What we learn from arthritis models to benefit arthritis patients

BaillieÁre's Clinical Rheumatology Vol. 14, No. 4, pp. 599±616, 2000

doi:10.1053/berh.2000.0102, available online at http://www.idealibrary.com on

1 What we learn from arthritis models to bene®t arthritis patients Wim B. van den Berg Department of Rheumatology, University Hospital Nijmegen, Geert Grooteplein Zuid 8, 6500 HB Nijmegen, The Netherlands

Chronic arthritis is characterized by persistent joint in¯ammation and concomitant joint destruction. Animal models have been of great value in understanding potential pathogenetic pathways and studying therapeutic principles. The ®rst models were based on T cell-driven pathways and taught us that arthritis can be induced by a variety of stimuli. This suggests that the involvement of a single (auto)antigen in rheumatoid arthritis is unlikely and suggests that the regulation of arthritis can best be approached via bystander suppression. Insight into the pivotal role of TNFa and IL-1 has emerged from studies employing a range of common and also novel transgenic models. Combination treatment with both TNF and IL-1 blockers is warranted to control both joint in¯ammation and joint destruction. Novel approaches with viral gene constructs of cytokines and cytokine inhibitors teach us that ecient gene therapy is a possibility for small joints. Key words: chronic arthritis; animal models; cartilage destruction; cytokines; gene transfer.

A major goal in the ®eld of arthritis research is to identify a treatment that selectively inhibits the progression of destructive arthritis yet leaves host defence mechanisms virtually intact. This requires a critical understanding of the cells and mediators involved in the destructive process in articular cartilage and bone, as well as a better insight into factors involved in the initiation, maintenance and remission of arthritic disease. In principle, studies on the pathogenesis of rheumatoid arthritis (RA) should be performed on patients, but such approaches are seriously hampered by the fact that the precise time of onset of the condition is often unknown. In addition, most patients have been receiving numerous drugs. Moreover, lesional tissue is easily obtained from end-stage disease, at the time of joint replacement therapy, but remains scarce during the early stages of arthritis. Animal models of arthritis o€er a useful alternative, not only for further understanding the pathogenesis of chronic, destructive arthritis, but also for the evaluation of novel therapeutic modalities. Therapies that are promising in animals will subsequently be applied to human disease, their ecacy in clinical trials then indirectly proving the predictive value of such model systems. No single animal model as yet fully resembles human RA. In fact, the wide variety of agents that can induce an experimental arthritis with features in common with human RA suggests that RA may have a variety of causes and that characteristic clinical 1521±6942/00/040599+18 $35.00/00

c 2000 Harcourt Publishers Ltd. *

600 W. B. van den Berg

features re¯ect common end-points. The large variation in cellular patterns and cytokine expression, as recently observed in early synovial biopsies from various RA patients, further supports the notion that RA should be considered to be a heterogeneous disease. The challenge for the near future will be to identify subgroups of RA patients and to achieve tailor-made therapy. Current therapy with biological response modi®ers is based mainly on common principles observed in various animal models as well as in in vitro culture systems of synovial cells and joint tissues. A further analysis of aspects peculiar to an individual arthritis model is probably of limited value for general treatment, but it may help to identify diverse pathogenetic pathways akin to subgroups of patients. In the following sections, attention will be focused on concepts in classical rodent animal models of arthritis, their relevance to the identi®cation of pivotal cytokines involved in the arthritis process, the usefulness of novel transgenic models and the development of advanced therapeutic approaches in these models, including gene therapy. Features of the human disease will, however, be brie¯y summarized ®rst. CURRENT CONCEPTS AND FEATURES OF RA RA is characterized by the chronic in¯ammation of many joints and the concomitant, progressive destruction of bone and cartilage. Its pathogenesis is still largely unknown. The synovial tissue contains variable amounts of macrophages and T cells, often also showing ®brosis and synovial lining hyperplasia. Current concepts of chronicity include the persistent stimulation of T cells by as yet unknown (auto)-antigens and the T cellindependent, direct activation of synovial cells by (non)-antigenic triggers. The latter might imply a continuous activation by bacterial and viral stimuli or locally generated immune complexes, but it might also re¯ect deranged cell function and tumour-like growth in the absence of obvious triggers. The relative involvement of T cells and macrophages may di€er at various stages. The involvement of cartilage autoantigens remains an attractive theory: in itself, it explains the precipitation of the disease process in the joints, and it ®ts with the clinical observation of waning arthritis in joints with fully destroyed cartilage as well as in joints undergoing replacement therapy. It can be observed clinically that there is no need for the surgical removal of synovial tissue; when an arti®cial joint is introduced, the in¯amed synovial tissue will disappear automatically in such a joint. Cartilage can supply autoantigens or function as a reservoir of trapped immune complexes of broad speci®city. Another theory compatible with the joint speci®city of the in¯ammatory process is the deranged behaviour of the characteristic lining cells of the synovial tissue. Whatever the triggering mechanism, chronic synovitis is characterized by the generation of a vast amount of cytokines, chemokines and growth factors in the in¯amed synovial tissue. Apart from a focus of continued therapy on the general suppression of T cell and macrophage function, much e€ort has been put into the identi®cation of key mediators such as tumour necrosis factor-a (TNFa) and interleukin-1b (IL-1b), as well as into the generation of intelligent biological inhibitors of these mediators. Animal models have greatly contributed to recent progress in this ®eld. Given the variations found between RA patients, and the di€erence between various stages of the disease in one patient, it is obvious that no single animal model can be pinpointed as being the best model of RA, re¯ecting all its features. In fact, current animal models of arthritis do not fully mimic human RA but only represent certain aspects of human disease and should be treated as such.

Lessons from arthritis models 601

CLASSICAL MODELS OF ARTHRITIS: T CELL DEPENDENCE In line with historical concepts in RA, the models most widely studied over the past decades1±6 are those of adjuvant arthritis (AA), collagen-induced arthritis (CIA), antigen-induced arthritis (AIA) and streptococcal cell wall arthritis (SCW-A). These models are based on the principle of a T cell-driven process against a persistent stimulus in the joint (Table 1). The models teach us that the latter can be variable in nature: a planted protein antigen trapped in avascular joint tissues (AIA), poorly degradable bacterial cell wall fragments retained in synovial tissues (SCW-A), autoantigenic stimuli, such as cartilage-derived collagen type II (CIA) or as yet unidenti®ed autoantigens (AA). Flare-ups can be induced in AIA and SCW-A by a rechallenge with antigen, linked to local T cell hyperreactivity. With a higher dosage, and depending on the phlogistical properties of the rechallenging agents, ¯are-ups can also be based on macrophage reactivation. Models using streptococcal cell walls or zymosan (yeast particles) probably start as an acute, macrophage-driven disease, but chronicity is based on the generation of T cell reactivity.6 Another lesson from these models is the fact that chronicity of the arthritis is only achieved under conditions of hyperimmunization and/or loss of tolerance against autoimmune targets. An intriguing hypothesis was put forward that T cell reactivity elicited against bacterial components (AA, SCW-A) displayed cross-reactivity against common bacterial fragments in the synovium or proteoglycan structures in the articular cartilage. Although structural mimicry provides an attractive concept of how reactivity against infectious agents could lead to joint-speci®c autoimmune processes, ultimate proof that this principle plays a signi®cant role in human RA is still awaited. Adjuvant e€ects Apart from antigen-speci®c cross-reactive responses, plain adjuvant e€ects of bacteria or oil preparations used in the immunization process of the animal models might provide yet another mechanism of action, contributing to pathogenesis in AA and SCW-A. Strong adjuvant activity can disturb the tight regulation of immunity/ tolerance against any autoantigens, including joint-speci®c ones. The fact that those models can be induced only in rat strains, which show general susceptibility to a range of autoimmune diseases, argues in favour of such a mechanism. In fact, the more novel models of oil-induced arthritis and pristane arthritis7,8 are also based on this principle, in that immunization is performed with plain oil, in the absence of a speci®c antigen, yet the animals develop an autoimmune response against a variety of autoantigens. The recently developed autoimmune arthritis model, based on transgenic T cell receptor over-expression directed against major histocompatibility antigen molecules, re¯ects an intriguing variant of skewed regulation of tolerance. In contrast to the models discussed above, in which T cells remain a dominant, directing element in the chronicity of the arthritis, it was shown that arthritis persisting for 1 month could be transferred to naive animals using a small amount of antibody.9 The autoantigen involved in this model appears to be a glycolytic enzyme10, the B cell response being triggered by the autoreactive T cells. Intriguingly, joint-speci®c T cell targets are probably not involved. The general lesson that can be deduced from the above is that it is unlikely that one single reactivity against a particular autoantigen plays a dominant role in human RA. Model studies in CIA clearly demonstrate that a loss of tolerance against cartilagespeci®c type II collagen results in a chronic, destructive arthritis with many features in

AA CIA AIA ± SCW-A ± ± ± KRN MRL SCID ± ±

Adjuvant arthritis Collagen-induced arthritis Antigen-induced arthritis Flare of AIA Streptococcal cell wall arthritis Flare-up of SCW-A Oil-induced arthritis Pristane arthritis KRN transgenic mice MRL lpr/lpr mice SCID mice TNF transgenic mice IL-1 transgenic mice

TNF ˆ tumour necrosis factor; IL±1 ˆ interleukin±1. * Dependent on the dose of the stimulus.

Abbreviation

Model Autoantigen unknown Cartilage autoantigen Antigen retention in cartilage Local T cell hyperreactivity Poorly degradable walls T cell/macrophage mediated Adjuvant e€ect Range of autoantibodies Autoantibody Fas defect, spontaneous Engraftment of arthritic cells TNF overproduction IL-1 overproduction

Characteristic ‡‡ ‡‡ ‡‡ ‡‡‡ ‡ ‡ ‡ ‡ ‡ +

T cell

Table 1. Common animal models of arthritis.

‡‡ ‡‡‡

‡‡‡ ‡‡

Immune complexes

‡‡ ‡‡

‡‡

‡‡ ‡*

Macrophage/ ®broblast

Dominant principle

1 2, 5 3 6, 43 4 6, 34 7 8 9, 10 19 22, 23 27, 55 46

Reference

602 W. B. van den Berg

Lessons from arthritis models 603

common with human RA. This has led to a thorough search for such reactivity in RA patients, and although signi®cant reactivity can be found in some patients, it is far from proven that this is a dominant element in RA. In principle, any component from articular cartilage may function as a potential arthritogenic autoantigen, given that tolerance can be broken. The recent demonstration of similar arthritis models in mice upon hyperimmunization with other cartilagederived antigens6,11,12, such as proteoglycan, HC-gp-39, P78 and minor collagens (types IX and XI), provides further suggestive evidence of a multi-antigenic pathogenesis underlying RA. The ultimate proof that a given T cell reactivity is of pathogenetic importance in RA can only be provided by antigen-speci®c immunomodulation studies in patients. When it can be shown that such treatment downregulates speci®c T cell reactivity and concomitantly a€ects the course of human RA, this will identify the relevant triggers. Tolerance induction The ®rst studies of the induction of tolerance to type II collagen in RA patients looked promising13, but follow-up trials did not provide convincing evidence. A problem encountered in these studies was the low reactivity noted against collagen type II, which made it dicult to conclude that the tolerization per se had been successful, apart from potential arthritis suppression. Animal model studies have contributed to a further understanding of stimulating tolerance through the observation that it is relatively easy to induce tolerance before the expression of arthritis but dicult to achieve it in existing arthritis (the clinical situation). It is noteworthy that inducing tolerance against most antigens only appeared to be e€ective over a small dose range for the orally or nasally applied antigen, overdosing being largely ine€ective. The dose `window' might di€er between RA patients, making general application dicult. Cross-tolerance Aside from antigen-speci®c tolerance, it can be argued that the induction of crosstolerance is the way forward. The beauty of this approach is that the antigens involved in RA do not have to be identi®ed since T cell reactivity will be downregulated in a nonspeci®c manner through the generation of suppressive cytokines by tolerogenic T cells, reactive against joint-speci®c stimuli.14 This concept is based on the assumption that arthritogenic T cell reactivity in RA synovial tissue is of the T helper-1 (Th1) type, and that this activity can be suppressed at the site by mediators generated by Th2 or Th3 cells, such as IL-4 and transforming growth factor-b (TGF-b) (Figure 1). Evidence for cross-reactive suppression in animal models (AA, CIA) has recently been provided.15,16 For example, arthritis induced using type II collagen could be suppressed by inducing tolerance against HC-gp-39, whereas adjuvant arthritis could be suppressed by tolerance against heat shock proteins. IL-17-producing T cells Although there is variable belief in the relative contribution of a T cell-driven reaction to RA, there is no doubt that local T cell reactivity found in synovial tissue is mainly of the Th1 subtype and that there is a striking paucity of Th2 cells and IL-4. The low level of interferon-g (IFNg) identi®ed in T cells in the synovium has been regarded as a token of

604 W. B. van den Berg

Bystander suppression Synovial lining Arthritogenic epitopes

Th1 IL-4 Th2

TGFb

Tolerogenic epitopes

Th3

Hsp, Planted ‘Ag’

Cartilage ‘Antigens’ Proteogl, CII, HC-gp-39......

Figure 1. Simpli®ed overview of cytokine interactions in synovial tissue, including T cell- and direct synovial cell-driven pathways, and potential control by bystander suppression.

limited impact. In addition to IFNg production by Th1 cells it has, however, now become clear that IL-17 produced by synovial Th1-like cells is a feature in a considerable number of RA patients.17 IL-17 is a potent cytokine with IL-1-like properties, and the proper regulation of this reactivity is probably needed as well. Initial studies with IL-17-producing T cell clones from RA synovial tissue identi®ed these cells as being highly e€ective in the stimulation of pro-in¯ammatory cytokine production by synovial tissue cells18 and furthermore indicated that IL-17 is the major inducer of tissue-destructive, catabolic events. Additional animal model studies considering the therapeutic applicability of cross-tolerance, reducing both IFNg- and IL-17-driven pathways, are highly warranted.

NOVEL MODELS: LIMITED T CELL DEPENDENCE Apart from the belief in Th1 cells as a crucial therapeutic target, recent interest has been focused on the possibility that arthritis might develop in the absence of targetable immune elements and is in fact linked to deranged synovial cells. A ®rst indication of such pathways emerged from studies in compromised MRL±lpr/lpr mice, which showed spontaneous arthritis, among other abnormalities.19 Changes in the joint were synovial and mesenchymal cell hyperplasia, with late T cell in®ltration. The ®rst signs are synovial cells with a transformed appearance and the invasion of these cells into cartilage and bone, resulting in a rheumatoid arthritis-like pannus. Although initial studies argued that viral infection was the underlying trigger, it appeared that these mice had an Fas defect and displayed impaired apoptosis. The latter ®ts with uncontrolled cell growth in the synovial tissue. The potential involvement of retroviral antigens in chronic arthritis was underlined by the occurrence of arthritis after 2±3 months in mice transgenic for the human T cell leukaemia virus.20 In addition, transgenic mouse models were constructed that re¯ected activated/deranged cells. The over-expression of c-fos in synovial cells did not itself lead to arthritis. However, when AIA or CIA was elicited in these c-fos mice, a more severe and more destructive arthritis was noted.21 Remarkably, the cellular in®ltrate in these

Lessons from arthritis models 605

mice contained hardly any lymphocytes, yet marked cartilage destruction was found. This again stressed the potential role of mesenchymal cells in joint damage. The expression of c-fos coincides with the enhanced expression of stromelysin and collagenase, enzymes involved in cartilage destruction. SCID mice A ®nal development in model systems to be discussed here is the analysis of the arthritic potential of cells and/or tissues in severe combined immunode®ciency (SCID) mice. These animals allow engraftment and the in vivo study of cells taken from animal models or RA patients.22 For this purpose, cells or pieces of synovial tissue are transferred to SCID mice and the ensuing pathological changes are analysed. An interesting variation on design is the combination of cells or tissue with cartilage as a target tissue in order to obtain further insight into the mechanism of cartilage destruction. Recent studies with cells from RA patients show both extremes. It was convincingly demonstrated that RA synovial ®broblasts keep their transformed appearance and maintain their cartilage invasive and destructive behaviour over a prolonged period of time in the absence of T cells.23 On the other hand, T cells isolated from the in¯amed joints of patients with RA can induce a severe, destructive arthritis in SCID mice.24 It is of interest that the transfer of T cells isolated from the peripheral blood failed to induce arthritis, suggesting that arthritogenic T cells had accumulated in the RA joint. This illustrates that both T cells and ®broblasts show this destructive behaviour. The challenge remains of identifying the relative contribution of various cells with destructive potential at the site of the RA joint in the presence of regulatory cells. The latter are absent in designs with isolated cells, and SCID mice allow for the uncontrolled activity of such cells, potentially overinterpreting the destructive capacities. An advantage of the model is the practical suitability for the evaluation of gene therapy approaches. Cells can easily be manipulated with viral gene constructs before engraftment into mice, and the applicability to the synovial tissue of patients can be mimicked.

MACROPHAGE DERIVED CYTOKINES AS A THERAPEUTIC TARGET: TNF/IL-1 In line with a growing emphasis on synovial macrophages and ®broblasts, instead of lymphocytes, as a therapeutic target, major e€orts have been directed towards identifying key mediators released from these cells in the arthritis process. Despite a whole plethora of mediators that could be found in increased quantities in arthritic synovia, it appeared that so-called master cytokines, for example TNFa and IL-1, could be pinpointed. Animal model studies have greatly contributed to this identi®cation. The initial studies analysed the arthritogenic potential of recombinant cytokines when directly injected into the knee joints of rabbits and rodents. This provided the ®rst suggestive evidence that TNFa was an in¯ammatory mediator, whereas IL-1 was a crucial cytokine in both arthritis and cartilage destruction. TNFa alone was hardly destructive, but it could enhance in a synergistic way the destructive behaviour of IL-1.25,26 The next developments were crucial to our understanding: the identi®cation of arthritis as a major phenotype in human TNFa transgenic mice27 and the observation that common models of arthritis could be blocked using selective TNFa inhibitors.

606 W. B. van den Berg

TNFa a transgenic mice Follow-up studies in TNFa transgenics further underlined the fact that TNFa overexpression, in the absence of functional T and B cells, was arthritogenic. Recent observations28 clari®ed that there is no requirement for soluble TNFa but that the full expression of arthritis can occur even with a membrane-bound form of TNFa (mTNFa). The consequence of this is that therapies focused on TNF blockade should preferably make use of antibodies or scavenging soluble receptors that have excellent access to cell surfaces. Moreover, it implies that attempts to interfere with TNF-driven arthritis by the inhibition of TNF-converting enzyme should be considered with scepticism since such a treatment will accumulate TNF on the surface of cells, where it can still be arthritogenic. The development of arthritis in TNF transgenic mice could be prevented with antibodies to TNFa, which seems obvious. More interestingly, however, pathology could also be fully blocked with antibodies against the IL-1 receptor.29 The latter brings two important messages: IL-1 is the secondary mediator responsible for the arthritic changes, and TNFa alone is neither arthritogenic nor destructive towards joints. TNFa a in collagen arthritis Meanwhile, studies with neutralizing antibodies against TNFa have been instrumental in the elucidation of TNF as a major target in more natural arthritis models, with a T cell-driven pathogenetic pathway, compared with the plain over-expression of a single mediator. Ample studies have been performed in the generally accepted autoimmune model of CIA in the mouse. The suppression of arthritis was achieved both with neutralizing antibodies against TNFa and with soluble TNF receptors.30,31 Intriguingly, it was found that TNFa was crucial at the onset of the arthritis but appeared less dominant in the later stages.32 In fact, studies in TNF receptor knockout mice demonstrated that the incidence and severity of arthritis were less in such mice; once the joints became a€ected, however, full progression to erosive damage was noted in an apparently TNFindependent fashion.33 TNFa a in other models of arthritis Apart from analyses in CIA, studies with neutralizing antibodies have been carried out in a range of other arthritis models, including AIA in rabbits and mice, as well as SCW-A in rats and mice. The dominance of TNFa in joint in¯ammation was found in SCW-A34±36 but appeared to be less impressive in AIA37,38, implying that overkill by other mediators can occur and that the stimulus, the type of process and probably also the phase of arthritis are important determinants. In the unilateral form of these models, the reactivation of smoldering, chronic arthritis with homologous antigen was analysed, the lesson from these investigations being that such ¯are-ups do not become more TNFa dependent, but instead show an increasing contribution of IL-1b in the sustained in¯ammatory process (see below). Role of IL-1 in the chronicity of joint in¯ammation and tissue destruction As stated above, IL-1 is a potent cytokine in the induction of cartilage destruction25,26 and is a pivotal secondary mediator in arthritis and tissue destruction in TNF transgenic

Lessons from arthritis models 607

over-expression models.29 In addition, it has been found that IL-1 is not necessarily a dominant cytokine in the acute, in¯ammatory stages of most arthritis models, but plays a crucial role in the propagation of joint in¯ammation and concomitant cartilage and bone erosion in all models studied so far (Table 2). Table 2. Cytokine dependence in various murine arthritis models. Acute in¯ammation Model AIA AIA ¯are-up CIA SCW-A SCW-A ¯are-up ICA

Chronic in¯ammation

Cartilage destruction

TNFa

IL-1

TNFa

IL-1

TNFa

IL-1

+ ‡ ‡‡ ‡‡ ‡ +

+ ‡ ‡‡‡ ± ‡ ‡‡

±

‡

+ ± +

‡‡ ‡‡ ‡‡

± ± + ± ± ±

‡‡ ‡‡ ‡‡ ‡‡ ‡‡ ‡‡

TNFa ˆ tumour necrosis factor-a; IL-1 ˆ interleukin-1. AIA ˆ antigen-induced arthritis; CIA ˆ collagen-induced arthritis; SCW-A ˆstreptococcal cell wall arthritis; ICA ˆ passive immune complex arthritis. AIA ¯are-up is induced by antigen rechallenge at day 21. SCW-A ¯are-ups re¯ect the situation after three consecutive SCW-A ¯are-ups with a 7-day interval.

In CIA, it was shown that treatment with a set of neutralizing antibodies against both IL-1a and IL-1b was still highly e€ective in established arthritis, reducing both in¯ammation and the progression of cartilage destruction. Studies with antibodies to the separate IL-1 isoforms revealed that IL-1b is more crucial.32,39 This is in line with the clear ecacy in this model of ICE (IL-1b-converting enzyme) inhibitors and the observation of reduced CIA in ICE-de®cient mice.40 Corresponding studies with cytokine blockers in the models of AIA and SCW-A demonstrated a limited role of IL-1 in early joint swelling but again a pivotal role in late cell in®ltration in synovial tissue and advanced cartilage destruction.34±38,41±43 This was further substantiated by an analysis of SCW-A in TNFa- and IL-1b-de®cient mice.36,44 Of prime interest is the fact that the propagation of joint in¯ammation and erosion was not prevented in TNFa-de®cient mice, in contrast to the impressive arrest in IL-1b-de®cient mice. This was in line with the demonstration of TNF-independent IL-1 production in TNF-de®cient mice in this model. It should be noted that the transgenic over-expression of IL-1 also produced erosive arthritis.45,46 In conclusion, animal model studies in favour of IL-1 as a potential therapeutic target are as good and as old as the demonstration of the crucial role of TNFa in arthritis. IL-1 as a clinical target has not been pursued to the same extent, for reasons detailed below. Combination therapy In line with the identi®cation of TNFa and IL-1b as separate targets in animal models of arthritis, it has been convincingly demonstrated that combination therapy with both TNF and IL-1 blockers provides optimal protection. In SCW-A in mice, it was found that combination therapy with both soluble TNF receptors and anti-IL-1 antibodies was highly e€ective in both reducing joint swelling and preventing cartilage damage.35 An additional control of late erosion with the combination, compared with anti-IL-1 treatment alone, could not be identi®ed,

608 W. B. van den Berg

especially due to the fact that anti-IL-1 treatment alone was already fully e€ective in this respect. Dose±response titration, to see whether a synergistic e€ect could be achieved, was not pursued. In AA in rats, comparable studies with the combination of soluble TNF receptors and IL-1 receptor antagonist (IL-1ra) provided evidence for synergy. Soluble TNF receptors or IL-1ra alone were not fully suppressive47, but marked synergy was noted when the treatments were combined. In vitro studies have suggested that IL-1ra has to be present in at least a 1000-fold excess compared with IL-1 to be able to block the e€ects of IL-1 on IL-1 receptor-expressing cells. The limited half-life of the pegylated IL-1ra, at least compared with the neutralizing antibodies, places doubt on the full control of IL-1 action in the above experiments in AA, and this might explain the synergistic e€ect of combined TNF/IL-1 blockade. It is our own observation that CIA could not be controlled with the repeated, daily injection of IL-1ra, but that marked suppression was achieved when IL-1ra was supplied via an osmotic minipump.32 Similarly, the local overexpression of IL-1ra by retroviral gene transfer in in¯amed knee joints was e€ective at the site.48 Recent studies in AIA in rabbits provided further evidence for the ecacy of combination treatment targeting both TNF and IL-1.38

Clinical trials with anti-TNF/anti-IL-1 Recent studies in RA patients have provided impressive evidence for a marked antiin¯ammatory e€ect of treatment with anti-TNF neutralizing antibodies or soluble TNF receptors. Both joint swelling and pain were highly reduced.49,50 The potential e€ects on joint damage were not evaluated in the ®rst studies. Extended studies, however, have now also proved the arrested progression of joint erosion. At present, it cannot be excluded that some of the e€ect is due to cytotoxicity of the complement-binding blockers used, not only neutralizing TNF but also a€ecting TNF-bearing cells and general cytokine production. This may contribute to the anti-erosive character. The initial studies targeting IL-1 were carried out using soluble IL-1 type I receptor. Clinically relevant e€ects were not seen, which was disappointing at the time and questioned the relevance of IL-1 as a therapeutic target in human RA.51 It is now understood, however, that the choice of the type I receptor was unfortunate since this soluble receptor shows a high anity for IL-1ra, thus scavenging endogenous IL-1ra. In that sense, the decoy type II receptor might make a better inhibitor but as a downside has a lower anity for IL-1. Studies are awaited with optimal, engineered soluble IL-1 receptors. Apart from studies with soluble receptors, clinical trials have also employed IL-1ra, the e€ects on joint in¯ammation being limited. A signi®cant reduction of joint erosion was, however, observed, which is encouraging.52 Remarkably, if comparisons are allowed with animal model studies, it is doubtful whether the level of IL-1ra reached in these clinical trials was sucient fully to control IL-1, and further optimization is warranted. Intriguingly, proper neutralizing antibodies against human IL-1 are apparently not yet available, hampering a fair comparison with TNF blockade studies. It is expected that combination therapy with TNF and IL-1 inhibitors will be more bene®cial, not only in animal models of arthritis, but also in RA patients. Such studies have to be awaited before a proper conclusion can be drawn on the relative role of TNF and IL-1 in RA patients.

Lessons from arthritis models 609

Variability of TNF/IL-1 staining in early synovial biopsies of RA patients Synovial tissue samples are now available through arthroscopic approaches as well as the taking of blind needle biopsies in early RA patients. It is of interest that TNF is not always found in such specimens, large variations being noted.53,54 The latter includes our own observations, since we found signi®cant TNF staining in specimens of roughly 50% of our RA patients. Interestingly, IL-1 was present in all the specimens, distinct IL-17 staining being found in 70% of the samples. Anti-TNF therapy is not e€ective in all RA patients, and not all of the joints of responsive patients improve. It may therefore be argued that RA is a heterogeneous disease. Moreover, the cytokine interplay may vary in di€erent stages of the process in the same patient, and repeated sampling in large groups of RA patients is needed to shed more light on this issue. In conclusion, when interpreting the animal model data as well as the clinical data, it is suggested that TNF over-expression may result from separate pathways, one being disturbed synovial cell function with deranged TNF production55, the other being T cell or direct macrophage stimulation by as yet unidenti®ed triggers (Figure 2). Animal model studies provide cogent evidence that, with a range of stimuli, both TNF and IL-1 are produced, but not necessarily through a TNF-driven cascade. In such conditions, the separate blocking of IL-1 is warranted. If the major defect in most RA patients is deranged cell function, with a mere over-production of TNF, the selective targeting of TNF is probably sucient. OTHER CYTOKINES AS POTENTIAL THERAPEUTIC TARGETS It is fortunate that TNF and IL-1 appear to be suitable therapeutic targets in chronic arthritis. Animal model studies have identi®ed IL-10 as an endogenous modulator of TNFa overproduction in RA Stimulus X

T cell

‘Macrophage’

Macrophage

TNF

IL-1

‘Deranged cell’ Figure 2. Schematic presentation of ways to generate tumour necrosis factor (TNF) overproduction in synovial tissue. When the defect is in the synovial macrophages/®broblasts, a TNF±interleukin-1 (IL-1) cascade is logical, TNF blockade being sucient to control synovitis and IL-1-mediated destruction. When T cells and/or macrophages are triggered, model systems learn that this will generally lead to separate IL-1 production, which needs additional control.

610 W. B. van den Berg

arthritis and made it clear that IL-4 is hardly present. The elimination of IL-10 with neutralizing antibodies augmented CIA and SCW-A. In contrast, treatment with IL-4 and, even better, the combination of IL-4 and IL-10, suppressed arthritis as well as cartilage and bone destruction.56,57 In these studies, IL-10 appeared to be a potent suppressor of TNF, whereas only the combination of IL-10 and IL-4 suppressed both TNF and IL-1. In line with this, the initial clinical trials with IL-10 were disappointing, and it is expected that, in the treatment of RA patients too, IL-10 and IL-4 have to be combined. Apart from an understanding of the role of suppressive cytokines, further insight into novel pro-in¯ammatory cytokines has emerged from recent animal model studies. IL-12 potentiates Th1-driven processes, and IL-15 stimulates IL-17 production by Th1like cells. Both cytokines are the product of macrophages, being released in an enhanced quantity after bacterial stimulation (Figure 3). As such, these cytokines may link bacterial infection with the potentiation of Th1-driven immune reactions, including those against joint-speci®c autoantigens. The elimination of IL-12 and IL-15 so far appears to be e€ective in the early stages of CIA.58,59 It remains to be seen whether IL-12, IL-15 and IL-17 will provide a novel target in human RA. Major interest is now also focused on IL-18. This IFNg-inducing factor promotes Th1 reactivity and has also been shown to induce TNF and IL-1 in macrophages. As such, it might lie at the base of the TNF/IL-1 cascade. Recent studies on RA synovial tissue have identi®ed an increased quantity of IL-18, and the addition of IL-18 augmented CIA.60 Studies with neutralizing antibodies are in progress in arthritis models, the ®rst data suggesting that such treatment is protective. Intriguingly, IL-18 shares characteristics in common with IL-1, including the need of a converting enzyme (ICE) for its activation. In addition, an IL-18-binding protein has been identi®ed, which may be used as a therapeutic tool. Synovium X

Regulation of arthritis

X

APC

Bact. IL-15

IL-4

Cartilage erosion

Virus?

T

enzymes

IL-12

Ch

Macrophage Th2

Th1

IFN

g

Bystander epitopes T2

TNFa IL-1 IL-17

Fibro -

IL-4

IL-10, TGFb

M

Ch

+ T3

Bystander epitopes

Figure 3. Epitopes from cartilage or synovial tissue can be used to generate suppressive cytokines ± interleukin-4 (IL-4) and transforming growth factor-b (TGFb), which will control T helper cell-1 (Th1) reactivity against unidenti®ed (auto)antigens in a non-speci®c way (so-called bystander suppression). A prerequisite is the accumulation of Th2/Th3 cells with such reactivity at the site. Apart from using epitopes arising from cartilage or synovial tissue, suitable epitopes or antigens can be arti®cially planted into the joint.

Lessons from arthritis models 611

NOVEL APPROACHES IN ANIMAL MODELS In some of the above sections, attention was drawn to the contribution of knockouts and transgenic over-expression models in understanding cytokine interplay in arthritis. Transgenes are often placed under tissue-speci®c promoters in order to achieve selective over-expression at the site. Both TNF and IL-1 over-expression underline the arthritogenic potential of mediators27,46 and the role of membrane forms. The knockouts de®nitely con®rmed suggestive ®ndings obtained with blockers, avoiding a potential di€erence in ecacy, half-life or selectivity of the various blockers. A remarkable element of transgenic approaches is the possibility of the over-expression of human cytokines in mouse systems. This may identify the potencies of human molecules, but, perhaps more importantly, it can also be used to screen the therapeutic ecacy of blockers engineered for use in humans. The accessibility of targets becomes a critical issue, particularly in the light of the dominant role of membrane-bound forms of the cytokines, as well as the probable requirement of access to articular cartilage in order to control local cytokine or enzyme production by activated chondrocytes. General comments to be made here include the future need for inducible transgenes, thus avoiding the absence or presence of crucial mediators during development, and reducing the risk of unexpected ®ndings as a result of major disturbances in joint tissue formation or the maturation of normal immunity. As an example, TNF de®ciency has a major impact on the architecture of lymphoid organs, which might seriously ¯aw studies in immune models. Systems have now been developed, and are already in use, in which the transgene of interest is targeted at a desired point in time by the addition or elimination of drugs (e.g. tetracycline, the so-called tet-on/o€ system) that interact with intelligent promoter systems. Apart from the advantage of evaluation in relatively normal animals, such a conditioned system might also o€er the possibility of subtle transgene levels rather than the current extreme all-or-none situation. Some recent examples of further insight being gained into the role of mediators are arthritis studies in stromelysin- and inducible NO synthase- (iNOS-) de®cient mice. Using the speci®c knockout mouse, it was identi®ed that stromelysin is a crucial enzyme in erosive cartilage damage but not in early proteoglycan loss.61 Earlier studies with inhibitors of stromelysin were hampered by poor bio-availability and insucient speci®city. This can be elegantly circumvented with the use of knockout mice. The other example is the iNOS-de®cient mouse. Inhibitors developed to block nitrous oxide have so far been potent but not fully selective, also a€ecting other processes. Thus the ®ndings in arthritis models have been confusing. The study in iNOS-de®cient mice avoided this and provided cogent evidence for a crucial role of nitrous oxide in cartilage destruction.62 FEASIBILITY OF GENE TRANSFER AND THERAPY Apart from the control of arthritic disease by the systemic administration of TNF/IL-1 blockers, it is envisioned that control could be optimized by gene therapy, thus providing a consistent level of inhibitors at the site. Both retroviral and adenoviral approaches have been suggested63, the latter having the advantage that all the cells will be infected, whereas retroviral constructs will infect only dividing cells. A disadvantage of adenoviral transfection is the transient expression. A promising alternative has, however, now been provided by the use of adeno-associated viruses. A detailed discussion of viruses goes beyond the scope of this chapter63, but it is expected that viral

612 W. B. van den Berg

constructs will be markedly improved, hence avoiding immune clearance and health risks. An elegant development is the generation of intelligent gene constructs, using promoter constructs that are able to sense the need for a particular inhibitor under in¯amed or tissue-destructive conditions.64 Model systems in rodents and larger animals will then be instrumental in ®ne-tuning their future application in human RA. Some examples of successful approaches in animal models are summarized in Table 3; these include the over-expression of de®ned inhibitors such as IL-1ra, soluble TNF receptors and the modulatory cytokines IL-10 and IL-4. The SCID mouse model provides an elegant test system for the analysis of ecacy of transduced cells under in vivo engrafting conditions. Table 3. Examples of recent gene transfer studies in experimental arthritis. Gene construct

Model

Vector

IL-1ra IL-1ra IL-1ra IL-1ra IL-1/TNF receptor IL-10 IL-10 IL-4 TGF-b Fas Galectin

CIA AIA SCW SCID AIA AIA CIA CIA CIA CIA CIA

Retrovirus Retrovirus Retrovirus Retrovirus Adenovirus Adenovirus Adenovirus Adenovirus Plasmid Adenovirus Plasmid

Reference 48 65 66 67 38 68 69 70 71 72 73

For abbreviations, see text.

It has already been shown that synovial lining macrophages are crucial in the onset and propagation of arthritis, since the elimination of these cells with toxic liposomes prevents disease.74 Gene therapy can also be used in this direction, for example by enhancing the cell death of activated Fas-expressing cells through the over-expression of Fas-ligand, or the apoptosis of activated T cells through galectin-1 delivery.72,73 Another promising development is the application of engineered T cells overexpressing suppressive cytokines such as TGF-b71 and capable of tracking to the site of interest. Elegant improvements are the use of T cells with the right chemokine receptor make-up, as a recipient of the TGF-b construct, and the additional engineering of relevant chemokine receptors on recipient cells, facilitating speci®c homing. It is as yet unclear whether manipulated cells remain at the site for a prolonged period of time. A ®nal observation in many of the above studies is the protective e€ect not only in the joint where the transgene is over-expressed, but also in the ipsilateral and contralateral joints. The mechanism of this spread to nearby joints remains to be explored. Whereas gene transfer to synovial tissue seems possible, further research is needed for the application of gene transfer technology to the treatment of cartilage destruction. Current constructs with viral elements are too large to allow for the infection of diseased chondrocytes in the tight cartilage matrix, and other delivery systems such as liposomes or plasmids may be preferable. The latter is so far still hampered by poor transfer eciency. The control of cytokines and degradative enzymes, as well as the application of cartilage-speci®c growth factor to enhance the repair reactivity of the arthritic chondrocytes, is a challenging option.

Lessons from arthritis models 613

PERSPECTIVES It is expected that the human genome project and the use of gene chips, to evaluate di€erential gene expression in in¯amed synovial tissue and arthritic cartilage, will provide many novel genes to be investigated. Although current approaches generally try to identify the function of novel genes by transgenic over-expression or the use of knockout phenotypes, it seems obvious that the viral over-expression of novel gene constructs at selected sites will be a major methodology for the future. Although it is of scienti®c importance to see that the knockout of a gene causes major abnormalities in normal development or displays a lethal phenotype, it is faster and more informative for the understanding of joint disease to accomplish a local over-expression in joint compartments and to study the speci®c impact of novel genes at the local level. Given the promising results of cytokine blockade, it is expected that the challenge for the future will be in the engineering of cartilage. It is probable that we will be able to arrest the further progression of in¯ammation and joint destruction in most RA patients, but we will still be confronted with damaged cartilage. This condition will proceed to an osteo-arthritic lesion when further treatment is lacking. It has long been recognized that articular cartilage has a poor intrinsic repair capacity. Present attempts in cartilage engineering include the transfer of mature chondrocytes or mesenchymal stem cells to cartilage lesions, combined with the supply of slow-release systems of growth factors. It is challenging to investigate whether tissue regeneration can be improved by the transfer to such cells of genes encompassing a de®ned set of growth factors. The clinical application of cell transfer in cartilage repair has so far been limited to the restoration of traumatic cartilage lesions. Late stages of current arthritis models, undergoing cytokine-directed therapy and displaying various degrees of irreversible cartilage damage, would o€er future test systems to examine its applicability for diseased cartilage. Practice points . animal models of arthritis should be used to study aspects of human disease . the involvement of just one single (auto)antigen in RA is unlikely . the regulation of arthritis by bystander suppression circumvents antigen identi®cation . TNFa is a major therapeutic target in early arthritis . IL-1b is an additional target in chronic, destructive arthritis . combination therapy with anti-TNF/IL-1 is warranted . tailor-made anti-cytokine therapy is useful in de®ned RA patients . gene therapy is plausible for small joints REFERENCES 1. Pearson CM. Development of arthritis, periarthritis and periostitis in rats given adjuvants. Proceedings of the Society of Experimental Biology 1956; 91: 95±101. 2. Trentham DE, Townes AS & Kang AH. Autoimmunity to type II collagen: an experimental model of arthritis. Journal of Experimental Medicine 1977; 146: 857±868. 3. Dumonde DC & Glynn LE. The production of arthritis in rabbits by an immunological reaction to ®brin. British Journal of Experimental Pathology 1962; 43: 373±383.

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