The usefulness and the limitations of animal models in identifying targets for therapy in arthritis

Best Practice & Research Clinical Rheumatology Vol. 18, No. 1, pp. 47–58, 2004 doi:10.1016/j.berh.2003.09.007 available online at http://www.sciencedirect.com

5 The usefulness and the limitations of animal models in identifying targets for therapy in arthritis Paul H. Wooley*

PhD

Professor of Orthopaedic Surgery Department of Orthopaedic Surgery, Wayne State University School of Medicine, 1 South, Hutzel Hospital, 4707 St. Antonie Blvd, Detroit, MI 48201, USA

Animal models have played a critical role in the history of modern drug development for rheumatoid arthritis (RA). In this chapter I examine the contributions of animal models in arthritis therapy from adjuvant arthritis and COX-1 inhibitors to transgenic mice and biological response modifiers. Advances in knowledge of the mechanisms of connective tissue disease are frequently derived from the study of animal models, and these findings frequently identify therapeutic targets that are subsequently evaluated in animal models. Hence a critical relationship between insights into the pathology of arthritis and the development of novel therapeutic approaches exists around the study of animal models of arthritis. In particular, we examine how the study of collagen-induced arthritis in rodents led to pioneering work in cytokine inhibitors for the successful therapy of RA. Key words: rheumatoid arthritis; animal models; biological response modifiers; drugs; immunotherapy.

The last 25 years of arthritis research have seen marked advances in our understanding of the basic pathology of connective tissue disease. This may be attributed in large part to the development of molecular techniques to address basic disease mechanisms, but it is not accidental that the initiation of this period coincides with the discovery of type II collagen-induced arthritis (CIA).1 This observation does not imply that highly significant findings were not discovered using adjuvant arthritis or antigen-induced arthritis (AIA), but merely contends that the coincidence of an attractive model with significant advances in technology inevitably will drive the state of the art in any research field. In more recent years, transgenic technology has risen to the forefront with the discovery that genetic manipulation at a single locus can generate useful models of experimental arthritis. Others may well look back in another decade * Tel.: þ 1-313-745-6828; Fax: þ1-313-993-0857. E-mail address: [email protected] (P.H. Wooley). 1521-6942/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

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and decide that the ‘induced’ arthritides simply paved the way for a golden age of transgenic models, but one must also ponder whether sufficient rheumatoid patients will abandon their cytokine inhibition therapy to provide clinical testing for the next generation of drugs. In this chapter I review the progress of therapeutic advances in arthritis and the relationship to the advances in our knowledge of connective tissue disease derived from the study of animal models of arthritis.

EARLY MODELS OF ARTHRITIS Adjuvant arthritis became the benchmark for the evaluation of non-steroidal antiinflammatory drugs (NSAIDs, particularly COX-1 inhibitors) during the early 1960s.2 Although a highly useful model for the development of this drug modality, its precision in this employ also reflects the limitation of the model. COX-1 inhibitors can essentially cure adjuvant arthritis, but in human disease the anti-inflammatory and analgesic effects have far less influence upon the progression of erosive rheumatoid disease. This demonstrates a major tenet of drug development in animal models– models can represent only a fragment of the complex pathology of a disease such as rheumatoid arthritis (RA). This is further complicated by an appreciation that the human disease is a syndrome, and may exist in patients owing to a spectrum of environmental insults and immunogenetic regulation. Inbred animals subjected to a specific arthritogenic stimulus cannot be expected to represent the complex modality of RA. It is up to the investigator to discern whether the model represents a relevant aspect of the human pathology and to draw appropriate conclusions. The strength of models is drawn from the absence of the confounding factors that may ultimately explain the limited efficacy of a therapy that can occur when applied to the human condition. Another positive feature of adjuvant arthritis may be the ‘non-specific’ nature of the immune stimulation that results in joint disease. The role of the mineral oil and the mycobacterial components of complete Freund’s adjuvant (CFA) in the elicitation of joint disease still remains unclear, which is reminiscent of the unknown aetiology of RA. Despite the observation that both incomplete Freund’s adjuvant (IFA) and the mineral oil pristane may also induce arthritis in both rats and mice3,4, the arthritogenic process appears to depend upon either exogenous or autologous antigens such as heat-shock proteins (Hsp).5 Autoimmune T and B cell populations may arise due to the systemic overstimulation of macrophages by the adjuvant activation process, and give rise to joint infiltration and the production of a spectrum of autoantibodies against cartilage antigens and Hsp. In addition, macrophage-derived cytokines act directly upon synovial cells and chondrocytes, resulting in synovial hypertrophy, abnormal expression of MHC class II antigens and adhesion molecules, and atypical production of matrix components. However, another limit of the adjuvant arthritis in modelling RA may be the rapid disease onset (16 – 19 days), which is scarcely representative of the insidious onset and chronic nature of rheumatoid disease. Nevertheless, a rapid onset of disease is viewed as a major advantage in the pharmaceutical industry where cost considerations may take precedence over the scientific accuracy of a model. Another early model, AIA also contributed to the development of therapeutic strategies. AIA is elicited in rabbits and most rodent species by the injection of a soluble protein antigen (typically methylated bovine serum albumin) into the knee joint of an animal previously hypersensitized by immunization with the same antigen in Freund’s Complete Adjuvant (FCA). The simplicity might suggest that AIA is the consequence of any intra-articular immune response, but this characterization appears to be an

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oversimplification. The antigen must persist within the synovial joint, and the immunogenetic regulation of the response to antigen appears to contribute to arthritis susceptibility.6 The model is limited to an arthritis confined to the injected joint7, although the histological picture of a predominantly T-lymphocyte (mostly CD4þ) joint infiltrate, with B lymphocytes, mast cells and macrophages does provide a reasonably accurate model of RA.

COLLAGEN-INDUCED ARTHRITIS It did not take long from the initial description of CIA in rats1 to the first appearance of papers documenting the performance of the model using established rheumatoid drug treatments.8,9 CIA is only weakly responsive to NSAIDs or methotrexate, and is essentially unaffected by gold salts, chloroquine, colchicine or levamisole. The observation of poor NSAID activity in CIA was viewed by many as a distinct advantage over the adjuvant arthritis model. The nature of CIA, with its MHC-restricted autoimmune reaction to a native joint protein, represents a logical disease mechanism which closely resembles several aspects of RA. Indeed, reactivity to type II collagen appears to be quite prevalent in RA patients10 – 14, although the significance of this response in the disease pathology has never been firmly established. The CIA model provided several potential therapeutic targets, and the association of MHC linkage in both CIA and RA remains an intriguing and underexplored approach to this day. One working hypothesis for the immunopathology of RA remains the idea that joint antigens are presented in context of HLA-DR4 to autoreactive T cells, which trigger the autoimmune connective tissue disease. Thus, interference with DR4-restricted antigen presentation should block disease development. Initial studies using CIA supported this approach, with antibodies to H-2q surface antigens resulting in a marked protection against disease induction. Unfortunately, the therapeutic application of anti-Ia antibodies was less effective, probably due to the established presence of high levels of autoantibody and activated T cells within the joint. However, this target might still be appropriate for RA which (unlike rodent CIA) is characterized by exacerbations and remissions, and might be susceptible to the prevention of renewed immune activation as the disease progresses through cycles. The concern that MHC interference would be strongly immunosuppressive may be countered by the observation that most patients are heterozygous for class II MHC, and homozygous RA patients could be easily identified and exempted from treatment. Unpublished rumours have circulated describing fatal immunotoxic effects of MHC blockade in primates, and it would be useful to know whether this complication is valid. This approach could circumvent the requirement for strict knowledge of the nature of the antigen, or the T cell subset involved in the MHC-restricted arthritogenic response, but the development of MHC blocking peptides (see below) will probably render the development of this therapy unlikely. Immunotherapy for autoimmune arthritis became focussed upon the T cell with the discovery that cyclosporin prevented the onset of arthritis and slowed the progression of established disease in CIA.15 This phenomenon was confirmed in AIA, pristaneinduced arthritis16, proteoglycan-induced arthritis17 and streptococcal-cell-wallinduced arthritis18, and ultimately led to trials of anti-CD4 antibody therapy in RA. The clinical findings were less than spectacular, but this does not (in my opinion) reflect upon a failure of our model systems. Most rodent T cell depletion studies were not overly concerned with large reductions in circulating CD4 T cell levels, and many

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experients achieved levels below 5% of normal cell numbers. This aggressive approach was considered risky for human therapy, although there were no reports of problems with opportunistic infections or malignancy in the animal models. Combined with weakly therapeutic doses, the initial systemic delivery of anti-CD4 (rather than direct injection into the joint) made the outcome of clinical trials19 – 21 unsurprising to many immunologists, and the lack of high efficacy of anti-CD4 therapy in RA probably reflects a failure to target autoimmune T cells effectively (particularly synovial resident T cells) rather than the failure of the conceptual approach. Recent investigations using a primatized non-depleting anti-CD4 injected into the knees of RA patients indicated efficacy using MRI, arthroscopic, and histological imaging22, and CD4 T cell targeted immunotherapy may yet prove of benefit in RA.23 Targeting specific T cell subsets, based upon observations of skewing of T cell populations in affected joints24 has produced mixed results in collagen arthritis. Immunization against Vb peptides using BUB mice elicited antibodies reactive with the self-TCR and prevented the induction of CIA by eliminating or down-regulating pathogenic T cells25, and antibodies to specific Vb subsets have also been successful in CIA26,27, although not all researchers have experienced success with this approach.28,29 Although there is some evidence of T cell subset skewing in patients with RA30 – 32 it appears unlikely that there is sufficient restriction in T cell receptor usage in the disease to make this approach to immunotherapy viable. The most obvious success for the CIA model in therapeutic development can be attributed to the evaluation of cytokine inhibitors. Protocols for inhibiting the activity of IL-1b using IL-1 receptor antagonist protein (IRAP or IL-1ra)33 or antibodies to IL-1b34 have proved successful in CIA, and strategies for inhibiting TNF-a activity using soluble TNF receptor, recombinant TNF receptor:human Fc fusion protein, or antibodies to TNF have demonstrated potent anti-arthritic activity in CIA.35 – 37 These pre-clinical findings contributed to the successful development of anti-cytokine therapy for RA38 – 40, arguably the most significant advance in treatment for this disease since the discovery of steroids. Beyond the inhibition of inflammatory cytokines, CIA has indicated potential effects for other immune regulatory cytokines. It has been suggested that transforming growth factor (TGF)-b might be a suitable candidate36,41, but reports that TGF-b exerts an anti-arthritic effect in CIA are controversial, with some researchers finding that TGF-b may accelerate disease.42 It may prove problematic to utilize TGF-b effectively in RA because the effects of this cytokine on chondrocytes, as well as on immune cells, may make the outcome unpredictable. Antibodies to IL-6 have been shown to be efficacious in primate CIA43, and human trials using this approach are reported to show early success.44,45 Because the delivery of a cytokine inhibitor (which typically has a remarkably short plasma half-life) to the site of joint inflammation raises several practical problems, researchers have employed gene therapy techniques to examine the potential of this novel approach in arthritis.46 Systemic delivery of a TNFaR gene via adenoviral vectors was shown to reduce the severity of collagen-arthritis in rats, but was limited in intraarticular use due to the pro-inflammatory nature of the vector.47 Systemic delivery of chimeric human p55 TNFR-IgG fusion protein via adenovirus vector to mouse CIA had a short-term beneficial effect48, but again the outcome may have been compromised. It has been suggested that IL-4 might be an appropriate therapeutic for use in RA49 because this cytokine may exert an influence on the Th1/Th2 ratio within the rheumatoid joint. IL-4 introduced via an adenovirus vector into joints of mice with CIA caused over-expression of IL-4 in the mouse knee joint50, accompanied by enhanced onset and aggravation of the synovial inflammation. However, the histological analysis

Animal models and arthritis therapy 51

suggested prevention of chondrocyte death and cartilage erosion, with enhanced proteoglycan synthesis in articular cartilage. There have been several recent reports of success in CIA using gene therapeutic techniques to deliver IL-451 – 53, and it appears that this approach may have promise for the treatment of RA. The injection of viral IL10 via an adenoviral vector carrying the IL-10 gene into paw suppressed the development of collagen arthritis and reduced the progression of disease to noninjected paws, despite the absence of detectable levels of viral IL-10 in serum. Antiheterologous CII antibodies were unaffected, but IL-10 suppressed autoantibodies to murine type II collagen.54 Recently, vectors containing the murine IL-18 binding protein isoform c gene55, IL-1056, and IL-1ra57 have been demonstrated to be effective following intra-articular injection using CIA. Success using IRAP-mediated gene therapy in several models of arthritis58 has led to the first human trials of gene therapy in RA59, but it remains to be determined whether this approach represents the new future for arthritis therapy. Gene therapeutic approaches are now extending beyond the delivery of anti-inflammatory cytokines and inhibitors. A novel antigen-specific T-cell-mediated gene therapy has been demonstrated60 using T cell hybridomas specific for type II collagen, transduced to express an IL-12 antagonist and injected into collagenimmunized DBA/1 mice. The cell transfer was without effect on systemic T and B cells responses to CII, although antigen-elicited gIFN production was decreased while IL-4 responses in vitro were augmented. Collagen-reactive T cells migrated to inflammed joints, with a subsequent reduction or blockade of arthritis. Because these results could be reproduced in non-transformed CD4þ CII-reactive T cells it is possible that this approach could be applied to clinical trials. Collagen arthritis has also provided powerful evidence for a role of tolerance induction in the treatment of arthritis. Modulating the autoimmune response by the induction of tolerance might represent a key to the therapy of rheumatoid disease, and administration of type II collagen prior to the induction of CIA has proved to be effective in both rats and mice.61 Tolerant rats exhibited lower anti-collagen antibodies, as well as lower T cell proliferation responses to collagen in vitro. Oral administration of CII appears to alter the subsequent immune response to the arthritogenic challenge via an antigen-driven active suppression mechanism that affects both T cells and B-cells (rather than inducing clonal deletion or anergy) and may be implemented by the regulatory cytokines IL-4, IL-10 and TGF-b. Studies on the induction of oral tolerance therapy with type II collagen suggest that nasal exposure may be even more effective in tolerance induction.62 However, the results of oral collagen in patient trials63 have not reflected the degree of success observed in animals and may reflect both the complexity of the pathology of RA and the difficulty in regulating, let alone modulating, the human diet. Combination therapy, where cytokine modulators are used to reinforce the induction of tolerance64, may prove to be a promising approach to human disease, particularly as the appropriate toleragen for RA still remains poorly defined.

TRANSGENIC MODELS The use of transgenic mice to examine the contributions of specific genes or proteins to the pathogenesis of arthritis represents a novel, powerful approach to the development of anti-arthritic therapies. Early in this technology, the appearance of arthritis in transgenic mice for no immediately obvious reason generated surprise, and fostered the hypothesis that genetic alterations expressed within the joint might lead to non-specific arthritis-like conditions. One such early model occurred in mice

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transgenic for the env-pX region of the human T cell leukaemia virus (HTLV) type 1 genome which developed a spontaneous chronic arthritis due to the expression of the tax gene.65 Ankle joints were most frequently affected, and pannus formation leading to severe erosions of cartilage and bone was observed after several months of disease. In affected joints, cytokine genes (IL-1b, IL-6, TNF-a TGF-b1, g-IFN and IL-2) were activated, leading to elevated serum cytokine levels, elevated IgG levels and production of rheumatoid factor and antibodies against type II collagen, Hsps, nuclear antigens and cardiolipin. Other abnormal immune responses include the increased expression of class II MHC antigens, and reactivity against type II collagen and Hsp. It is now thought that reactivity to connective tissue antigens may well be key in this model66, because collagen immunization can provoke arthritis onset or exacerbation, and the T cells infiltrating skin and joint lesions appear to be oligoclonal in nature and share a similar T cell receptor Vb profile with T cells isolated from joints of mice with CIA.66 However, this model has added to speculation that a retrovirus could be involved in the pathogenesis of RA, possibly by affecting synovial cell growth or regulation67 – 69 which could ultimately influence immune responses to cartilage antigens. Interestingly, the prevalence of HTLV-I Tax positivity among patients with RA has been reported to be three times higher than among healthy blood donors.70 Cytokine-directed immunotherapy for RA was considerably strengthened by the discovery that mice carrying 30 -modified hTNF-a transgenes exhibit deregulated TNFa expression and developed chronic inflammatory polyarthritis.71 At 2 weeks of age in TNF-a (Tg197) transgenic mice, moderate synovitis was present in rear paws and cartilage changes occurred in the absence of synovial infiltrate, with chondrocyte hyperproliferation, cartilage malformation and marked proteoglycan loss. Clinical disease was visible in rear paws from 3 weeks of age, with swelling of the ankle joints, while front paws became involved later. Synovitis, bony erosions and periosteal inflammation worsen, and eventually result in impaired mobility.72 TNF-a transgenics do not exhibit a broad autoimmune syndrome, but Tg197 mice have a markedly shortened lifespan and appear to die from cachexia. Because the arthritis develops as a result of the systemic overproduction of TNF-a, it was gratifying to find that anti-TNFa immunotherapy ameleriotes the joint disease and also extends the life of the Tg197 transgenic mice.71 Recent findings suggest that anti-TNFa in Tg197 mice may reverse existing structural damage, including synovitis and periosteal bone erosions, and this finding may correlate with the reduction in signs and symptoms of RA and the inhibition of the progression of structural joint damage seen in patients treated with inhibitors of TNFa.19,73,74 Because the roles of TNFa and IL-1b appear to be rather similar in inflammatory arthritis, the TNF transgenic model predicted that over-expression of IL-1 would also generate a model of joint disease. Transgenic mice that constitutively produce human IL-1a do indeed develop a chronic, inflammatory, symmetrical, erosive polyarthritis, with synovial hyperplasia, pannus formation and erosion of both cartilage and bone.75 An unusual feature of this murine model is the marked polymorphonuclear neutrophil (PMN) infiltration of the synovial joint. These cells expressed the Gr-1high phenotype, which has been associated with tissue damage and may account for the accelerated loss of cartilage in this model. The supporting observation that IL-1 receptor antagonist knockout mice develop arthritis and autoimmunity76 adds to the importance of this cytokine in the disease process. To date, preclinical studies involving IL-1 transgenic mice have not been reported, but cumulative evidence in other animal models has led to the development of IL-1 inhibitors that show efficacy in RA.77,78

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Transgenic mice have also proven useful in investigating the mechanisms of immunoregulation in RA. Transgenic mice expressing the DQA1 and DQB1 genes from an RA-predisposing (DQ8/DR4/Dw4) haplotype and the autologous murine class II genes ‘knocked out’ developed autoimmune responses and a severe polyarthritis following immunization with type II collagen.79 – 81 Control HLA-DQ8-H-2Ab0 mice developed neither disease nor anticollagen immunity. This finding implies that human class II HLA-DQ antigens may present type II collagen in an arthritogenic manner, and considerably strengthens the murine CIA model as representing a relevant feature of pathology of arthritis in man. HLA-DR4 (DRB1*0401) may also act to present either human or bovine type II collagen in an arthritogenic manner in transgenic mice.15 T cell and B cell autoimmune responses were seen against murine CII, and the DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 known to be key to collagen arthritis. Similar findings were observed in DR1 (DRB1*0101) transgenic mice immunized with human CII81, with the development of severe autoimmune and erosive arthritis accompanied by DR1-restricted immune responses to human CII. Recent findings show that DR1 and DR4 bind the CII(263-270) peptide in a nearly identical manner82, indicating that both HLA-DR4 and HLA-DR1 are capable of binding peptides derived from human CII, which could be significant to the pathology of RA. Myers et al83 have recently built on this observation to develop peptides capable of altering the immune response to collagen by disrupting the DR1restricted immune response. These peptides greatly reduced the incidence and severity of CIA in HLA-DR1 transgenic mice, and thus we may be positioned to see the development of an ‘RA vaccine’ in the near future. The obvious limitation to this approach is the (as yet unknown) prevalence of a DR1-restricted autoimmune response to collagen in the pathology of a general RA population. However, the feasibility of this vaccine therapy raises exciting prospects for the future. A surprising novel transgenic model has provoked many researchers to re-examine some of the basic concepts of autoimmunity in RA84, and might revolutionize our thinking on arthritis immunotherapy. The K/B £ N T cell receptor mouse model appeared when the KRN TCR transgenic mouse was crossed with the NOD (nonobese diabetes) strain.85 The progeny developed an aggressive spontaneous, symmetrical arthritis in peripheral joints with a pathology resembling human RA. Histological features included synovial infiltration, pannus formation, erosive disease and bone remodelling. The critical autoimmune reactivity has been shown to reside in the serendipitous specificity of the KRN Vb6 T cell receptor. Although initially characterized as reactive with a bovine ribonuclease peptide in context of the MHC antigen I-Ak, the KRN T cell receptor recognizes glucose-6-phosphate isomerase (G6PI) in the context of the NOD-derived I-Ag7 class II MHC molecule.86 This immune recognition gives rise to anti-G6PI autoantibodies, which appear critical to the development of joint disease, as passive transfer of anti-G6PI antibodies can result in the appearance of transient joint disease. The precise pathological relevance of an immune response to G6PI in the aetiology of arthritis is unclear87,88, because G6PI is ubiquitously expressed in all cells. The significance of this autoantibody in joint disease was further enhanced by the discovery of anti-G6PI autoantibodies in RA patients89, suggesting a common mechanism that may prove highly relevant to joint disease. However, recent findings suggest that no disease-specific pattern of antibody positivity to GPI exists in RA patients, and that the frequency of anti-G6PI immunity in RA is lower than previous estimates.90 – 92 It is too early to generalize upon the significance of the KB/N model as a useful tool in the study of the immunopathology of RA, and we may yet see therapeutic approaches based upon this discovery. But the recent doubts

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again emphasize the problems with interpreting animal models of human disease and raise the issue of whether transgenic manipulation (and the subsequent selective processes that permit a genetically altered animal to survive) can always represent true single-gene events. This concern will affect novel therapy development, as exemplified when the surprising discovery that disruption of the stromelysin-1 gene was without effect on susceptibility to collagen-arthritis in B10.RIII transgenic mice.93 This observation caused a marked loss of enthusiasm for this matrix metalloproteinase as a useful therapeutic target.

SUMMARY This chapter is not intended to represent a complete picture of the role of animal models in the development of anti-arthritic therapies, and there are several significant omissions beyond the scope of this current article. The list of publications alone that cite novel drugs and modalities from herbal medicines to stress exposure is beyond easy recollection. Hopefully, I have addressed several of the high points in drug discovery that have occurred during the last quarter century and seen the contribution of animal models to significant advances in the array of pharmaceutical agents now available to rheumatologists. Nevertheless, significant challenges remain: RA is still a disease of unknown aetiology, and none of the therapies described represents a cure. It will be interesting to see whether progress in animal models will eventually provide the profound insights required to conquer this condition. However, it appears highly likely that animal models will continue to generate novel targets for intervention in RA for the next 25 years.

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