Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology 12. Once established, immune reactions against joint components contribute significantly to the pathological hallmarks of the disease, being synovial hyperplasia (pannus formation) and a variable degree of destruction and remodeling of joint cartilage and bone. RA affects approximately 1% of people in Western countries, with a 2:1 prevalence in females over males. The ageing societies in the developed countries create a growing need for safer and more effective therapies to treat chronic diseases such as RA. The advent of biotechnology has fuelled the search for drugs that act more specifically to overcome the considerable side effects of nonspecific anti-inflammatory and immunosuppressive drugs. Especially for immune-mediated diseases, biotechnology-based therapies have a great therapeutic potential. The preclinical development of immunomodulatory compounds often begins with an observation in vitro, after which proof of therapeutic principle is obtained in animal models, usually in inbred strains of rats or mice.

Unfortunately, the promising effects of new therapeutics observed in rodents are often not reproduced on testing in patients. There is a growing awareness that the evolutionary gap between inbred rodent strains and the human population is too wide for direct translation of data from rodents to humans 3. Because of the closer evolutionary and immunological proximity to humans, nonhuman primates may help to bridge this gap 456. Trans-species antigen presentation of human antigen-presenting cells to rhesus T cells and vice versa 78 nicely illustrates the immunological proximity of rhesus monkeys and humans 91011.

It is of critical importance for preclinical safety testing that the selected animal model is sensitive to the pharmacological action of the tested drug and that the tissue distribution and pharmacological properties of the molecules targeted by the treatment are comparable to those observed in patients 12. Parallel to the advent of biotechnology in recent decades, the interest in nonhuman primate models of human disease, in which highly specific new treatments can be tested, has increased. It is remarkable that whereas in transplantation research nonhuman primates are considered an essential preclinical model in the development of new therapies, the selection of therapies for a chronic disease such as RA relies mainly on inbred rodent models 6. Many new therapeutic reagents, such as antibodies, cytokines, and cytokine antagonists but also more specifically acting small molecules, are active only in humans and some closely related nonhuman primate species.

Nonhuman primates spontaneously develop several of the arthritic diseases that affect the human population 913. However, spontaneous manifestations of arthritis in a large outbred population of rhesus monkeys (>1,000 individuals) kept at the Biomedical Primate Research Centre in Rijswijk (the Netherlands) are rare. The low incidence and unpredictable nature of spontaneous arthritis prompted us to develop a model that can be induced at will and that is suitable for testing new therapies for safety and efficacy.

Initial attempts were aimed at the reproduction of well-established arthritis models in rats and mice, to test whether these were experimentally feasible and would be compatible with ethical and practical standards. Widely used models, such as streptococcal-cell-wall-induced or mycobacterium-induced reactive arthritis in Lewis rats, could not be reproduced in rhesus monkeys 14.

A frequently used model of joint inflammation in rodents is antigen-induced arthritis (AIA). In a preliminary experiment, intra-articular injection of methylated ovalbumin (OVA) into OVA-sensitized rhesus monkeys induced macroscopic arthritis in one of two monkeys (MPM Vierboom, personal observation). The AIA model may provide a useful model, causing less discomfort to the animals than systemic polyarthritis, for the assessment of the immunogenic properties of new products to assist in the repair of the joint under local inflammatory conditions or therapeutics that are administered locally to suppress inflammation.

The clinical expression of arthritis induced by collagen type II (CII) in rodent strains is strongly influenced by their genetic background 151617. Immunization with heterologous CII induces reproducible autoimmune-mediated arthritis in a variety of genetically susceptible strains of mice and rats and in macaques 1819. Interestingly, immunization with bovine CII induced spondylitis without joint involvement in Buffalo rats (RT1^b), while Wistar rats (RT1^u) developed chronic joint inflammation without marked involvement of the spinal column. The F₁ offspring of both strains developed inflammation at both locations (B 't Hart, personal observation).

While inbred rodent strains are genetically uniform and essentially represent a single individual in an outbred population, an outbred colony of rhesus macaques more closely resembles the human population in its heterogeneity. Predictably, the incidence and clinical presentation of collagen-induced arthritis (CIA) in a random sample (more than 50) of the large, outbred rhesus monkey colony at our institute (more than 1,200 animals) appeared heterogeneous, as is observed for human RA. In about half of randomly selected animals from the outbred colony of genetically typed rhesus monkeys at our institute, CIA could be induced. On the basis of these data, the CIA model in rhesus monkeys was further developed as a preclinical model of human RA.

CIA is induced in rhesus monkeys by immunization with 3 to 5 mg bovine collagen type II (b-CII) dissolved in 0.5 ml 0.1 M acetic acid and emulsified in an equal volume of complete Freund's adjuvant (CFA). This emulsion is injected into the dorsal skin, distributed over 10 spots to reduce the formation of ulcerative skin lesions.

The time of onset and severity of clinical signs varies, most likely reflecting the outbred nature of the colony. Whereas RA susceptibility, more precisely the disease severity, in the human population maps to the major histocompatibility complex (MHC) class II alleles HLA-DR1 to HLA-DR4, no apparent association with the MHC class II region has been observed in rhesus monkeys thus far. This was rather unexpected, because 'shared epitopes' that confer a high risk to RA (QKRAA and QRRAA) are present at the correct location in several Mamu-DRB1 alleles (see the IPD-MHC sequence database 20). In contrast, a strong influence of the MHC class I region on the susceptibility to CIA was found. Young animals (of Indian origin) from our colony that were positive for the Mamu-A26 serotype appeared completely resistant to the disease even after several booster immunizations. This resistance may be age dependent, since Mamu-A26⁺ monkeys more than 20 years old developed CIA, though the disease was less severe than in animals lacking this marker. Furthermore, the resistance is specific for the immunizing antigen, since Mamu-A26⁺ and Mamu-A26^- monkeys are equally susceptible to experimental autoimmune encephalomyelitis (EAE) induced with human myelin basic protein (Table 1). Selection of A26^- monkeys thus allows reproducible induction of CIA in >95% of animals. Furthermore, the Mamu-A26-associated CIA resistance was mainly observed in rhesus monkeys of Indian origin (B 't Hart, RE Bontrop, personal observation). We recently found that the A26 serotype defines a region configuration encoding multiple Mamu-B molecules, which has been renamed Mamu-B26 21. Hence it is possible that this resistance is defined by a particular combination of MHC class I molecules or by a closely linked gene. A gender bias as observed in humans for the risk of developing RA was not found for CIA in rhesus monkeys, although a prevalence in females has been found in the closely related cynomolgus macaque.

Several surrogate markers for CIA have been developed, which reflect different pathological aspects of the model, that is, markers for inflammation, bone degradation, and clinical wellbeing. These markers help to determine the therapeutic efficacy of a new therapy. Consistent improvement of a biomarker in the experimental group versus a control group without a direct clinical effect can nevertheless indicate a therapeutic effect. The relation between various biomarkers, the clinical manifestation of arthritis in the model, and the response to treatment is illustrated by data collected over the past decade.

A clear contribution of autoantibodies to the immunopathogenesis of CIA was found in arthritic animals. The resistance to CIA observed in Mamu-B26 (formerly A26)-positive animals is most likely associated with the failure to produce adequate levels of immunoglobulin (Ig)M antibodies against the immunizing antigen 2627. Interestingly, CIA-resistant animals mount a normal collagen-specific IgG response, both quantitatively and qualitatively, that is, reactivity profile with epitopes in the CB11 fragment of collagen 28. Unpublished data indicate that the subclass of anticollagen IgG antibodies in resistant monkeys may resemble human IgG4, which does not efficiently fix complement. As the IgG4-like antibodies bind to the same epitopes as the complement-fixing IgG1/3-like antibodies in CIA-susceptible animals, these IgG4-like antibodies may protect the joint cartilage against opsonization.

For routine histology, the patellae of both knee joints and the proximal interphalangal and distal interphalangeal joints of the third digit of the hand and foot were processed and analyzed. Figure 3 shows different phases of the destructive erosion of cartilage and subchondral bone in an arthritic finger joint. We use the pathology grading system published by Pettit and colleagues 29. This system quantifies the degree of inflammation, bone destruction, and cartilage degradation on an arbitrary scale from 0 to 5.

The arthritic joints of CIA-affected rhesus monkeys display essentially the same histopathological hallmarks as RA joints. In the early phase of active CIA, hyperplasia of the synovium and pannus formation were already observed 30. These preceded the dramatic destruction of cartilage and bone in advanced CIA (Fig. 3, bottom left).

The ethical management of the rhesus monkey CIA model relies on a semiquantitative clinical scoring system that represents the overall disease status of the animals (Table 2). Clinical signs that were monitored daily are body weight, body temperature, and the amount of pain relief used. Macroscopic signs of inflammation, that is, the number of joints showing soft-tissue swelling, warmth, and redness, were recorded twice weekly. Medication to minimize the discomfort during the experiment was given at the indication of the institute's veterinarians. Pain relief medication consisted of buprenorphine (Temgesic; an opiate). Ulcerative skin lesions developing at the immunization sites were sprayed daily with disinfectant wound spray (Acederm) to prevent further contamination.

Prophylactic treatment with a promising compound can result in a marked reduction of the clinical score, signifying improved clinical wellbeing, as was recently described for a low-molecular-weight CCR5 antagonist 31.

All these markers can be used to evaluate various aspects of the disease, allowing us to differentiate between disease-modifying drugs affecting bone degradation or therapies affecting inflammation.

The variety of intervention studies performed in the past decade have provided insight into the pathogenic mechanisms operating in the rhesus monkey CIA model. As proposed for RA by Choy and Panayi 1, we like to distinguish three phases in the etiopathogenesis of the CIA model (Fig. 4).

In the later stages of the disease, the cartilage is severely damaged, requiring repair of the damage for restoration of function. A possible strategy to treat this condition is the introduction of a matrix that provides a scaffold for chondrocytes and that stimulates the regeneration of the cartilage. The main obstacle will be the introduction of such a matrix under inflammatory and destructive conditions. Conceptually, the regenerating substrate not only provides a scaffold for rebuilding the cartilage but also provides immunomodulatory signals that help to restore homeostatic mechanisms maintaining tolerance to joint antigens. Implantation of a tolerogenic collagen matrix, for example, would obviate the need for further immunosuppressive regimens.

A still-unresolved question is why the inflammation in RA is chronic. We have postulated a model in which the dendritic cell (DC) plays a pivotal role in governing the response to released self-antigens. The reasoning is that DC maturation is regulated by the interaction of C-type lectin receptors (CLRs), binding glycan epitopes on self-antigens and pathogens, and Toll-like receptors (TLRs), which recognize conserved molecular patterns on pathogens 47. This notion has led to the Yin-Yang hypothesis for the regulation of autoimmunity and tolerance by DCs 48. In the concept, testable hypotheses were formulated for the maintenance of tolerance to self-antigens in a resting immune system and the induction of reactive or chronic inflammation by infection. The three depicted paradigms mentioned below describe extreme situations. In clinical reality, subtle variants may occur.

One paradigm, the tolerance paradigm, postulates that in a resting immune system, immature DCs present in lymhoid organs, and their equivalents in the joint, such as the type A synovial lining cells, continuously sample glycoproteins released from the normal tissue turnover via their CLRs. When presented in the absence of DC maturation signals, T cells recognizing the presented glycoproteins attain a regulatory function. By this mechanism, the nonresponsiveness of the immune system is maintained. This mechanism may explain why a pig collagen matrix implanted into the knee joint of a healthy rhesus monkey does not evoke anticollagen autoimmunity (our own unpublished observations). In addition, the robust tolerance that is induced when rhesus monkeys are pretreated with attenuated collagen in incomplete adjuvant – that is, in the absence of a TLR ligand – may be explained by this model. Interestingly, the observation that rats injected with T cells from rats immunized with attenuated CII produced lower anti-CII antibody levels suggests a regulatory function of the transferred cells 41.

The infection paradigm postulates that disturbance of the CLR/TLR balance by a viral or bacterial infection induces DC maturation. Consequently, self-antigens sampled by the DCs will be presented in the context of costimulatory signals expressed by mature DCs and induce Th1 cell activation. Immunohistochemical analysis of the arthritic joints of RA patients reveals that high quantities of the TLR2/Nod1,2 ligand peptidoglycan are present in the arthritic joint 49. Peptidoglycan is arthritogenic by itself in susceptible rodent strains and can enhance a specific autoimmune reaction to central nervous system myelin, giving rise to monophasic autoimmune encephalomyelitis 50. Importantly, when the TLR stimulus is cleared, the CLR/TLR balance is restored, and tolerance can be restored by the induction of regulatory T cells (Treg) specific for joint components. This paradigm could explain why in monkeys that have recovered from CIA it is almost impossible to induce exacerbation of the arthritis by a second immunization with bovine collagen type II 2651.

Another paradigm is that of altered glycosylation. Glycosylation is the most important post-translational modification of secreted proteins, which are expressed on the cell membrane or part of the extracellular matrix. Glycosylation of self-glycoproteins is not constant, but varies with time (ageing) and place (organ-specific glycosylation). Moreover, the normal glycosylation patterns can change under pathological conditions that cause stress to cells, such as infection, or on exposure to certain hormones or cytokines 5253. The disturbance of the normal glycosylation under these conditions may affect the restoration of the delicate CLR/TLR balance after clearance of the TLR ligand and thus impair remission of the inflammation. RA is one of the clinical disorders in which abnormal glycosylation of self-antigen (agalactosyl IgG) has been suggested as a cause of autoimmunity 54. It was shown in a mouse CIA model that the arthritogenic and tolerogenic capacity of CII depends on the glycosylation of the immunizing autoantigen. We hypothesize that the disturbed glycosylation may not be confined to IgG but may also affect other self-antigens in the joint, such as CII.

CIA in rhesus monkeys is a very useful preclinical model of human arthritis, but it is suboptimal in a number of respects. First, the arthritis can be very severe. In addition, the large size of the monkeys, which weigh 6 to 10 kg at adulthood, is an advantage for blood collection or invasive techniques (arthroscopy). However, this implies that large amounts of test compound are required to observe a clinical effect. Another disadvantage of using CIA in rhesus monkeys is the aggressive nature of these monkeys, which usually have to be sedated for each handling, limiting the frequency of experimental interventions. Moreover, the model is usually monophasic, although in some cases exacerbation could be induced by booster immunization 26. Finally, because of the high susceptibility to mycobacterial components of adjuvant, rhesus monkeys develop severe ulcerating skin lesions where the CII/CFA inoculum is injected.

We have encountered similar phenomena with the rhesus monkey EAE model, which was developed as a model of multiple sclerosis 37. To overcome this problem, we have developed an EAE model in a New World nonhuman primate species, the common marmoset 55. This is a small animal (weighing approximately 400 g), which is ideal for efficacy testing in an early development phase of a new drug, when only limited amounts of the compound are available. The new EAE model appeard to lack all the negative aspects of the rhesus monkey EAE models and to represent the human disease multiple sclerosis much better. A unique aspect of marmosets is that they normally give birth to nonidentical twins or triplets, which, because they share the placental bloodstream, are complete and stable bone marrow chimeras. The resulting allo-tolerance between twins allows the exchange of cells and tissues without rejection. We expect that the same differences observed for the EAE models also hold true in CIA and have started to explore the susceptibility of marmosets to arthritis induction with bovine collagen type II. Whether the success of the marmoset model for multiple sclerosis can be reproduced in RA will become clear in the coming years.

AIA = antigen-induced arthritis; AP = alkaline phosphatase; CFA = complete Freund's adjuvant; CIA = collagen-induced arthritis; CII = collagen type II; CLR = C-type lectin receptor; CRP = C-reactive protein; DC = dendritic cell; EAE = experimental autoimmune encephalomyelitis; HP = hydroxylysylpyridinoline; HPLC = high-performance liquid chromatography; IFN = interferon; Ig = immunoglobulin; IL = interleukin; LP = lysylpyridinoline; MHC = major histocompatibility complex; OVA = ovalbumine; RA = rheumatoid arthritis; Th1 = T helper type 1; TIMP = tissue inhibitor of metalloproteases; TLR = Toll-like receptor.

The author(s) declare that they have no competing interests.