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Inflammation & Allergy - Drug Targets, 2008, 7, 000-000 1
1871-5281/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.
Molecular Targets of Rheumatoid Arthritis
Hiroshi Okamoto*, Daisuke Hoshi, Akiko Kiire, Hisashi Yamanaka and Naoyuki Kamatani
Institute of Rheumatology, Tokyo Women’s Medical University, Tokyo, Japan
Abstract: Rheumatoid arthritis (RA) is a multifactorial disease characterized by chronic inflammation of the joints. Both
genetic and environmental factors are involved in the pathogenesis of joint destruction and disability. In the inflamed RA
joint, the synovium is highly infiltrated by CD4+ T cells, B cells, and macrophages. Furthermore, the intimal lining be-
comes hyperplastic due to the increased numbers of macrophage-like and fibroblast-like synoviocytes. This hyperplastic
intimal synovial lining forms an aggressive front, called pannus, which invades cartilage and bone structures, leading to
compromised function and/or destruction of affected joints. RA pathology is mediated by a number of cytokines (TNF-,
IL-1, IL-6, IL-17, IFN, et c.), chemokines (MCP-1, MCP-4, CCL18, etc.), cell adhesion molecules (ICAM-1, VCAM-1,
etc.) and matrix metalloprotein ases. Currently, treatment strategies targ eted again st TNF-, IL-1 and IL-6 are available. In
this review, we will summarize the use of biologics, the pros and cons of the use of biologics, and discuss on the potential
molecular targets of RA.
Rheumatoid arthritis (RA) is a systemic autoimmune
disease characterized by symmetric polyarticular joint disor-
ders that primarily affect the small joints of the hands and
feet. Both genetic and environmental factors are involved in
the pathogenesis leading to joint destruction and disability.
In the inflamed RA joint, the synovium is highly infiltrated
by CD4+ T cells, B cells, and macrophages, and the intimal
lining becomes hyperplastic owing to the increased number
of macrophage-like and fibroblast-like synoviocytes. This
hyperplastic intimal synovial lining forms an aggressive
front, called pannus, which invades cartilage and bone struc-
tures, leading to compromised function and/or destruction of
affected joints. It is widely accepted that hyperplasia of
synovial cells is dependent on dysregulated proliferation and
apoptosis. Preceding synovial hyperplasia, inflammation
takes place at the site of the affected joint, followed by im-
munological dysfunction. This process is mediated by a
number of cytokines (TNF-, IL-1, IL-6, IL-17, IFN, etc.),
chemokines (MCP-1, MCP-4, CCL18, etc.), cell adhesion
molecules (ICAM-1, VCAM-1, etc.) and matrix metallopro-
teinases. In this review, we will provide an overview of bio-
logical agents targeted against cytokines and cell surface
molecules and also discuss on the potential molecular targets
of RA (Table 1, Fig. 1).
BIOLOGICAL AGENTS TARGETED AGAINST SPE-
CIFIC CYTOKINES
1) Tumor Necrosis Factor (TNF)-
Many cytokines have pathological roles in the develop-
ment of RA. They are activated in the synovium by various
cell populations such as macrophage-like cells and fibro-
blast-like cells. They include TNF- and interleukin 1(IL-1),
both of which induce another proinflammatory cytokine,
interleukin 6 (IL-6). These cytokines probably contribute to
the pathogenesis of rheumatoid arthritis through induction of
*Address correspondence to this author at the Institute of Rheumatology,
Tokyo Women’s Medical University, 10-22 Kawada-cho, Shinjuku, Tokyo
162-0054, Japan; Tel: +81-3-5269-1725; Fax: +81-3-5269-1726;
E-mail: hokamoto@ior.twmu.ac.jp
antibody-producing B cells and activation of T cells, macro-
phages, and osteoclasts. Of these, TNF- has emerged as a
dominant proinflammatory mediator and an important mo-
lecular target for therapy. A recent advance in the manage-
ment of RA is the use of biological agents which block cer-
tain key molecules involved in the pathogenesis of the disor-
der.
Recent studies in animal models support the use of anti-
TNF- strategies in the treatment of arthritis. For example,
anti-TNF- therapy has proven efficacious in collagen-
induced arthritis. Such therapy significantly attenuates in-
flammation and reduces joint destruction [1]. Additional
evidence of the role of TNF- in arthritis comes from studies
of transgenic mice. Mice that over-express TNF- spontane-
ously develop an erosive, inflammatory arthritis [2]. The
arthritis can be effectively abrogated by blocking TNF-.
Note that in this model, inflammation can also be attenuated
by blocking IL-1, suggesting the pivotal role of TNF- in the
induction of the inflammatory cytokine cascade. Therefore,
these data indicate that TNF- is an important therapeutic
target in RA [3].
Currently, three TNF--blocking agents are clinically
available: infliximab (Remicade), etanercept (Enbrel) and
adalimumab (Humira). Infliximab and adalimumab are
monoclonal antibodies directed against TNF- while etaner-
cept is a construct of two molecules of TNF- receptor (p75
receptor) linked to the Fc portion of IgG1), giving rise to an
immunoglobulin-like molecule. Clinical trials with the three
agents show high efficacy in RA patients who failed tradi-
tional non-biologic disease-modifying anti-rheumatic drugs
(DMARDs). The anti-TNF- agents can be used alone or in
combination with methotrexate (MTX) [4-5], but the combi-
nation has superior efficacy [6-9]. As infliximab is a chi-
meric monoclonal antibody containing murine amino acid
sequences, concerns about immunogenicity of this therapy
were investigated by measurement of the frequency of hu-
man anti-chimeric antibody (HACA) responses following
repeated infliximab administration. Evidence of the forma-
tion of infliximab HACA complexes has been reported [10].
Clinical strategies to reduce the frequency of the formation
2 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 Okamoto et al.
of HACA include the induction of immunologic tolerance
through the use of a regular injection regimen (every eight
weeks) and concomitant immunosuppressive therapy with
MTX. However, temporary withdrawal of infliximab treat-
ment is sometimes required because of severe infection,
pregnancy, or the need for surgery. Withdrawal of treatment
may induce the production of HACA owing to the interrup-
tion of immunologic tolerance. As previously pointed out,
further clinical studies are needed to determine the appropri-
ate interval for resumption of infliximab treatment [11].
In a recent trial, infliximab, etanercept and adalimumab
were used for 12 months in combination with MTX. All
three agents achieved the same ACR 20 (American College
of Rheumatology 20% improvement criteria), 50 and 70 re-
sponses compared to placebo plus MTX: 60% versus 25%,
40% versus 10%, and 20% versus 5%, respectively. Modi-
fied Sharp scores were even more impressive, indicating that
these agents prevented joint damage (as assessed by serial X-
rays) out of proportion to their ability to reduce clinical signs
and symptoms of disease [12]. There were also improve-
ments in function as assessed by the Health Assessment
Fig. (1). Schematic representation of the potential therapeutic targets in RA.
Table 1. List of Currently Available Biological Agents and Potential Molecular Targets Introduced in this Review
Biologic Agents Generic Name Route and Standard Dosing
Adalimumab Subcutaneous; 40 mg every other week
Etanercept Subcutaneous; 50 mg per week
Anti-TNF therapy
Infliximab Intravenous: 3 mg/kg at 0, 2 and 6 weeks followed by maintenance every 8 weeks thereafter
Anti-IL-1 therapy Anakinra Subcutaneous; 100 mg daily
Anti-IL-6 therapy Tocilizumab Intravenous: 8 mg/kg every 4 weeks
B cells (ant-CD20) Rituximab Intravenous: 375 mg/m2 infusion weekly for 4 to 8 weeks
T cells (CTLA-4 Ig) Abatacept Intravenous: 500 to 1000 mg dosed by weight repeat at 0, 2 and 4 weeks and then every 4 weeks there-
after
Chemokines CCL18/PARC, MCP-4/CCL13, CCR5, MCP-1/CCL2, Fractalkine/CX3CL1, etc
Potential Targets
Transcription Factors
NF-B, NFAT, AP-1, JAK/STATs, etc.
Molecular Targets of Rheumatoid Arthritis Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 3
Questionnaire (HAQ). Other studies have shown that the
combined use of these agents with MTX early in th e course
of RA has the highest efficacy and highest suppression of
disease and radiographic progression compared to using
MTX alone (study of DEO19, ASPIRE, PREMIER, etc.).
This further supports the concept of treating RA aggressively
early in the disease in order to achieve better long-term out-
comes. The three TNF- blocking agents have also been
approved in several countries for the treatment of ankylosing
spondylitis and psoriatic arthritis. Although the therapeutic
effects of anti-TNF- agents are superior to conventional
DMARDs, there are still nonresponders. Patients who fail
one TNF- inhibitor may still respond well to either of the
other two agents.
Infections are serious potential side effects of any drug
that modifies the immune response, and tuberculosis (TB) is
a special concern. Screening for TB before starting treatment
is prudent since there is a high risk for reactivation of latent
TB early in the course of an ti-TNF- therapy. Any latent TB
should be treated (preferably for a month) before initiating
anti-TNF- therapy [13]. Other data have documented exac-
erbations of hepatitis in chronic hepatitis B patients and car-
riers [14]. Prolonged suppression of the immune response
may allow the re-activation of slow viruses such as the JC
virus. Two cases of a fatal brain disease caused by JC virus,
called progressive multifocal leukoencephalopathy (PML),
have been reported in patients with systemic lupus erythema-
tosus who were administered rituximab. Therefore, anti-
TNF- agents have an associated risk for the dev elopment of
PML. However, anti-TNF- agents are thought to be safe in
patients with hepatitis C virus. Many of the usual signs of
sepsis may be suppressed during treatment with TNF- in-
hibitors, since a patient’s ability to mount an inflammatory
response may be seriously compromised. Thus, physicians
looking after such patients must be aware of other signs of
infection, such as dyspnea and general malaise.
It is uncertain whether anti-TNF- agents increase the
risk of lymphoma, but the currently available evidence sug-
gests that this is the case, although the magnitude of in-
creased risk is very small [15]. The association of anti-TNF-
agents with higher risks for solid malignancies requires
further analysis.
There are early indications that sustained treatment with
biologic agents may reduce the risk of premature mortality in
RA and that the higher risk of cardiovascular disease and
strokes may be ameliorated [16]. Understanding the differ-
ences between these drugs in terms of pharmacokinetics is
an important challenge for future assessment and optimiza-
tion of anti-TNF- therapeutic strategies.
2) Interleukin-1 (IL-1)
IL-1 is a key modulator of immune and inflammatory
processes: it mediates inflammation by recruiting neutro-
phils, activating macrophages, and stimulating growth and
differentiation of T cells and B cells [17]. Moreover, IL-1
induces proteolytic enzymes that degrade extracellular ma-
trix, resulting in tissue d estruction. Given that IL-1 is a key
mediator of inflammation, bone resorption, and cartilage
destruction, regulation of this cytokine is important for
modulating the joint damage that is characteristic of RA.
There is strong evidence that links IL-1 to RA [18]. Ani-
mal studies demonstrated that intra-articular administration
of IL-1 causes arthritis and synovitis [19]. Following gene
transfer to rabbit synovium, constitutive intra-articular ex-
pression of human IL-1 elicited synovial changes that were
similar to those seen with RA [20].
Clinically, IL-1 is detectable in the plasma and synovial
fluid of patients with RA and its concentration in plasma has
been correlated with RA disease activity [21]. When RA
patients’ synovial tissues were explanted, the in vitro pro-
duction of IL-1 correlated positively with arthroscopic re-
sults that quantified the extent of inflammation [22]. These
findings suggested that inhibition of IL-1 should provide
benefits in signs and symptoms of disease as well as in pre-
vention of joint destruction.
IL-1 receptor antagonist (IL-1 Ra) occurs naturally and is
a specific inhibitor of IL-1. Endogeneous production of IL-1
Ra plays an important anti-inflammatory role by modulating
IL-1-induced proinflammatory processes. When present in
excess, IL-1 Ra blocks the association of IL-1 with its recep-
tor, and IL-1 proinflammatory signaling is inhibited. IL-1 Ra
decreases the production of proteolytic enzymes that are in-
volved in tissue destruction and reverses the inhibitory ef-
fects of IL-1 on tissue repair.
Animal models demonstrated that the balance of IL-1 and
IL-1 Ra is important in the pathophysiology of chronic in-
flammatory diseases, such as RA. For instance, IL-1 Ra
knockout mice spontaneously develop chronic inflammatory
polyarthropathy that resembles RA and administration of IL-
1 Ra blocked the exacerbation of arthritis in animal models
that were exposed to IL-1 [23]. Furthermore, when soluble
IL-1 Ra was administered intra-articularly to rabbits which
had antigen-induced arthritis, cartilage degradation and white
blood cell infiltration was greatly reduced [24].
A quantitative investigation of the balance of IL-1 and
IL-1 Ra demonstrated that RA patients have a low ratio of
IL-1 Ra to IL-1 in synovial cell samples (range: 1.2 - 3.6).
This ratio is believed to be too low to block IL-1 activity
[25]. A 10- to 100-fold excess of IL-1 Ra is needed to inhibit
IL-1 bioactivity [26]. The inadequate production of IL-1 Ra
in patients who have RA is believed to be due, in part, to
alternative splicing of IL-1 Ra mRNA and defective IL-1 Ra
production by synovial macrophages [25]. Thus, the admini-
stration of IL-1 Ra to patients who have RA might restore
the balance between IL-1 and IL-1 Ra and improve their
disease status.
Anakinra (Kineret, Amgen Inc., Thousand Oaks, Califor-
nia) is a recombinant, nonglycosylated form of native human
IL-1 Ra produced in an Escherich ia coli bacterial expression
system [27]. It differs from native human IL-1 Ra by the
addition of a single methionine residue at its amino terminus.
Anakinra antagonizes the biologic activity of IL-1 by com-
petitively inhibiting IL-1 binding to its type 1 receptor,
thereby blocking cell signaling. In animal models of arthritis,
anakinra suppressed IL-1 -induced responses, including
swelling, chronic inflammation, osteoclast activation, and
bone resorption, if adequate levels were maintained in the
blood [28].
In a large, multicenter trial in Europe, 472 adult patients
who had severe, active RA were randomized to receive pla-
4 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 Okamoto et al.
cebo (n = 121) or anakinra at daily subcutaneous dosages of
30 mg (n = 119), 75 mg (n = 116), or 150 mg (n = 116) for
24 weeks [29]. Patients were required to have typical fea-
tures of active RA, including the presence of at least ten
swollen joints for at least six months but less than eight
years, and to have failed no more than two DMARDs.
DMARD therapy had to be discontinued for at least six
weeks before enrollment and dosages of NSAIDs or oral
corticosteroids remained constant for the duration of the
study. Statistical analyses of ACR end points and composite
scores were based on a modified intent-to-treat population,
with last observation carried forward as the 24-week value.
After completion of the active phase of the study, patients
continued on a 24-week extension (347 completers) [30] or
52-week extension (218 completers) [31].
A significantly higher proportion of patients who were on
the 150 mg/day dosage of anakinra responded to treatment
by ACR criteria (43% reached an ACR 20 response) than did
the placebo-treated patients (27%; P = 0.014) at 24 weeks
[29]. Although higher proportions of patients who were on
the 30 mg/day (39%) and 75 mg/day dosages (34%) of anak-
inra achieved an ACR20 response than did placebo-treated
patients (27%), the improvements did not reach statistical
significance (P = 0.054 and 0.258, respectively). A compari-
son of the placebo-treated group with the combined anak-
inra-treated groups revealed that significantly more anakinra-
treated patients reached an ACR20 response than those who
were treated with placebo (P = 0.020). The group that re-
ceived anakinra at a dosage of 150 mg/day also had signifi-
cantly fewer swollen and tender joints (P < 0.01), lower in-
vestigator and patient assessments of disease activity (P <
0.01), and decreased pain and HAQ scores (P < 0.001) at 24
weeks compared with the placebo-treated group. All three
active treatment groups had statistically significant im-
provements in erythrocyte sedimentation rate (ESR) and
CRP values at 24 weeks (P < 0.01). Improvement with anak-
inra was rapid; statistically significant differences were seen
soon after initiation of treatment.
The analysis of long-term data showed that the 24 week
efficacy of anakinra was maintained at 48 weeks [29,31]. For
those patients who were administered anakinra during the
initial, placebo-controlled phase of the study, 51% demon-
strated an ACR20 response at 24 weeks compared with 46%
at 48 weeks (all dosages combined). In addition, 18% of pa-
tients who continued anakinra treatment demonstrated an
ACR 50 response and 3% demonstrated an ACR70 response
at 48 weeks (all dosages combined). These studies demon-
strated the efficacy of anakinra monotherapy in the treatment
of RA.
3) Interleukin-6 (IL-6)
IL-6 is a proinflammatory cytokine that is abundantly
expressed in patients with RA and is detectable in the joints
and circulation of such patients during active phases of the
disease [32]. IL-6 binds to its soluble and membrane-bound
receptors, and their interaction with gp130 transduces intra-
cellular signals that mediate gene activation and a wide range
of biologic activities [33]. Preclinical and human studies
have demonstrated that IL-6 activities are relevant to RA, as
it induces differentiation of B cells into immunoglobulin-
secreting plasma cells, activates T cells, induces acute-phase
proteins by hepatocytes, and enhances production of platelets
[34,35]. IL-6 also induces the recruitment of chemokines and
leukocytes and has been shown to induce proliferation of
synovial fibroblasts [36]. In addition, IL-6 has profound ef-
fects on bone, inducing osteoclast differentiation and activa-
tion in vitro [37]. IL-6 decreases chondrocytes’ productions
of aggrecan protein and type collagen and thereby damages
cartilage [38]. The inflammatory and destructive effects o f
IL-6 have been validated, with data in animals suggesting an
important role of IL-6 in the induction and maintenance of
chronic synovial inflammation [39,40]. For example, IL-6
gene-knockout mice are protected against arthritis [41]. Be-
cause of the key role played by IL-6 in several phenomena
typical of RA, IL-6 is a candidate target for therapeutic inhi-
bition as a novel approach to the treatment of RA.
Tocilizumab is one of the molecules in development that
offers a new mechanism of action. Tocilizumab, a human -
izied anti-human IL-6 receptor antibody of the IgG1 sub-
class, was developed collaboratively by Osaka University
and Chugai Pharmaceutical Company, Ltd. (Japan). Tocili-
zumab is humanized by the grafting of complementarity-
determining regions of a mouse anti-human IL-6 receptor
monoclonal antibody on to human IgG1, using recombinant
DNA technology. Tocilizumab competes for both the mem-
brane-bound and soluble forms of the human IL-6 receptor,
thus inhibiting the binding of IL-6 and its proinflammatory
activity. Short-term clinical trials of tocilizumab in adult-
onset RA and systemic-onset juvenile idiopathic arthritis
have demonstrated acceptable safety and significant efficacy
[42].
Choy et al. have conducted a British phase study with
45 patients treated with a single dose of 0.1, 1, 5, or 10
mg/kg tocilizumab, or placebo [43]. This study evaluated
tocilizumab safety and RA disease activity in the second
posttreatment week using the ACR improvement criteria.
The ACR20 rate was 56% for the 5 mg/kg tocilizumab group
and 0% for the placebo group, indicating a significant differ-
ence. However, no significant difference in ACR20 rate was
observed between other tocilizumab groups and the placebo
group. The results of this study also confirmed an adequate
level of safety with no serious side effects.
In a Japanese 24 week phase / study, those patients
who did not show serious side effects, and had improved
CRPs or erythrocyte sedimentation rates (ESR) in the second
week following the third dose, were further treated with to-
cilizumab if the patients w ere willing to continue the tocili-
zumab therapy, and also if the principal investiga-
tor/physician decided that the patients required further treat-
ment. Of all the patients, the ACR20 response rate at the
sixth week was 60%, and 86% at the sixth month. The find-
ings of this open study suggest that tocilizumab has signifi-
cant beneficial effects on RA disease activity, which led to a
phase study.
A randomized, multicenter, double-blind, placebo-
controlled trial of tocilizumab was conducted in a Japanese
late phase study, with 164 patients who previously had an
insufficient response to one or more doses of anti-rheumatic
or immunosuppressive drugs. Patients underwent intrave-
nous therapy with either 4 or 8 mg/kg tocilizumab, or pla-
cebo given at four week intervals for a total of three times.
RA disease activity was evaluated in the fourth posttreatment
Molecular Targets of Rheumatoid Arthritis Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 5
week [44]. The ACR20 response rate for the 8 mg/kg tocili-
zumab dose was 78%, while that for the placebo was only
11%, thus confirming the clinical utility of tocilizumab in a
double-blind study.
Evaluation of drug safety showed no significant differ-
ences in adverse event rates between 4 and 8 mg/kg tocili-
zumab, and placebo groups. Laboratory findings, however,
indicated a dose-dependent increase of total cholesterol val-
ues in 44% of patients treated with tocilizumab. This in-
crease stabilized at around 240 mg/dl, and HDL cholesterol
also increased in a similar way. Long-term safety evaluation
is necessary to know whether or not the increased total cho-
lesterol value indicates a higher risk of cardiovascular dis-
ease. It has been reported that TNF inhibition also resulted in
an increase in total cholesterol, suggesting that this effect
may be due to a decrease in RA disease activity [45].
Tocilizumab monotherapy, without MTX, resulted in an
insignificant number of patients developing antinuclear anti-
bodies or anti-DNA antibodies, which are frequently ob-
served with anti-TNF antibody treatment. Moreover, anti-
tocilizumab antibodies were observed in only 2% of the pa-
tients treated with tocilizumab, reconfirming the advantage
of humanized antibodies. As IL-6 induces antibody produc-
tion, IL-6 inhibition may reduce the production of neutraliz-
ing antibodies. In terms of treatment strategy, it is highly
advantageous that MTX is not required.
Maini et al. conducted a European phase study on the
combined use of tocilizumab with MTX [46]. This study
yielded results that confirmed the effectiveness of tocilizu-
mab as a monotherapy. No difference was observed in the
ACR20 rate between monotherapy with 8 mg/kg tocilizumab
and combined treatment with 8 mg/kg tocilizumab and
MTX. However, a synergetic effect was observed in com-
bined treatment with 4 mg/kg or a lower dose of tocilizumab
and MTX.
A Japanese phase trial was conducted to examine the
preventive effects of tocilizumab on progressive joint de-
struction. This study evaluated the one year change in the
van der Heijde’s modified Sharp score (a quantitative radio-
graphic evaluation of bone erosion and joint space narrowing
in hand and foot joints of RA patients). The results indicated
that tocilizumab is also effective in preventing progressive
joint destruction [47].
BIOLOGICAL AGENTS TARGETED AGAINST CELL
SURFACE MOLECULES
1) B Cells (CD20)
Although the precise contribution of B cells to the im-
munopathogenesis of RA is not well characterized, a number
of reports have suggested their involvement. It is well known
that rheumatoid factor (RF) and anti-citrullinated protein
antibodies contribute to immune complex formation and
complement activation in the affected joints. Rheumatoid
synovial membrane contains abundant B cells that produce
the RF antibody. These auto-antibodies are produced by
plasma cells. The B cells in RA not only work as a source of
auto-antibodies but also work as highly efficient antigen-
presenting cells (APCs) by processing and presenting anti-
genic peptides to T cells. Furthermore, activated B cells can
synthesize cytokines (such as IL-4, IL-10, etc.) and mem-
brane-associated molecules that provide non-specific help to
adjacent T cells [48]. Therefore depletion of B cells might
modify the pathology of RA. CD20 is a B cell-specific sur-
face antigen which is expressed by early pre-B cells and re-
tained through development to mature B cells. CD20 is lost
prior to differentiation into plasma cells. Rituximab is a chi-
meric mouse/human monoclonal antibody targeted against
CD20. Several mechanisms have been proposed to explain
the mechanism by which Rituximab depletes B cells. First,
antibody-dependent, cell-mediated cytotoxicity might be
triggered through the binding of membrane-associated
CD20/Rituximab to Fc receptors on natural killer cells,
macrophages and monocytes. Second, complement-
dependent cytotoxicity might be induced by the interaction
between membrane-associated CD20/Rituximab and C1q.
Finally, it may promote apoptosis of CD20+ B cells. Rituxi-
mab-induced down-regulation of CD40 and CD80 on B cells
might inhibit the activation and development of T helper 1
dominance by preventing CD40/CD40L and CD80/CD28-
mediated downstream interactions [49].
Based on the success of a pilot study, at least three dou-
ble-blind, placebo-controlled clinical trials of rituximab have
been conducted and significant improvement of RA has been
reported. A phase IIa trial demonstrated that a single course
of two 1,000 mg infusions of rituximab, alone or in combina-
tion with either cyclophosphamide or continued MTX, sig-
nificantly improved disease symptoms by weeks 24 and 48
[50]. DANCER (International Clinical Evaluation of Ri-
tuximab in Rheumatoid Arthritis) was an international, phase
IIb, randomized, double-blind, double-dummy, placebo-
controlled, multi-factorial trial. A total of 465 patients were
randomized into nine treatment groups: the rituximab groups
(placebo [n = 149], 500 mg [n = 124], or 1,000 mg [n = 192]
on days 1 and 15) each also taking either placebo glucocorti-
coids, intravenous methylprednisolone premedication, or
intravenous methylprednisolone premedication plus oral
prednisone for two weeks. All patients received MTX (10 -
25 mg/week); no other DMARDs were permitted. Patients
who receiv ed two 500 mg or two 1,000 mg infusions of ri-
tuximab met the ACR20 at week 24 (55% and 54%, respec-
tively) compared with placebo (28%). ACR50 responses
were achieved by 33%, 34%, and 13% of patients, respec-
tively, and ACR70 responses were achieved by 13%, 20%,
and 5% of patients. Changes in the Disease Activity Score in
28 joints (DAS28) was significantly greater in the patients
treated with two 500 mg or two 1000 mg rituximab infusions
than in the patients treated with placebo. In comparison with
placebo, moderate or good European League Against Rheu-
matism (EULAR) responses were achieved by a significantly
higher proportion of patients treated with two 500 mg
(P < 0.0001) or two 1000 mg rituximab infusions
(P < 0.0001), with a trend toward a higher proportion of pa-
tients achieving a good EULAR response with two 1000 mg
rituximab infusions. The authors concluded that both rituxi-
mab doses were effective and well-tolerated when added to
MTX therapy in patients with active RA [51].
The phase III REFLEX study (Randomized Evaluation of
Long-Term Efficacy of Rituximab in RA) compared rituxi-
mab (1 g x 2) with placebo (MTX 10 – 25 mg/week and
standard 14 day steroid induction was used in both arms) in
patients inadequately responding to anti-TNF- therapy. A
24-week ACR20 response was achieved by 51% of the pa-
6 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 Okamoto et al.
tients on rituximab and 18% of those receiving placebo;
moderate or good EULAR responses were obtained in 65%
and 22% (P < 0.0001), respectively. ACR50 and ACR70
responses were also significantly more frequent in the ri-
tuximab-treated patients (27% and 12% vs 5% and 1% in
controls). The rate of serious infections was not significantly
high compared with the control group [52]. Meta-analyses of
randomized placebo-controlled trials did not reveal a signifi-
cant increase in the risk of serious infections during rituxi-
mab therapy in RA patients [53].
Recently, an open-label extension analysis of RA patients
previously treated with rituximab was conducted [54]. Pa-
tients who had participated in any of three double-blind trials
were eligible for additional courses (two infusions of 1,000
mg given two weeks apart) if they exhibited a swollen joint
count and tender joint count of > 8 with > 16 weeks elapsing
after the previous course. A total of 1,039 patients received >
1 course of rituximab. Of these, 570 received two courses,
191 received three courses, and 40 received four courses, for
a total of 1,669 patient-years. Irrespective of prior TNF in-
hibitor exposure, ACR20 responses were comparable after
course one and after course two (65% versus 72%), as were
ACR50 and ACR70 responses. Moderate/good EULAR re-
sponses were also comparable in course two relative to
course one (88% versus 79%), with EULAR remission oc-
curring in a two-fold higher proportion of patients after
course two than after course one (13% versus 6%). The seri-
ous infection rate after course one (5.1 per 100 patient-years)
remained stable through additional courses. The proportion
of patients with circulating IgM and IgG levels below the
lower limit of normal (LLN) increased with subsequent
courses; however, serious infection rates in these patients
were comparable with those in patients with immunoglobu-
lin levels above the LLN. The authors concluded that pa-
tients treated with repeated courses of rituximab have sus-
tained clinical responses with no new adverse events. Even
though there are optimistic reports on the adverse effects of
this treatment, clinicians should be aware that the use of ri-
tuximab could allow for the development of severe infec-
tions, especially in cases in which functions of antigen pres-
entation play an important role in disease protection [55].
2) T Cells (CD80/86)
RA-associated antigens are probably presented to T cells
by professional APC such as dendritic cells, macrophages, or
activated B cells. This process entails binding of antigenic
peptides to class II MHC molecules, and more than 80% of
RA patients carry the so-called shared epitope of the HLA-
DRB1*04 cluster [56]. Disease-associated HLA-DR alleles
are thought to present arthritis-related peptides leading to the
stimulation and expansion of autoantigen-specific T cells in
the joints. The first signal for T cell activation is b inding of
peptide-MHC complexes to T-cell receptors, followed by the
binding of CD80 and CD86 on APC and T cells expressing
CD28. These two signals are required for the full activation
of T cells [57,58]. T cells in the synovial membrane usually
belong to the T-helper 1 subset which induce activation of
macrophages, B cells, fibroblasts, and osteoclasts. To pre-
vent T cell over-activation, the cells express cytotoxic T
lymphocyte antigen (CTLA) 4, which has a higher affinity
for CD80 and CD86 than for CD28 and conveys inhibitory
signals.
Abatacept, a recombinant fusion protein, consists of the
extracellular domain of human CTLA 4 and part of the Fc
domain of human IgG1 (59). This fusion protein has a high
binding affinity for CD28 and competes with it for binding
of CD80 and CD86, thus interfering with T cell activation
[60]. In a six-month, double-blind, randomized, placebo-
controlled study of abatacept therapy in patients with RA,
patients treated with 10mg /kg abatacept wer e more likely to
have an ACR20 than were patients who received placebo
(60% vs 35%, P < 0.001) [61]. A phase III trial (AIM) sup-
ported these results. While continuing their methotrexate
treatment (at about 15 mg a week), 433 patients received
intravenous abatacept (about 10 mg/kg bodyweight) and 219
received placebo, on days 1, 15, and 29, and every four
weeks thereafter. At six months, ACR20, 50, and 70 re-
sponses were seen in 68%, 40%, and 20%, respectively, of
patients treated with abatacept, levels which were signifi-
cantly higher than with placebo. Similar differences were
also seen for DAS28, the health assessment questionnaire
disability index, and health status. After one year, abatacept
had slowed the progression of structural joint damage and
the results were statistically significantly compared with
placebo. Compared with the placebo group, abatacept-treated
patients had a similar incidence of adverse events but a
higher incidence of prespecified serious infections and infu-
sion reactions [62]. Recently, results from the long-term ex-
tension of the AIM trial were reported. In patients treated
with abatacept for two years, there was a greater reduction in
the progression of structural damage than in year one. The
mean change in total Genant-modified Sharp scores was re-
duced from 1.07 units in year one to 0.46 units in year two
and similar reductions were observed in erosion and joint
space narrowing scores [63]. In another phase III trial (AT-
TAIN), 393 patients with active RA and an inadequate re-
sponse to TNF inhibitors were studied; these individuals
received abatacept (10 mg/kg) or placebo until day 141. Par-
ticipants were required to cease treatment with etanercept at
least 28 days in advance of enrollment; infliximab treatment
was stopped 60 days in advance. They also had to be stable
on disease-modifying antirheumatic drugs, with 75–82%
continuing methotrexate treatment (about 15 mg a week).
After six months, the rates of ACR 20 responses were 50.4%
in the abatacept group and 19.5% in the placebo group (P <
0.001); the respective ACR50 and ACR70 response rates
were also significantly higher in the abatacept group than in
the placebo group [64]. In another trial, patients with active
rheumatoid arthritis despite etanercept therapy received
abatacept (2 mg/kg) or placebo while continuing etanercept
treatment. The results indicated only marginal benefit of the
combination (ACR20 and 50 responses vs placebo: 48.2% vs
30.6% and 26% vs 19%, respectively). No major further im-
provements were achieved on switching to abatacept at 10
mg/kg. Importantly, the frequency of infections, including
serious infections, was higher in patients with the combina-
tion treatment than those with etanercept alone [65]. There-
fore, a combination of abatacept with biological agents is not
recommended.
POTENTIAL MOLECULAR TARGETS OF RA
1) Chemokines
Chemokines are chemotactic cytokines that regulate leu-
kocyte migration during inflammatory responses, as well as
Molecular Targets of Rheumatoid Arthritis Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 7
homeostatic trafficking of lymphocytes and dendritic cells
[68,69]. In the setting of RA, chemokines and their receptors
appear to play an important role in cell migration and the
positioning of leukocytes within the inflamed rheumatoid
synovium. The specific roles of chemokines and chemokine
receptors in the chronic inflammatory process in synovial
tissue are under intense scrutiny [66,67]. Recently, the im-
portance of chemokines in autoimmune diseases, neoplasia,
and cardiovascular diseases has been demonstrated.
The chemokine superfamily includes at least 50 low mo-
lecular weight proteins (6-14 kDa). Their primary structure
is characterized by the presence of four conserved cysteine
residues. Based on the positioning of the two NH2-terminal
cysteines, the chemokine family can b e structurally divided
into CC chemokines, CXC chemokines, CX3C chemokines
and C chemokines. Chemokines activate leukocytes via G-
protein coupled receptors, which mediate most of their bio-
logical activities [70]. Inflammatory cells infiltrating RA
synovial tissue express chemokine receptors including
CXCR3, CCR5, CCR3, CCR2, and CXCR2 [71]. The CC
chemokines, such as monocyte chemoattractant protein-1
(MCP-1)/CCL2, macrophage inflammatory protein-1
(MIP-1)/CCL3, MIP-1/CCL4, and RANTES/CCL5, are
implicated in RA pathogenesis via recruitment and retentio n
of monocytes and T lymphocytes in the joints [72]. In addi-
tion to cell traffic regulation, several chemokines have been
reported to enhance the migration and proliferation of FLS
and to up-regulate their production of gelatinase and colla-
genase [73]. Thus, chemokines are key molecules in the
pathogenesis of RA and are potential therapeutic targets for
RA.
CCL18/PARC
CCL18/PARC is a CC chemokine that is structurally
related to CCL3/MIP-1, a well-ch aracterized proinflamma-
tory chemokine. CCL3/MIP-1 binds to at least two recep-
tors, i.e. CCR1 and CCR5, and thereby exerts chemoattrac-
tant activity on several types of leukocytes [74-76]. How-
ever, human CCL18/PARC receptors (and the murine
equivalent) have yet to be identified. CCL18/PARC is either
constitutively expressed or induced in mono-
cytes/macrophages and dendritic cells, and chemoattracts T
and B lymphocytes [77,78]. Thus far, CCL18/PARC expres-
sion has been reported in a number of unrelated pathologies
including atherosclerosis, hypersensitivity pneumonitis, al-
lergic contact hypersensitivity, and ovarian carcinoma.
There have been only two reports discussing
CCL18/PARC in the context of arthritis. Schutyser et al.
reported a four-fold enhancement of CCL18/PARC levels in
synovial fluids from RA patients compared to those with
crystal-induced arthritis and osteoarthritis. In addition, im-
munochemical analyses revealed that CD68+ mono-
cytes/macrophages were the main sources of CCL18/PARC
in arthritic synovial tissues [79]. Radstake et al. assessed the
expression of various chemokines by immature and mature
dendritic cells and examined their regulation by the Fc
gamma receptor in RA patients and healthy donors. They
provided evidence for elevated CCL18 expression by den-
dritic cells in RA patients and demonstrated that this expres-
sion was regulated in part by FcR triggering. They sug-
gested a potential role for CCL18/PARC-producing dendritic
cells in RA pathogenesis [80]. We found that CCL18/PARC
mRNA was expressed at significantly higher levels in carti-
lage from RA patients compared to OA and control patients
(P < 0.001). Cartilage tissue from patients with OA showed
significantly higher expression of CCL18/PARC than control
patients (P = 0.0085). CCL18/PARC mRNA was also de-
tected at much higher levels in synovial membranes from
RA patients than from OA patients (P = 0.0001). Immuno-
histochemical study revealed high expression of
CCL18/PARC in cartilage and synovial tissues from RA
patients [81].
CCL18/PARC may be overexpressed in the joints, carti-
lage and synovial tissues of RA patients and detection in
their synovial fluid and serum is possible. To this end, we
analyzed CCL18/PARC in the synovial fluids and sera from
RA patients as well as those with OA. There were signifi-
cantly higher levels of CCL18/PARC (156.21 ± 125.73
ng/ml, n = 71) in the serum of RA patients compared to OA
patients (64.54 ± 40.90 ng/ml, n = 12) and healthy donors
(28.04 ± 10.96 ng/ml, n = 20). Moreover, when
CCL18/PARC was measured in the synovial fluid of RA and
OA patients, the average CCL18/PA RC concentrations
(275.20 ± 228.16 ng/ml, n = 15) were significantly higher in
the RA patients than the OA patients (33.13 ± 14.84 ng/ml, n
= 6). Our results indicated that CCL18/PARC and the down-
stream signaling pathways may be considered novel thera-
peutic targets in RA [81].
MCP-4/CCL13
MCP-4/CCL13 is a CC chemokine recently identified
from a human cDNA library. It directs the migration of eosi-
nophils, monocytes, and T lymphocytes through several
chemokine receptors, including CCR-2 and CCR-3 [82]. The
role of MCP-4/CCL13 in disease is not well defined, but
recent studies have suggested that it is involved in inflamma-
tory cell recruitment in allergic disorders such as asthma and
atopic dermatitis [83]. We found that the concentration of
MCP-4/CCL13 protein in synovial fluid from RA patients
(mean 208.07 ± 462.6 pg/ml) was significantly higher than
from OA patients (mean 29.6 ± 45.7 pg/ml) (p = 0.0000112).
Immunohistochemical study revealed that MCP-4/CCL13
protein was expressed in cartilage from RA patients, but was
not detected in cartilage from OA patients. We found that
IFN- significantly stimulated MCP-4/CCL13 production in
a dose-dependent manner, while IL-1 and TNF- had no
significant effects. Interestingly, IFN- induction of MCP-
4/CCL13 was significantly and remarkably enhanced when
combined with IL-1 or TNF-. We also found that MCP-
4/CCL13 could activate extracellular signal-regulated kinase
(ERK), widely known to play a major role in cell prolifera-
tion. ERK activation peaked around 10 - 20 minutes and
returned to basal levels within 60 minutes. Incubation with
100 μM PD98059, a specific inhibitor of ERK activation,
abolished upregulation of ERK by MCP-4/CCL13 in RA
FLS. In addition, MCP-4/CCL13 enhanced the proliferation
of FLS in a dose-dependent manner, and PD98059 com-
pletely inhibited the stimulatory effect of MCP-4/CCL13 on
FLS proliferation. These results demonstrated that MCP-
4/CCL13 secreted from chondrocytes in the joints plays an
important role in the development of aggressive synovial
tissues in RA. Our data showed that not only synovial cells
but also chondrocytes are actively involved in RA patho-
8 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 Okamoto et al.
genesis. Thus, MCP-4/CCL13 merits further study as a new
approach to anti-RA therapy [84,85].
CCR5
Regulated upon activation, normal T cell expressed and
secreted (RANTES)/CCL5 (a ligand for CCR1, CCR3, and
CCR5) has been implicated in RA pathogenesis. Histological
examination of affected rheumatoid joints revealed extensive
RANTES/CCL5 expression in sites of the synovial lining
and sublining layers [86]. The expression of RANTES/CCL5
in cultured synovial fibroblasts was elevated in both a time-
and dose-dependent manner following stimulation by TNF-
and IL-1 [87]. The chemokine receptor CCR5 is preferen-
tially expressed on Th1 lymphocytes. There have been re-
ports that RA is a Th1-dominant disorder, and that CCR5-
and/or CXCR3-expressing cells are enriched in affected RA
joints [88]. Yang et al. demonstrated that a CCR5 antagonist
inhibited the development of arthritis in a collagen-induced
arthritis (CIA) mouse model [89]. They showed that inhibi-
tion of arthritis development was not caused by an effect on
generation of collagen-sensitized T cells, but by interference
with migration to joint lesions. Recently, Vierboom et al.
reported that systemic administration of SCH-X, a small
molecule antagonist of CCR5, inhibited development of CIA
in rhesus monkeys [90].
We studied the effects of systemic administration of
TAK-779, a low molecular weight (Mr 531.13) nonpeptide
[91], on the development of adjuvant-induced arthritis in
rats. We found that TAK-779 suppressed the progression of
clinical arthritis compared with control rats treated with
PBS, as demonstrated by paw volume and arthritis score. In
the TAK-779-treated rats, statistically significant effects
were observed at higher doses (P < 0.01). By day 18, his-
tological analyses of the ankle joints in TAK-779 rats treated
at 1 or 2 mg/kg/day, and control rats given PBS, showed that
TAK-779 inhibited mononuclear cell infiltration and pannus
formation in synovial tissue. These data suggested that TAK-
779 has anti-arthritis effects in vivo and further supports the
evidence shown by others that CCR5 plays an important role
in the development of arthritis in animal models of human
RA. Thus, therapy using a CCR5 antagonist may serve as a
new strategy for treatment of RA [92].
MCP-1/CCL2
Monocyte chemoattractant protein (MCP)-1/CCL2 (a
ligand of CCR2) can attract monocytes, T cells, NK cells,
and basophils [93]. MCP-1/CCL2 is highly expressed in
synovial tissue and synovial fluid from RA patients, and
synovial tissue macrophages are the dominant source of
MCP-1/CCL2 production [94]. MCP-1 levels in culture su-
pernatants were significantly correlated with the amounts of
IL-1, IL-6, and IL-8 following incubation of RA patient’s
synovial cells. Furthermore, the expression of MCP-1
mRNA by cultured synovial cells was stimulated by IL-1
and TNF- [95]. Analysis of FLS from patients with RA
showed that MCP-1/CCL2 enhanced IL-6 and IL-8 produc-
tion [96]. We found that angiotensin II activated NF-B in
synovial cells to induce MCP-1/CCL2 [97]. In an MRL/lpr
mouse model of arthritis, administration of anti-CCL2/MCP-
1 monoclonal antibodies before disease onset prevented the
onset of arthritis [98]. However, a randomized controlled
study with an anti-CCL2/MCP-1 monoclonal antibody
(ABN912) failed to detect clinical benefit of this antibody
compared with placebo. Unexpectedly, there was a dose-
related increase in total CCL2/MCP-1 bound to ABN912 in
peripheral blood, up to 2,000-fold [99]. Further studies using
other anti-CCL2/MCP-1 agents is required to evaluate the
importance of CCL2/MCP-1 as a therapeutic target of RA.
Fractalkine/CX3CL1
Fractalkine/CX3CL1, a chemoattractant for monocytes
and lymphocytes, was significantly elevated in RA SF com-
pared with SF from patients with OA. RA synovial fluid
(SF) and peripheral blood contained monocytes expressing
Fractalkine/CX3CL1 [100]. Recombinant human Frac-
talkine/CX3CL1 significantly induced migration of human
dermal microvascular endothelial cells, suggesting that it
may mediate angiogenesis in RA [101]. Frac-
talkine/CX3CL1 secretion by RA FLS was regulated mainly
by TNF-. Stimulation of RA FLS with Frac-
talkine/CX3CL1 led to cell migration dependent on the acti-
vation of ERK-1/2. In a study of murine collagen-induced
arthritis, anti-Fractalkine/CX3CL1 monoclonal antibody
ameliorated arthritis by inhibiting infiltration of inflamma-
tory cells into the synovium [102]. Although clinical studies
are required, neutralization of Fractalkine/CX3CL1 might
represent a new approach to the treatment of RA.
2) Transcription Factors
The expression of cytokines (TNF-, IL-1, IL-6, IL-17,
IFN, etc.), chemokines (MCP-1, MCP-4, CCL18, etc.), cell
adhesion molecules (ICAM-1, VCAM-1, etc.) and matrix
metalloproteinases is controlled at the transcriptional level
and all these factors play pivotal roles in the pathogenesis of
RA. Thus, using biologic or pharmacologic agents to modu-
late cytokine production at the level of transcription is an
attractive strategy.
Individual genes contain DNA sequences essential for
basal or regulated gene expression. The presence of specific
DNA sequences that can bind particular proteins (transcrip-
tion factors) determines a gene’s response to stimulatory or
inhibitory signals. We will focus on the role of transcription
factors in the pathology of RA and discuss the possibility of
targeting those proteins for the treatment of RA.
NF-
B (Nuclear Factor
B)
The NF-B proteins belong to a family of ubiquitously
expressed transcription factors that play an essential role in
most immune and inflammatory responses. In mammals, the
NF-B family consists of five members: RelA (p65), RelB,
c-Rel, NF-B1 (p50 and its precursor p105), and NF-B2
(p52 and its precursor p100). They form a variety of ho-
modimers and heterodimers, each of which activates its own
characteristic set of genes [103 - 105]. The NF-B proteins
are retained in an inactive form in the cytoplasm through
their interaction with inhibitor of NF-B proteins (IB). Cel-
lular stimulation by cytokines (e.g., TNF- and IL-1) acti-
vates inhibitor of NF-B kinase (IKK complex) which phos-
phorylates IB, leading to its ubiquitination and subsequent
proteosomal degradation. Degradation of IB enables NF-B
to translocate to the nucleus, stimulating the transcrip tion of
genes containing the consensus B sequence 5’-
GGGPuNNPyPyCC-3’ (Pu: purine, Py: pyrimidine). The
genes containing the B sequence include those for cytoki-
Molecular Targets of Rheumatoid Arthritis Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 9
nes and chemokines (TNF, IL-1, IL-2, IL-6, MIP1, MIP2,
RANTES, etc.), adhesion molecules (E-selectin, ICAM-1,
VCAM-1, etc.), anti-apoptosis genes (XIAP, c-IAPs, Fas-
associated death domain-like interleukin 1 converting en-
zyme inhibitory protein (c-FLIP), Survivine, Bcl-2, Bcl-xL,
etc.), NF-B family genes (p52/p100, p50/p105, c-Rel,
IB, etc.), cell proliferation associated genes (Cyclin D1, c-
Myc, BMP-2, etc.), viral genes (HIV-1, SIV, EBV, etc.) and
others (MMPs, VEGF, iNOS, COX-2, etc.) (Fig. 2). Some of
these genes appear to have important roles in the pathogene-
sis of RA [103]. In addition, the NF-B family of genes is
highly expressed and activated in RA-affected tissues and
several interventions, such as dominant-negative IKK and
antisense NF-B oligonucleotides have effectively prevented
the expression of cytokines and the development of arthritis
in vitro and in animal models. Furthermore, NF-B report-
edly contributes to the intense proliferation of synovial cells.
Several lines of evidence suggest that RA synovial cells pro-
liferate aggressively and this plays an important role in the
pathogenesis of RA. Synovial hyper-proliferation has been
reported to be caused, at least in part, by impaired apoptosis
of synovial cells due to up-regulation of anti-apoptotic mole-
cules such as bcl-2, and FLIP [106,107]. Thus NF-B con-
tributes to the hyper-proliferation of synovial cells in RA by
regulating gene expression of FLIP and bcl-2.
NF-B is activated by various inducers including cytoki-
nes (TNF-, IL-1, IL-2, IL-17, etc.), mitogens (BAFF,
CD40 ligand, etc.) and stress/carcinogens (ultraviolet light,
hypoxia, PMA, etc.). Another inducer is serum amyloid A
(SAA) protein. SAA is an acute-phase protein produced by
hepatocytes in response to proinflammatory cytokines, and
its expression is up-regulated during the course of the in-
flammatory process [108]. Although a wealth of information
Fig. (2). Schematic representation showing NF-B and NFAT activation pathway.
10 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 Okamoto et al.
concerning the diagnosis and pathogenesis of AA amyloido-
sis has been accumulated, the biological role(s) of SAA in
the pathogenesis of RA is still not fully understood. We stud-
ied the effects of SAA on cytokine production from FLS and
found that SAA induced expression of the proinflammatory
cytokines IL-6 and IL-8 in a dose-dependent manner. SAA
stimulated the activation of NF-B signaling as determined
by a reporter gene assay and western blot analysis of IB.
In order to determine whether the effect of SAA on NF-B
activation is mediated through the binding of SAA to the
receptor for advanced glycation end products (RAGE) on
synovial cells, we pre-incubated SAA with various concen-
trations of soluble recombinant RAGE protein before adding
it to the FLS. We observed a dose-dependent inhibition of
SAA-induced IB degradation. Immunofluorescent studies
revealed that SAA stimulation promoted nuclear transloca-
tion of NF-B, whereas preincubation of SAA with RAGE
inhibited nuclear translocation [109]. These data suggested
that SAA of RA joints is actively involved in the pathogene-
sis of RA through the SAA-RAGE-NF-B signaling path-
way.
We found that angiotensin II is also an inducer of NF-B
activation in FLS. We have shown that angiotensin II acti-
vated NF-B in synovial cells to induce MCP-1 and that the
angiotensin receptor blocker inhibited this activation [97].
It is noteworthy that some of the anti-RA drugs (includ-
ing corticosteroids) block the NF-B activation cascade.
Among the drugs currently used for the treatment of diseases
other than RA, such as diabetes, hyperlipidemia and hyper-
tension, there are some drugs which have potential to inhibit
NF-B activation. To seek other candidate compounds for
use in an anti-RA strategy, we studied several drugs with
pleiotropic actions on the NF-B activation cascade, includ-
ing ligands for peroxisome proliferator-activated receptors
(PPARs).
We found that production of IL-6, IL-8 and GM-CSF by
FLS was suppressed in a dose-dependent manner in the pres-
ence of the PPAR- ligand, fenofibrate. Fenofibrate did not
inhibit basal level expression of these cytokines and these
compounds are not toxic to FLS. Fenofibrate inhibited IL-
1-induced degradation of IB and nuclear localization of
NF-B. We tested the effect of fenofibrate in vivo on the
progression and severity of AIA (adjuvant-induced arthritis)
in female Lewis rats and found that fenofibrate suppressed
the progression of clinical arthritis compared with control
rats treated with PBS, as demonstrated by paw volume and
arthritis score. These data suggest that fenofibrate has anti-
arthritis effects in vivo [110]. Considering the wide array of
processes under the control of NF-B, including expression
of cytokines and COX-2, osteoclast differentiation and apop-
tosis, and the impact of these processes on the pathogenesis
of RA, NF-B may be an efficient therapeutic target for RA.
Thus, therapy with fenofibrate may serve as a new anti-NF-
B strategy for the treatment of RA. We also have shown
that fenofibrate is useful for the treatment of RA and auto-
immune hepatitis with case reports [111, 112].
Besides its involvement in immuno-regulation, NF-B is
associated with inhibition of programmed cell death (PCD)
and has an important role in the development and homeosta-
sis of the immune, hepatic and nervous systems. The embry-
onic lethality of RelA-deficient mice was one of the first
indications that NF-B contributes a crucial anti-apoptotic
effect during normal development. In fact, embryonic death
in this case was attributed to extensive apoptosis of develop-
ing hepatocytes. A similar phenotype is seen in mice lacking
both copies of IKK, or lacking IKK along with the IKK
regulator, NEMO. Many studies suggest that NF-B controls
anti-apoptotic mechanisms associated with oncogenesis and
extensive evidence demonstrates that compounds which
block NF-B activation can serve as an anti-cancer strategy
[113].
In the pathology of RA, it is widely accepted that the
progressive destruction of articular cartilage depends on the
development of hyperplastic synovial tissue, and that hyper-
plasia of FLS is dependent on dysregulated proliferation and
apoptosis [114]. MTX is now widely accepted as a standard
therapeutic strategy for RA, and the mechanism of action of
MTX is thought to be its inhibitory effects on hyperplasia of
synovial tissue. Therefore, any compound, which could in-
hibit synovial hyperplasia has the potential for a promising
anti-RA strategy. Lipophilic statins, such as fluvastatin, re-
portedly induce apoptosis in RA synoviocytes. Thus, their
use may constitute a new approach in the treatment of RA
[115]. Clinical studies also suggested that statins have bene-
ficial effects in reducing RA disease activity [116]. In addi-
tion, a cyclin-dependent kinase inhibitor, p16INK4a sup-
presses synovial cell proliferation, resulting in inhibition of
RA pathology in an animal model [117]. Vitamin K2
(menaquinone-4, MK-4) induces apoptosis in hepatocellular
carcinoma, leukemia and MDS cell lines. Thus, we investi-
gated the effect of MK-4 on the proliferation of rheumatoid
synovial cells and the development of arthritis in a CIA rat
model. Our results indicated that MK-4 inhibited the prolif-
eration of cultured synovial fibroblasts and the development
of CIA in a dose-dependent manner. We concluded that MK-
4 may represent a new agent for the treatment of RA in com-
bination therapy with other disease-modifying anti-
rheumatic drugs [118, 119]. Therefore compounds which
block NF-B activation can serve not only as an anti-cancer
strategy, but also as anti-RA strategy.
NFAT (Nuclear Factor for Activation of T Cells)
Ca2+ is used as an intracellular signal in a wide variety of
organs and cells. For example, Ca2+ regulates calcineurin,
which in turn dephosphorylates and induces the nuclear lo-
calization of the cytoplasmic components of NFAT tran-
scription complexes. In the nucleus, NFAT transcription
complexes assemble on target DNA to activate expression of
genes such as IL-2, IL-3, GM-CSF, IL-4, IL-5, IL-13, IFN-,
TNF-, CD40L, and FasL etc. Ligand binding of various
receptors results in the activation of PLC, release of IP3, and
a transient release of Ca2+ from intracellular stores through
IP3 receptors. This initial release of Ca2+ is not sufficient to
activate NFAT target genes and an influx of Ca2+ through
CRAC(Ca2+ Release-activated Ca2+) channels is required
(Fig. 2) [120]. Pharmacologic inhibitors of NFAT transloca-
tion, such as FK506 and CsA, are clinically used for trans-
plant therapy because of their ability to prevent an immune
response against transplanted tissue. These compounds bind
to two intracellular proteins, FKBP and Cyclophilin. The
drug–protein complex then binds to the interface of the cal-
cineurin A/B complex, and blocks its phosphatase activity by
preventing substrate access. The importance of NFATs in the
Molecular Targets of Rheumatoid Arthritis Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 11
pathogenesis of RA is confirmed by the clinical observation
that treatment with cyclosporin A (CsA) is effective in oth-
erwise refractory RA. In addition, tacrolimus (FK-506) is
now widely used in the treatment of RA [121].
NFAT plays multiple roles in the destruction of bone in
RA. Bone destruction is caused by abnormal activation of
the immune system in RA. Osteoclasts are cells of the
monocyte/macrophage lineage and key players in the control
of bone metabolism. Receptor activation of NF-B ligand
(RANKL) induces osteoclast differentiation in the presence
of macrophage colony-stimulating factor. RANKL activates
the TNF receptor-associated factor 6, c-Fos, and calcium
signaling pathways, all of which are indispensable for the
induction and activation of NFAT1. NFAT1 is the master
transcription factor for osteoclast differentiation and thereby
regulates many osteoclast-specific genes. As NFAT plays
important roles not only in inflammation but also in osteo-
clast differentiation and bone destruction, NFAT should be
considered a possible target of anti-RA strategy.
Other Transcription Factors
Other transcription factors implicated in the pathogenesis
of RA are activator protein-1 (AP-1), the signal transducer
and activator of transcription (STAT) family of proteins,
interferon regulatory factors (IRFs), the Forkhead (Fox) fam-
ily of proteins, T-box transcription factor 21 (TBX21)/ T-
box expressed in T cells (T-bet), the CCAAT-enhancer-
binding protein family, and the Ets transcription factor fam-
ily [122]. Extensive genetic studies of RA have revealed an
association between RA and single nucleotide polymor-
phisms (SNPs) in the Runt-related transcription factor
1(Runx1) binding site of the SLC22A4 gene, in the major
histocompatibility complex class II transactivator
(MHC2TA) gene, and in the STAT4 gene [123 - 126].
Transcription factors play critical roles in regulating the
functions of immune effector cells, including expression of
cytokines/chemokines and also in the control of synovial cell
apoptosis. Growing experimental evidence emphasizes the
importance of NF-B, NFAT, JAK/STAT and other tran-
scription factors in RA. Therefore, signaling cascades asso-
ciated with these transcription factors are possible targets for
a comprehensive anti-RA strategy. New therapeutic strate-
gies may target transcription factor activity by controlling
their synthesis or modulating protein-protein interactions in
the activating signaling cascade. Specific inhibitors have
already reported, for example: a small molecule inhibitor of
NFAT, decoy oligonucleotides for NF-B, interfering RNAs
targeting components of the STAT pathway, and inhibition
of Toll-like receptor signalling pathway by Chaperonin 10
[127 - 131]. However, most of the transcription factors in-
volved in RA have pleiotropic roles in other biological proc-
esses and therefore, inhibition of these transcription factors
might invite unexpected side effects in vivo. Clinical and
molecular biological studies must work in tandem for the
development of effective and safe therapeutic strategies
against RA.
CONCLUDING REMARKS
RA is a multifactorial disease and no single process fully
explains its pathophysiology. Both genetic and environ-
mental factors are involved in the pathogenesis of joint de-
struction and ultimate disability. Therefore, many biological
pathways are involved and any molecule in these pathways
represents a potential molecular target for the treatment of
RA. However, most of these factors are involved in the
maintenance of normal biological functions in humans.
Therefore, inhibition of these factors might invite unex-
pected side effects in vivo. Cooperative contributions from
both clinicians and laboratory molecular biologists will be
required for the development of effective and safe treatments
for RA.
ABBREVIATION S
ACR = American College of Rheumatology
ARB = Angiotensin receptor blocker
APCs = Antigen-presenting cells
CIA = Collagen induced arthritis
CsA = Cyclosporin A
CTLA = Cytotoxic T-lymphocyte antigen
DAS28 = Disease activity score in 28 joints
DMARDs = Disease-modifying anti-rheumatic drugs
ESR = Erythrocyte sedimentation rate
EULAR = European League Against Rheumatism
ERK = Extracellular signal-regulated kinase
FLIP = Fas-associated death domain-like interleukin
1 converting enzyme inhibitory protein
FLS = Fibroblast-like synoviocytes
HAQ = Health assessment questionnaire
HACA = Human anti-chimeric antibody
IB = Inhibitor of NF-B proteins
IKK = Inhibitor of NF-B kinase
IRFs = Interferon regulatory factors
IL-1 Ra = IL-1 receptor antagonist
LLN = Lower limit of normal
MTX = Methotrexate
MCP-1 = Monocyte chemoattractant protein-1
NFAT = Nuclear factor for activation of T cells
PPARs = Peroxisome proliferator-activated receptors
PCD = Programmed cell death
PML = Progressive multifocal leukoencephalopathy
Pu = Purine
Py = Pyrimidine regulated upon activation
RANTES = Normal T-cell expressed and secreted
RAGE = Receptor for advanced glycation end
products
RANKL = Receptor activation of NF-B ligand
RA = Rheumatoid arthritis
RF = Rheumatoid factor
12 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 Okamoto et al.
SAA = Serum amyloid A
STAT = Signal transducer and activator of
transcription
SNPs = Single nucleotide polymorphisms
SF = Synovial fluid
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Received: March 1, 2008 Revised: March 10, 2008 Accepted: March 12, 2008
