, and seem to function in a manner similar to TLRs [119]. Mutations of the Nod2 gene have been described in patients with Croh s disease, but the role of Nods in inflammatory disease is otherwise poorly characterized. As previously shown in SCW arthritis, inhibition of NF- B by a proteosome inhibitor can decrease the severity of arthritis [93,120]. Recently, a novel TLR2 fusion protein has been designed to block TLR2 mediated signaling and thus inhibit the activation of rheumatoid synovial fibroblasts in vitro [121]. If effectiveness of such treatment can be demonstrated in animal models, novel TLR ligand based therapies for RA may be developed in the future Role of mononuclear phagocytes In addition to T cells, a variety of antigen-presenting cells infiltrate the synovium and have been shown to play an important role in RA [ ]. The mononuclear phagocytes are considered important in models of inflammation [125] since they have the potential to play a two-fold role, first in the initial innate response and later in the destructive process of the chronic phase [126]. The innate response sets the stage for the cognate response by releasing the pro-inflammatory cytokines that determine the Th1/Th2 response and the extent of the T cell response. Our previous reports have strongly implicated the role of monocytes in the PGPS model. Activation of monocytes was observed during the acute, non-t cell-dependent phase, and later during the active T cell-dependent phase [31]. Similar changes in leukocyte number are seen in the AIA rat model [127]. Clodronate depletion of phagocytes has been used in CIA [127] and AA [128] to demonstrate the role of these cells Cell trafficking and adhesion molecule expression One of the characteristics of RA is the infiltration of the synovium with leukocytes, and particularly with CD4 + T lymphocytes. It is currently held that after activation by APC, activated T cells migrate from the lymphatic system and blood stream into target tissues such as the joint, but this does not address the mechanics of how this accumulation occurs. Do selected activated T cells traffic to synovial tissue, do they pass through randomly during surveillance trafficking and become induced by local factors to adhere and accumulate in synovium, or do they proliferate in the synovium? One or more of these mechanisms may be operational. Sophisticated intravital microscopy studies have been performed to more clearly define the unique immunologic events occurring in intact lymph nodes [129]. Such techniques have commonly been applied to CNS, cremaster muscle, and mesentery, but few have addressed synovial microcirculation [130]. Endothelial cell adhesion molecules expression is perhaps the best studied aspect of leukocyte trafficking. In the acute BALB/c SCW model we have seen increases in P-selectin (CD62P), ICAM-1, and VCAM-1 in synovial tissue [62]. Single blocking mabs had minimal impact on the inflammation in this model (Kimpel, unpublished observations) likely due to the redundancy in the inflammatory cascade, thus a cocktail of antibodies to different adhesion molecules may be necessary to impact a complex disease such as arthritis. In the acute monoarticular SCW model Schimmer reduced ankle edema using P-selectin or ICAM-1 blockade [130].