Current Rheumatology Reviews, 2008, 4, 277-287 277

1573-3971/08 $55.00+.00 2008 Bentham Science Publishers Ltd.

What Animal Models are Best to Test Novel Rheumatoid Arthritis
Therapies?
Paul H. Wooley
*

Orthopaedic Research Institute, Via Christi Healthcare System and Wichita State University, Wichita, KS, USA
Abstract: The relevance of animal models of rheumatoid arthritis and their relationship to the development of anti-
arthritic therapies is reviewed in depth. Different mechanisms for the induction of experimental arthritis, including
infectious processes, non-specific inflammation, autoimmune responses to cartilage components, and genetic
manipulation are discussed in context of pathological pathways relevant to rheumatoid arthritis. A variety of species,
including rats, mice, dogs, pigs, and monkey are examined for advantages and drawbacks in preclinical development of
anti-arthritic agents, and their capacity to mimic critical aspects of arthritis pathology. The history of anti-arthritic therapy
from non-steroidal anti-inflammatory agents to biological response modifiers is placed in context with the evolution of
animal models of arthritis. The potential of novel models based upon targeted gene manipulations and corresponding
pathway-specific drugs is examined as a new approach to identifying therapies relevant to rheumatoid disease.
Keywords: Adjuvant-induced arthritis, collagen-induced arthritis, rheumatoid arthritis, streptococcal cell wall arthritis,
transgenic mouse.
INTRODUCTION
The question of the best animal model of rheumatoid
arthritis (RA) has endured for decades, and is a pressing
issue for major pharmaceutical companies, start-up
biotechnologies, the FDA and basic researchers studying
the pathophysiology of arthritis. There is, of course, no
easy answer. First, no model truly resembles RA with
respect to the complete pathology, immunology, and
cellular biology of the human disease. Secondly,
rheumatoid arthritis is not a monolithic disease entity, but
rather a syndrome that varies widely from patient to patient
with no reliable indications of the disease etiology. The
many variations seen in clinical RA raise the question as to
whether we are actually investigating a single disease
entity, or a spectrum of signs and symptoms that may be
loosely grouped together as an inflammatory, erosive joint
disease with unknown origins. So the question must be
rephrased when a novel therapeutic strategy for RA is
considered for pre-clinical development. The query
becomes: Is the pathological pathway targeted for
therapeutic manipulation relevant to rheumatoid disease? If
so, which animal model is most relevant to this
pathological pathway?
RELEVANT PATHOLOGICAL PATHWAYS IN
ANIMAL MODELS OF RA
One useful feature of most animal models of arthritis is
that we do understand much of the underlying pathology of
the joint disease. These mechanisms can be considered in
four major categories:



*Address correspondence to this author at the Orthopaedic Research
Institute, 929 North St. Francis, Wichita, Kansas 67214, USA; Tel: 316
268 5486; Fax: 316 291 4998; E-mail: Paul_Wooley@Via-Christi.org

Infectious Arthritis and Reactive Arthritis
There are viruses, bacteria, and mycoplasma that have been
identified as causative agents in animal arthritis. Although an
infectious etiology for rheumatoid arthritis has not been
demonstrated, infectious and reactive arthritis are well
recognized in humans, and a pathogenic agent cannot be
completely ruled out in playing a role in RA. Second, the
immune response against pathogenic organisms can result in a
joint disease, termed reactive arthritis in patients. There are
several convincing models of this mechanism that have been
demonstrated in animals.
Autoimmunity to Joint Components
The most widely recognized example of this mechanism is
type II collagen-induced arthritis, although responses to
cartilage proteoglycans can provoke a similar joint disease.
These models do not arise spontaneously, but require
immunization using adjuvants to provoke an aggressive
immunological response to cartilage components.
Non-Specific Inflammatory Stimulants and Immune
Reactions
Adjuvant-induced arthritis (AIA) and pristane-induced
arthritis (PIA) are the classic models related to inflammatory
stimuli. Adjuvant arthritis is essentially confined to rats, and
represented the ideal model for the development of COX
inhibitors. The extreme sensitivity of the model to COX
inhibitors can be argued as a weakness of this model: since this
compound class can cure the rat arthritis, but not the human
condition, it is likely that the overall mechanism in AA is
distinct from RA. Animals with PIA exhibit a broad range of
autoimmune responses, which is a curious sequel to a non-
specific stimulus, and therefore may also represent a model of
systemic lupus erythematosous. The mechanism by which
non-specific inflammation provoked by a mineral oil results in
rheumatoid factor, anti-collagen antibodies, and hyper-
gammaglobulinemia remains unclear. AIA is non-specific
278 Current Rheumatology Reviews, 2008, Vol. 4, No. 4 Paul H. Wooley
immune response within the joint can also result in
inflammatory, erosive disease. Induction of this model
requires systemic immunity established by repeated
immunization against a soluble antigen (typically a foreign
serum albumin), and then joint inflammation is induced by
intra-articular challenge with the antigen. Arthritis is
confined to the injected joint, and this model can be
established in most laboratory animal species.
Transgenic Manipulation of Genes Relevant to
Inflammatory and Immune Responses
Transgenic animals represent a powerful tool when
considering appropriate models for developing biological
response modifiers. The use of the TNF transgenic mouse
in the development of TNF inhibitors represented a
milestone in novel strategies of pre-clinical research. In
this instance, Keffer et al. [1] demonstrated that
unregulated production of TNF in transgenic mice resulted
in a realistic inflammatory joint disease. This was
accompanied by surprisingly little tissue inflammation in
the major organs, although the mice exhibited signs of
cachexia and died early in life. This model was ideal for
the pre-clinical development of anti-TNF therapies,
particularly infliximab, since treatment of the transgenic
mice with this anti-TNF construct also permitted an
extension of the lifespan and facilitated breeding. Several
other transgenic mouse strains have exhibited joint disease,
and some might not have been predicted to exhibit signs of
arthritis from the genetic material under modification.
Notably, the K/BxN transgenic strain fits this category.
This category could also consider the chimeric model
established by surgical implantation of RA tissue into
immunodeficient animals. Although this does not provide a
model of an arthritic joint, it does represent a potential
model to examine of drug effects in viable human synovial
tissue.
SELECTION OF AN APPROPRIATE ANIMAL
SPECIES
In general, the FDA prefers the demonstration of pre-
clinical anti-arthritic effects in two animal species prior to
NDA approval. With the proliferation of murine collagen
arthritis and transgenic mouse strains tailored to the
proposed mechanism of action, it is common that mice will
occupy one of the requisite species. It is then considered
preferable to avoid rats (due to the overwhelming
similarities in biology, toxicology, etc) as a second species,
which can lead to a difficult choice to fulfill the
development requirement. Ideally, primates would serve as
the closest relevant species to humans, and if cost
containment presents no obstacle, primate models are
available. Squirrel monkeys, rhesus monkeys and cerebus
monkeys have all been reported to develop collagen-
induced arthritis [2, 3], and the resemblance to the patho-
logy of RA is fairly good. The systemic reaction to
immunization in primates (which may be related to the use
of complete adjuvant rather than actual autoimmunity to
type II collagen) appears to be very severe, and early
reports contained descriptions of debilitating and cachetic
effects. Despite these initial difficulties, and the fact that
primate use is frequently prohibitory expensive and a
minority of animal facilities can serve as primate centers, there
are several successful reports of primate use in novel therapy
development. Brok et al. [4] reported on the trial of
Daclizumab (a humanized monoclonal antibody against the -
chain of the CD25 IL-2 receptor) using CIA in rhesus
monkeys. When Daclizumab treatment was commenced before
the onset of CIA, a significant reduction of inflammation and
joint erosion was observed. In a therapeutic protocol, joint-
degradation was abrogated, and the overall trial indicated
positive pre-clinical findings. Yamamoto et al. [5] recently
reported on the treatment of CIA in cynomolgus monkey using
a chimeric anti-osteopontin antibody. Blocking of the
interaction of a cryptic epitope within osteopontin with its
integrins has been show to inhibit CIA in mice, and a murine
monoclonal antibody specific for a cryptic epitope of human
osteopontin, fused with human IgG1 constant region (C2K1)
was shown to ameliorate established collagen-induced arthritis
in cynomolgus monkeys. Inhibition of joint swelling by C2K1
was observed at 4 to 5 weeks after onset of arthritis,
coincidental with peak blood levels of C2K1. J oint swelling
reappeared following a sharp decline of C2K1 blood levels at
6 weeks. However, erosion of bone and cartilage in joints was
still significantly reduced, and this novel approach to therapy
appears promising. Vierboom et al. [6] used CIA in rhesus
monkeys to test a small molecular weight antagonist of CCR5
(SCH-X). Treatment was initiated on the day of CIA induction
and continued for 45 days. Monkeys were monitored for signs
of clinical arthritis and markers of inflammation and joint
degradation. Two of five animals in the SCH-X-treated group
displayed prominent soft-tissue swelling, compared with all 5
saline-treated monkeys, and SCH-X treatment resulted in an
suppression of the C reactive protein acute-phase reaction, a
reduction in the excretion of cartilage breakdown products and
blocking of the erosion of joint cartilage: Overall, the use of a
CCR5 antagonist appears to have potential for treatment of
active RA. Saito et al. [7] have used cynomolgus monkeys
with collagen-induced arthritis to examine the tissue
distribution of R-125224 (a humanized anti-human Fas
monoclonal antibody), and discovered a high signal in the
bone marrow, thymus, lungs, liver, adrenals, spleen, ovaries,
axillary lymph node and mesenteric lymph node compared to
the signal in plasma. Although the experiment demonstrated
that R-125224 cross-reacted with the monkey Fas antigen, the
influence on arthritis was not the focus of this study.
Feasibility studies for gene therapy in primate CIA have also
been published by Bessis et al. [8]. In addition to the close
genetic relationship of primates to humans, the physical size of
most monkeys is a considerable advantage if an intra-articular
injection is the most pertinent mode of administration. In this
report, cell cultures of skin fibroblasts were transfected with a
plasmid carrying the lacZ gene, and then injected into
metacarpophalangeal, metatarsophalangeal, and interphalan-
geal joints. Synovial tissue X-gal labeling showed significant
reporter gene activity in transfected cells up to 6 days after
intra-articular injection. These are clearly early indications,
and considerable work remains to perfect a sustained but
controllable vector expression system to develop an anti-
arthritic gene therapy approach. Primates have also been
employed to address potential side effects of biological
response modifiers, since the capacity to develop an adverse
immune response against the protein therapeutic (however
well engineered) remains a constant problem. Rojas et al. [9]
Novel Rheumatoid Arthritis Therapies Current Rheumatology Reviews, 2008, Vol. 4, No. 4 279
used cynomolgus monkeys to assess the in vivo formation,
distribution, and elimination of immune complexes
resulting from a low-level immune response to Infliximab
(IFX), a chimeric IgG1 monoclonal antibody to TNF.
They observed a rapid formation of immune complexes
comprised of IFX and radiolabeled anti-IFX IgG antibody,
with a terminal half-life of approximately 38 hours
compared with 86 hours for a non-immune antibody
control. There were no macroscopic or microscopic
histological findings, but the results indicate that immune
complexes between IFX and anti-IFX IgG antibodies do
form in vivo. This experiment was not conducted during
the treatment of primate CIA with IFX, and therefore may
not accurately represent the risk of a response to the
biological response modifier during an ongoing
autoimmune disease. However, the study illustrates the
potential of this approach for risk assessment, as well as
therapeutic evaluation, using animal model systems.
Given the desire for a non-rodent model of arthritis
with a large synovial joint, it is perhaps surprising the
porcine models have not been thoroughly investigated,
particularly since mini-pigs and micro-pigs are attractive
alternatives to large animal husbandry [10]. Beyond one
early description of carrageenin-induced arthritis in the
SPF pig [11], reports on porcine arthritis have been
confined to infectious joint disease [12, 13] and no
commonly accepted model of rheumatoid disease has
emerged. Caprine encephalitis/arthritis due to lentiviral
(CAEV) infection [14, 15] is an intriguing model that has
undergone steady investigation for two decades, but studies
are currently focused upon the molecular mechanisms
underlying the viral activity [16, 17]. Although there are no
current publications on the effects of anti-arthritis therapies
in this model, there are reports that indicate the
participation of TNF [18], IL-16 [19] and nitric oxide
[20] in the pathology of CAEV arthritis.
The existence of canine rheumatoid arthritis (CRA) has
been recognized for decades [21, 22], but is considered to
arise spontaneously and no breed of RA susceptible dog
has been established for experimental purposes. Ohashi et
al. [23] have shown that antigen-induced arthritis may be
elicited in the joints of beagles sensitized and challenged
intra-articularly with bovine serum albumin, but this model
has not been further developed for pre-clinical use. Early
studies of anti-arthritic therapy of spontaneous canine RA
made an important observation; while cytotoxic agents are
effective in canine RA, COX inhibitors are contraindicated
due to safety concerns [24]. The presence and significance
of canine rheumatoid factor remains controversial,
although the preponderance of publications suggested that
most canine RA is seropositive [25-28]. Inflammatory
cytokines appear to contribute to the joint disease [29],
elevated levels of MMP-9 [30], MMP-3 and TIMP-1 [31]
in arthritic joints have been described, and antibodies to
both type II collagen [32] and heat shock proteins [33]
have been reported. The immunohistochemistry of the
rheumatoid joint depicts an accurate model of human RA.
Hewicker-Trautwein et al. [34] reported that synovial
membrane biopsies from dogs with CRA exhibited
numerous diffusely and perivascularly distributed CD5
+

lymphocytes in the subsynovium, with CD4
+
cells
outnumbering CD8
+
cells in the perivascular areas. CRA
resulted in higher numbers of / TCR
+
cells compared with
/ TCR
+
cells, and a marked increase of MHC class II antigen
expression was noted, with 50% to 90% of the synovial lining
cells strongly MHC class II
+
. Overall, CRA appears to
resemble human RA to a great degree. No structured pre-
clinical trials of anti-arthritic agents have been reported in
CRA, and although the model appears both accurate and
relevant, it would difficult to design a trial given the low
frequency of CRA in the dog population, and the absence of an
available arthritis breed [24].
Rabbits may provide the best compromise between
reasonable joint size and readily available RA model systems
when intra-articular joint manipulations are required. For some
gene therapy approaches, it may be desirable to obtain
synovial cells for culture and ex vivo gene transfection, prior to
re-implantation within the synovial cavity to evaluate the
therapeutic benefits. The ex vivo approach provides useful
information on transfection efficacy, transgene stability, and
the rate of target protein production. The rabbit represents the
animal with the minimum donor joint size that can provide a
reasonable number of synovial cells upon harvest, with strains
that exhibit a sufficient degree of inbreeding to alleviate
allograft rejection upon re-introduction to a recipient. The
majority of researchers utilize AIA in the rabbit, a well
documented technique usually employing immunization and
intra-articular challenge to mBSA [35, 36], although
inflammatory arthritis has been elicited in the rabbit joint via
the intra-articular introduction of human gliostatin - platelet-
derived endothelial cell growth factor [37], transforming
growth factor -2 [38], recombinant human IL-8 [39, 40],
Phospholipase A2 (PLA2) enzymes [41] and lipopoly-
saccharide (LPS) [42]. A recent evaluation of rabbit AIA has
shown a significant concordance between the genes typically
seen activated in rheumatoid synovial tissue and joint tissues
from rabbits with AIA [43]. Four days after disease induction
rabbits were injected with glucocorticoids and 24 hours later
synovial tissue, menisci, and cartilage from the knee were
collected. Changes in mRNA levels were assessed using
reverse transcription-polymerase chain reaction with primers
for collagen I, collagen II, biglycan, decorin, matrix metallo-
proteinases-3 and -13 (MMP-3 and MMP-13), COX-1 and
COX-2, TNF, IL-1, inducible nitric oxide synthase (iNOS),
hyaluronan synthase-2 (HAS-2). While most genes were up-
regulated in arthritic tissues, iNOS, HAS-2, COX-1 were
down-regulated. Gene activities of collagen II, MMP-3, and
MMP-13 were further down-regulated by glucocorticoids in
both normal and arthritic tissues, but other genes showed a
variety of responses. Overall, the gene activity pattern
appeared promising for the evaluation of arthritic events, and
the ability to use gene array analysis suggests that this type of
approach may permit detailed analysis of the pathways
involved in novel therapeutics. Another advantage of the rabbit
in RA drug development is the potential for the use of novel
formulations that require accurate delivery of a reasonable
volume of material within the synovial cavity. The rabbit knee
has been recently employed for testing liposome, niosome,
lipogelosome and niogelosome formulations of diclofenac
sodium [44], and the size of the joint permitted drug retention
analysis by radiolabeling with Tc-99m and gamma
scintigraphy. The scintigraphic imaging studies revealed that
radiolabelled lipogelosome formulation containing DF-Na
retained much longer in the experimentally arthritic knee joints
280 Current Rheumatology Reviews, 2008, Vol. 4, No. 4 Paul H. Wooley
of the rabbits, and appeared to result in improved efficacy.
Microsphere delivery of methotrexate has been evaluated
using a similar model system [45], and this type of novel
technology may improve established therapeutics or permit
novel approaches by retaining the active mediator within
the joint space, thus both improving efficacy and reducing
the chance of unwanted systemic effects. While such a
technical approach is theoretically possible in mouse and
rat models, the maximum volume that may be injected into
a mouse knee using a Hamilton needle and 27g needle that
does not result in significant leakage (personal experience
using contrast medium) is 25 l. It would be predicted that
the rat knee would retain ~100ul without significant
leakage. Intra-articular injection is typically more difficult
to accomplish in normal mice compared with arthritic
animals, since the distended joints in the latter group
provide a larger target. However, the accumulation of
synovial fluid during joint edema probably increases the
rate of fluid loss from the injected arthritic joint. Due to the
limits of experimental accuracy of intra-articular injection
in the mouse, the term peri-articular injection is preferred
to describe the delivery of local factors or gene therapy to
the murine arthritis joint [46].
Rabbits also provide an opportunity for minimally
invasive procedures in the arthritic joint. Beischer et al.
[47] examined a technique for ablating inflamed synovium
in joints using photo-dynamic therapy. The photosensitizer
Haematoporphyrin Derivative (HpD) was injected into
periarticular tissues, and pharmacokinetic studies revealed
that inflamed synovium accumulated HpD. Activation of
HpD by intra-articular light administration resulted in
selective ablation of the inflamed synovium. Histological
examination of the knees indicated that selective
destruction of inflamed synovium was achieved at light
doses 100 joules/cm2 and above. Photodynamic therapy in
rabbit AIA was also utilized by Bagdonas et al. [48], who
demonstrated the accumulation of endogenously produced
porphyrins after administration of exogenous 5-amino-
levulinic acid (ALA), with the highest fluorescence
intensity of endogenously produced porphyrins occurring
in the tissues of the inflamed joints. The authors concluded
that this observation supports the application of
photodynamic therapy for the treatment of arthritis and the
use of optical non-invasive methods based on fluorescence
detection of endogenously produced porphyrins for
diagnostics of inflamed tissues. It appears that the rabbit
model does provide an opportunity to model these types of
techniques, which would be extremely challenging in mice
and rats. This is supported by the studies of radiation
synovectomy in the AIA rabbit model [49], where a
rhenium-188 (188Re)-tin colloid improved the macro-
scopic appearance and reduced the knee joint diameter,
which was consistent with a reduction of the histological
score compared to untreated arthritic controls. Yao et al.
[50] used rabbit AIA to demonstrate that intra-articular
injection of an adenoviral vector expressing human tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL)
stimulated synovial apoptosis and reduced inflammation.
Gene transfer of TRAIL induced apoptosis in cells within
the synovial lining, reduced leukocytic infiltration and
stimulated new matrix synthesis by cartilage. This study
[51] was extended using a rabbit arthritis model induced by
intra-articular introduction of allogeneic fibroblasts genetically
engineered to secrete human IL-1. This novel approach to
engineering a pathway-specific arthritis appears highly useful,
and demonstrated that recombinant chimeric human TRAIL
protein (rTRAIL) also induces apoptosis of proliferating rabbit
synovium and reduces inflammation. Apoptosis of the
synovial lining was demonstrated using TUNEL analysis, and
leukocyte infiltration into the synovial fluid of the inflamed
knee joints was reduced by more than 50% following rTRAIL
treatment. Apoptosis induction in the rabbit AIA model has
also been investigated by Mi et al. [52] using a synovial-
targeted transduction peptide, HAP-1, which is able to
facilitate specific internalization of protein complexes into
human and rabbit synovial cells. HAP-1 and a non-tissue-
specific cationic protein transduction domain, PTD-5, were
fused to an antimicrobial peptide, (KLAK) [2], to generate two
pro-apoptotic peptides termed DP2 and DP1. Intra-articular
injection of these peptides into arthritic rabbit joints induced
apoptosis of the hyperplastic synovium, and reduced cellular
infiltration and synovitis. These publications and other papers
indicate that human and rabbit key mediators of inflammation
do share sufficient homology to permit effective testing.
Podolin et al. [53] examined the effects of a non-peptide
antagonist of human CXCR2 that inhibits human IL-8 binding
to, and human IL-8-induced calcium mobilization mediated
by, rabbit CXCR2 (but not rabbit CXCR1). They concluded
that that the rabbit was an appropriate species to examine the
anti-inflammatory effects of human CXCR2-selective
antagonists, and investigated anti-arthritic activity using
experimental arthritis induced by knee joint injection of human
IL-8 or LPS, in addition to the AIA model. The antagonist
significantly reduced synovial fluid neutrophils, monocytes,
and lymphocytes, and levels of the soluble factors TNF, IL-8,
PGE
2
, leukotriene B
4,
and leukotriene C
4
.
Despite the overall promising use of rabbit AIA in recent
studies, the model also illustrates some of the difficulties of
pre-clinical anti-arthritic drug development. Marcinkiewicz et
al. [54] investigated the therapeutic potential of taurolidine in
various experimental arthritis models of synovitis.
Intraperitoneal treatment with taurolin failed to control the
development of CIA in mice with high serum level of IgG
anti-CII, but intra-articular application of 2% taurolin resulted
in amelioration of AIA in all rabbits with a reduced swelling
and SAA levels as compared to the control animals.
Histopathologic evaluation revealed extensive necrotic lesions
of synovial tissue treated with taurolin, suggesting a
synoviorthesis-like effect. This paper demonstrates that not
only that the appropriate model selection may be key to the
evaluation of an anti-arthritic agent, but also the formulation
and delivery can be critical in the trial outcome.
SELECTION OF THE APPROPRIATE ANIMAL
MODEL
The majority of potential anti-arthritic therapeutic
strategies today are driven by targeted mechanism of action
approaches at the molecular level. This stands in stark contrast
to the methods employed two decades ago, when the majority
of arthritis drugs were developed by major pharmaceutical
companies using classical medicinal chemistry, biochemistry
and animal testing. Rheumatoid arthritis was strictly
considered as an autoimmune disease (which it still is), and
Novel Rheumatoid Arthritis Therapies Current Rheumatology Reviews, 2008, Vol. 4, No. 4 281
immunosuppressive agents were the basic class of
compounds for preclinical testing. However, there was a
major weakness to this strategy since the majority of the
compounds under consideration for development were the
byproducts of anti-cancer programs. There has never been
sufficient evidence to conclude that dose modification of
anti-tumor agents would yield a compound capable of both
inducing remission in active rheumatoid arthritis, and then
being capable of maintaining remission with a low side-
effect profile over years of chronic administration. It could
be successfully argued that this strategy did yield
methotrextrate as a highly effective ant-arthritic agent, but
this drug can hardly be considered as side-effect free with
chronic administration, and requires considerable patient
management on behalf of the rheumatologist. The MOA
approach to drug development is not without problems
however, particularly with respect to the complex nature of
the human disease. In some respects, the more specific the
pathway antagonist leveled at RA, the greater the chance
that it will only work in a minority of patients. The history
of drug development in the available models may provide
useful indications for future drug development.
INFECTIOUS ARTHRITIS AND REACTIVE
ARTHRITIS
The selection of an infectious model for preclinical use
in RA may imply the belief that RA is an infectious
disease, a stance that has been highly controversial for
many years [55-58] although recently supported by some
DNA analysis studies [59-60]. Further, as Wang et al. [61]
observed studies directed at the pathogenesis of infectious
disease have twice led to the identification of an
endogenous mediator of arthritis. In this paper, they
promoted HMGB1 (a 30-kD nuclear and cytoplasmic
DNA-binding protein which is a necessary and sufficient
mediator of lethal sepsis) as a mediator of arthritis because
it is produced at the site of joint inflammation, it causes the
development of arthritis when applied to normal joints, and
therapies that inhibit HMGB1 prevent the progression of
collagen-induced arthritis in rodents. However, the use of
infectious models to develop agents for RA is uncommon,
although a wide variety of organisms may induce joint
disease in a broad range of animal species, including
rabbits, dogs, pigs, goats, rats, mice and hamsters. Most
models employ intra-articular inoculation to establish
disease, although some infectious agents may be
administered intravenously or via other extra-articular
routes to induce arthritis. The infectious agent may be
trophic for the joint due to expression of adhesins or other
molecules that promote sequestration within synovial
tissues [62], or promote selective migration of T cells to
synovial tissue [63]. Staphylococcal species produce a
range of receptors for connective tissue components,
including collagen and fibronectin. Chronic, persistent
infection of the joint can generate excessive local antigenic
stimulation, resulting in inflammatory and immune
complex-mediated reactions. Bacterial cell wall and
mycoplasma membrane components may also provide
nonspecific immune activation through mitogenic activity
[64, 65], and could provide a useful model for strategies
designed to target Toll receptor dependant mechanisms.
Viral infection of synovial cells, as seen in the CAEV
model, may elicit novel cell surface antigens or abnormal
expression of activation antigens and the overproduction of
autoantigens such as Hsps. Viral infection may also disrupt
immune regulation, increasing pro-inflammatory cytokines
and immune activity against the normally immunologically
privileged components of the joint. The histologic and
radiologic features of CAEV are characterized by progressive
lesions consisting of membrane villus hypertrophy with
extensive angiogenesis and mononuclear cell infiltration, and a
progression to marked erosive changes. J oint disease is
accompanied by an immune response to viral surface and
envelope glycoproteins, chronic B cell activation, and the
accumulation of activated macrophages and CD8
+
T-cells
within the synovium.
Experimental Staphylococcus aureus (S. aureus) arthritis in
mice, rats and rabbits illustrates a number of relevant pathways
in joint disease [66]. This organism can induce an exudative
arthritis in one or more joints, with extensive destruction of
articular cartilage and subchondral bone. The initial features of
S. aureus arthritis are synovial infiltration by polymorpho-
nuclear leukocytes (PMNs) and endothelial-like cells, followed
by extensive synovial hypertrophy with marked proliferation
by macrophage and fibroblastic cells and infiltration by B and
T-cells. Macrophage production of TNF and IL-6 appear to
be critical to the regulation of the cellular influx, due to effects
upon adhesion molecules and expression of chemokines [67].
Granulomatous tissue with bacterial foci are frequently present
in the joint, and extensive cartilage and bone destruction
occurs. S. aureus is usually recovered from the joint, and the
virulence factors that mediate arthritis may include the profile
of capsular polysaccharides, the production of adhesins and the
secretion of endotoxins with superantigenic properties (the
capacity to activate a broad range of T-cells by direct binding
to the V portion of the T-cell receptor rather than by
conventional antigen presentation). Superantigen involvement
in arthritis is also implicated in mycoplasma (M. arthritidis)-
induced arthritis in mice. T cell activation by the mycoplasma
superantigen appears crucial to the development of arthritis,
and may influence disease through cytokine activation and
skewing of the normal TH1/TH2 relationship [50, 68].
Persistence of antigens from micro-organism within the
joint appears to be critical for the induction of arthritis,
particularly for reactive arthritis models. Streptococcal cell
wall (SCW) arthritis in rats is induced by an aqueous
suspension of cell wall fragments injected intraperitoneally in
susceptible rat strains. A chronic, erosive polyarthritis results,
with a course of remissions and exacerbations, a feature rarely
observed in other animal models [68, 69]. The initial arthritis
occurs rapidly, with acute inflammation in rear paws peaking
approximately 4 days post-immunization. This inflammation
recedes, but chronic arthritis recurs between 2-4 weeks, with
synovitis, pannus formation and erosion of cartilage and bone.
Cycles of remission and exacerbation may persist over several
months and culminate in joint fibrosis and ankylosis. The
initial acute arthritis is T-cell independent, but requires an
intact alternative complement pathway. In contrast, the chronic
phase is complement independent, but mediated by T-cell-
dependent mechanisms. Disease exacerbations in SCW
arthritis may be provoked by the use of several pro-
inflammatory cytokines (IL-1, TNF, and -IFN), although
IL-2 alone does not mediate a flare reaction. SCW arthritis has
been used to test several novel therapeutic approaches,
282 Current Rheumatology Reviews, 2008, Vol. 4, No. 4 Paul H. Wooley
including a secretory leukocyte protease inhibitor (SLPI)
that shares the protease-reactive site found in human SLPI
[57], selective inhibitors of p38 MAP kinase [58, 70], and
N-butyryl glucosamine [61].
CARTILAGE AUTOIMMUNITY MODELS
Collagen-induced arthritis has essentially become the
standard pre-clinical development model for anti-arthritic
therapies during the last fifteen years. The MHC restricted
reaction to the major joint protein resulting in autoimmune
joint disease is intellectually attractive, and closely
resembles many aspects of RA. T cell directed immunothe-
rapy was initiated when cyclosporin was shown to prevent
the onset of arthritis and slow the progression of
established disease [71]. Targeting specific T cell subsets,
based upon observations of skewing of T cell poplations in
affected joints [72] has produced inconsistent findings.
Immunization agaist V peptides using BUB mice elicited
antibodies reactive with the self-TCR and prevented the
induction of collagen-induced arthritis by eliminating or
downregulating pathogenic T cells [73], and antibodies to
specific V subsets have also been successful in CIA [74,
75], although this finding is not universal [76, 77].
However, CIA was critical for the development of cytokine
inhibitors for clinical use in RA. Inhiibtion of IL-1 using
IL-1 receptor antagonist protein (IRAP or IL-1ra) [78] or
antibodies to IL-1 [79] was successful in collagen-
induced arthritis, and inhibition of TNF using soluble
TNF receptor, recombinant TNF receptor: human Fc fusion
protein, or antibodies to TNF all yielded suppression of the
clinical aspects of CIA [80-82]. These preclinical findings
contributed to the successful development of anti-cytokine
therapy for RA [83-85], which has now become the
accepted standard of care. Since the delivery of most
cytokine inhibitors raises several practical problems and
site injection complications, gene therapy is commonly
evaluated in CIA [86]. Systemic delivery of a TNFR gene
via adenoviral vectors was shown to reduct the severity of
collagen-arthritis in rats, but was limited in intra-articular
use due to the pro-inflammatory nature of the vector [87].
Systemic delivery of chimeric human p55 TNFR-IgG
fusion protein via adenovirus vector to mouse CIA had a
short-term beneicial effect [88], and again may have been
limited by vector related issues. Recent approaches using
the electrotransfer of plasmids encoding cytokine inhibitors
[56, 59] and intranasal delivery delivery [89] have
circumvented many of the inflammation-promoting
properties of the early vectors. It is surprising how readily
accomplished nasal delivery was in mice, who appear to
instinctively inhale small volumes of fluid placed on the
entrance to the nasal cavity. This tendency, and the lack of
a gag reflex to expel drugs delivered by oral gavage, are
highly useful when considering the appropriate method of
drug delivery. There have been several reports of anti-
arthtic effects in CIA using gene therapy to deliver IL-4
[90-92], and this strategy may have promise for the
treatment of RA. The injection of viral IL-10 via an
adenoviral vector carrying the IL-10 gene into paw
suppressed the development of collagen arthritis and
reduced the progression of disease to non-injected paws.
Anti-heterologous CII antibodies were unaffected, but IL-
10 suppressed autoantibodies to murine type II collagen
[93]. Vectors containing the murine IL-18 binding protein
isoform c gene [94], IL-10 [95], and IL-1ra [96] have been
demonstrated to be effective following intra-articular injection
using CIA. Gene therapy has extended beyond cytokine
inhibition in the CIA model to address specific T cell based
therapy. Antigen-specific T cell mediated gene therapy [97]
using T-cell hybridomas specific for type II collagen and
transduced to express an IL-12 antagonist were tested in
collagen immunized DBA/1 mice. The cell transfer was
without effect on systemic T and B cells responses to CII,
although antigen-elicited IFN 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. Recently, TCR / genes
from an arthritic paw resident T cell clone autoreactive (but
not specific for type II collagen) were tested for therapeutic
effects. TCR genes from transduced T cells accumulated in the
inflamed paw, and injection of cells cotransduced with the
TCR / genes and soluble TNFR-Ig genes resulted in a
significant suppression of CIA with reductions in TNF, IL-
17, and IL-1 expression [98]. Thus, it appears that CIA still
repesents an excellent overall model for new drug
development.
ARTHRITIS MODELS PROVOKED BY NON-
SPECIFIC INFLAMMATORY STIMULANTS AND
IMMUNE REACTIONS
Although adjuvant arthritis is usually associated with the
development of COX inhibitors, it remains a viable model for
novel anti-arthritic therapy [99]. In particular, AA serves as a
model for the role of heat shock proteins (HSP) in autoimmune
arthritis. HSPs are potential targets for regulatory T cells due
to over-expression in inflamed joints, and immunization
against hsp affords protection against the induction of AA
[100]. Cross-reactivity to microbial HSP has been promulgated
in the pathology of RA, and an association between increased
expression of HSP and susceptibility to AA has been
demonstrated [101]. The injection of anti-CD134 liposomes
(directed against T cells that responded to the disease-
associated epitope of mycobacterial heat shock protein 60)
subcutaneously in the hind paws of AA rats targeted
CD4
+
CD134
+
T-cells in the popliteal lymph nodes and resulted
in the amelioration of the experimental arthritis. It would
therefore appear that AA represents the ideal model to address
pre-clinical strategies directed towards hsp based therapies,
including such approaches as DNA vaccines [102]. Several
other approaches have been recently evaluated in AA,
including autologous stem cell transplantation [103], gene
therapy based upon artificial chromosome vector systems
[104], CCR-5 antagonists [105] and T
reg
cells (CD4
+
CD25
+
T-
cells). Overall, although AA is a somewhat old fashioned
model of RA, it possesses several autoimmune pathways that
are current for drug development. Pristane-induced arthritis,
despite its unique autoimmune profile elicited by a non-
specific entity, MHC associated susceptibility and excellent
pathology, remains somewhat underused in pre-clinical
development. This is probably due to the long latency period
for arthritis development, although it could be argued that this
drawback accurately resembles rheumatoid disease. Despite
the lack of popularity compared with CIA, PIA has been
successfully used to evaluate anti-arthritis strategies. These
include antibodies to CD4 and CD8 [106], anti-TNF [107],
Novel Rheumatoid Arthritis Therapies Current Rheumatology Reviews, 2008, Vol. 4, No. 4 283
immunization of mice with an immunodominant epitope
from heat-shock protein 65 (hsp 65) [108], antibo-dies to
Oncostatin M [109] and an extensive evaluation of
prednisolone, methotrexate, three nonsteroidal antiinfla-
mmatory drugs (celecoxib, diclofenac, and indomethacin),
a p38 MAPK inhibitor, SB242235, and human soluble
TNF receptor (sTNFR; etanercept) and murine sTNFR
fusion proteins [110].
As discussed in the previous section, AIA remains a
useful model that can be controlled from the standpoint of
joint selection, which can afford some advantages. A
recent study [111] has investigated the influence of the
phosphodiesterase (PDE) IV inhibitor rolipram, which
resulted in the inhibition of early AIA with downregulation
of OVA-specific splenocyte proliferation and decreased
IFN-gamma release, but no effects on IL-10, or levels of
anti-OVA IgG, IgG2a, and IgG1. Other investigations have
examined the effects of chondromodulin [112], the natural
weak androgen dehydroepiandrosterone [113], and human
anti-C5 single-chain Fv (scFv) [114].
ARTHRITIS MODELS FROM TRANSGENIC
MANIPULATION OF GENES RELEVANT TO
INFLAMMATORY AND IMMUNE RESPONSES
The capability of gene knockout (or positive
deregulation) to evaluate the effects of a specific pathway
on the susceptibility or resistance to arthritis is a useful tool
for evaluating potential pathway specific inhibitors for
arthritis. A number of transgenic mouse lines sponta-
neously develop arthritis, including mice transgenic for the
env-pX region of the human T-cell leukemia virus (HTLV)
type 1 genome, which develop a spontaneous chronic
arthritis due to T cell responses to the expression of the tax
gene [115, 116], knock-in mice (gp130DeltaSTAT/
DeltaSTAT), in which all STAT-binding sites were deleted
from their gene encoding gp130 but binding sites for both
J anus kinases (J AKs) and for the protein tyrosine
phosphatase-2 (SHP-2) were preserved [117], and mice in
which the src homology 2 domain-bearing protein tyrosine
phosphatase binding site of gp130, tyrosine 759, was
mutated to phenylalanine [118]. These models have not
been widely disseminated for use in drug development.
There are two major tactics that have been employed using
transgenic mice:
1) Examination of the transgene effect on collagen-
induced arthritis, including the engineering of hybrid mice
that express human MHC antigens [119] and, 2)
investigation of arthritis in an established transgenic line
using passively transferred arthritis. For the latter
approach, the development of an effective antibody for the
elicitation of arthritis [120] was a byproduct of a
serendipitous experiment that yielded an interesting murine
model of RA. The K/BxN T cell receptor mouse model
appeared when the KRN TCR trangenic mouse was
crossed with the NOD (non-obese diabetes) mouse [121].
The progeny from this cross developed a spontaneous,
symmetrical arthritis in peripheral joints with a pathology
closely resembling RA. Histological features include
synovial inflitration, pannus formation, erosive disease and
bone remodeling. The critical autoimmune reactivity
resides in the specificity of the KRN V6 T cell receptor.
Although initially characterized as reactive with a bovine
ribonuclease peptide in context of the MHC antigen I-A
k
, the
KRN TCR responds to glucose-6-phosphate isomerase (G6PI)
in context of the NOD-derived I-A
g7
class II MHC molecule.
This immune recognition gives rise to anti-G6PI autoanti-
bodies, which appear to be key in the development of joint
disease, since the passive transfer of these antibodies can result
in joint disease. The K/BxN model has be used directly for
therapeutic development, as illustrated by successful treatment
with an angiogenesis inhibitor [122]. However, the use of
K/BxN serum to passively transfer arthritis has been used to
investigate the effects of other transgenes, and Choe et al.
[123] reported that neither IL-1R nor MyD88-deficient mice
developed detectable arthritis after transfer of K/BxN sera, and
that serum transferred arthritis was not sustained in TLR-4
mutant mice.
The generation of transgenics on a CIA susceptible
background requires considerably more time and effort, but
has the advantage of examining the entire profile of the
arthritogenic response. B10.Q.IL-10(-/-) mice were shown to
have increased CIA incidence and more severe arthritis than
B10.Q.IL-10 (+/-) littermates [124], suggesting that IL-10 may
exert an anti-arthritic effect. Using a similar approach,
transgenic mice that harbored the tumor necrosis factor-
stimulated gene 6 (TSG-6) under the control of the T cell-
specific lck promoter were shown to exhibit reduced
susceptibility to CIA [125], mice over-expressing intracellular
interleukin-1 receptor antagonist (icIL-1Ra1) or secreted
interleukin-1 receptor antagonist (sIL-1Ra) were resistant to
CIA induction [126], and DBA/1J mice with mutations of the
Perforin (pfp) molecule (DBA/1J -pfp-/-) were less susceptible
to CIA [127].
Although not strictly a transgenic model, the SKG mutant
strain [128] has recently emerged as a potential model for
novel therapeutic strategies. J oint disease occurs due to a
spontaneous point mutation of the gene encoding an SH2
domain of the signal transduction molecule ZAP-70 in T cells.
The mutation results in changes in the threshold of thymic
selection, permitting the survival of autoimmune T cells.
Erosive arthritis and joint infiltration occurs after 8-12 months,
and is predominantly mediated by CD4
+
T cells. The
autoimmune profile includes production of rheumatoid factors,
antibodies to type II collagen, hypergammaglobulinemia, and
antibody to hsp-70. Disease may be adoptively transferred by
T cells (but not antibody), and there appears to be an
environmental factor as gnotobiotic mice do not develop
arthritis [129]. Overall, the representation of the broad aspects
of RA by a point mutation appears to represent a powerful tool
for the analysis of RA, and although cytokines appear to
contribute to the pathology [130], no therapeutic studies have
been published in this model to date.
However, transgenic mice can also illustrate some of the
paradoxes of the etiology of arthritis. Amano et al. [131]
demonstrated that mice overexpressing an E3 ubiquitin ligase
(Synoviolin/Hrd1) developed a spontaneous arthropathy, but
synoviolin/hrd1 (+/-) mice were resistant to collagen-induced
arthritis. The authors suggest that this paradox is explained by
the enhanced apoptosis of synovial cells, but there is a danger
in the over-reliance on transgenic mice to accurately depict the
total effects of the gene product under investigation. Despite
that cautionary note, it appears that transgenic models provide
284 Current Rheumatology Reviews, 2008, Vol. 4, No. 4 Paul H. Wooley
a novel means to address the specific mechanism of action
of a potential anti-arthritic strategy, and could eventually
lead to the best model for rheumatoid arthritis.
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Received: December 14, 2007 Revised: J anuary 23, 2008 Accepted: February 22, 2008