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NF-╨Ю╤ФBHoly Grail for rheumatoid arthritis.

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Vol. 50, No. 8, August 2004, pp 2381–2386
DOI 10.1002/art.20468
© 2004, American College of Rheumatology
Arthritis & Rheumatism
An Official Journal of the American College of Rheumatology and
NF-␬B: Holy Grail for Rheumatoid Arthritis?
Gary S. Firestein
The transcription factor NF-␬B might be the Holy
Grail of therapeutic targets for rheumatoid arthritis
(RA). It serves as a “master switch” for an inflammatory
cascade that encompasses many key genes involved in
synovitis, including cytokines, metalloproteinases, and
regulators of small-molecule mediators (1). NF-␬B suppression is demonstrably beneficial in a host of animal
models of inflammatory disease, and many therapeutic
agents, including corticosteroids, leflunomide, sulfasalazine, and aspirin, could mediate at least some of their
effects through NF-␬B blockade. The creativity of diverse investigators driven to block this pathway is impressive, and has led to experiments using smallmolecule inhibitors of regulatory enzymes, gene therapy,
decoy oligonucleotides, and various biologics. In this
issue of Arthritis & Rheumatism, Blackwell and colleagues (2) describe a novel approach to inhibiting
NF-␬B activation through the delivery of a fusion protein that includes components of the natural inhibitor of
NF-␬B, called I␬B, and a viral-derived chaperone that
escorts proteins through the plasma membrane into the
cytoplasm. This novel approach suppresses local cytokine production and the influx of leukocytes into sites of
Inflammatory and immune responses, especially
after activation of primitive pattern-recognition receptors by pathogens, are largely coordinated by
NF-␬B (3). For instance, the transcription of many
cytokine genes, including interleukin-6 (IL-6), IL-8,
tumor necrosis factor ␣ (TNF␣), and IL-1␤, is initi-
ated by NF-␬B activation. Induction of adhesion
molecules on endothelial cells (vascular cell adhesion
molecule 1 [VCAM-1], E-selectin, and intercellular
adhesion molecule 1 [ICAM-1]) with recruitment of
inflammatory cells to extravascular sites is also mediated by this transcription factor (4). NF- ␬ B–
dependent tissue remodeling and increased vascular
permeability through the expression of metalloproteinases, inducible nitric oxide synthase, and cyclooxygenase 2 contribute to local injury. Even antibody
production and T cell–dependent delayed-type hypersensitivity use this pathway.
NF-␬B is a complex group of heterodimeric and
homodimeric transcription factors. Key members of this
family include NF-␬B1 (p50/p105), NF-␬B2 (p52/p100),
RelA (p65), RelB, and c-Rel. These molecules are
trapped in the cytoplasm as an inactive complex by I␬B.
Heterodimers containing p65 and either p50 or p52 are
among the most frequently observed in various cell
lineages and, when freed from I␬B, can initiate gene
transcription through transactivating domains. Other
combinations, such as p50 homodimers, exhibit negative
regulatory effects on many genes. Cell activation
through cytokine stimulation, engagement of Toll-like
receptors (TLRs), or stress initiates a host-defense signaling pathway that can converge on an enzyme complex
containing two I␬B kinases, known as IKK-1 and IKK-2
(also called IKK␣ and IKK␤, respectively), as well as a
regulatory protein (IKK␥) that is required for IKK
activation (5). Upstream kinases, including members of
the MAPKKK family and NF-␬B–activating kinase
(NAK), can phosphorylate the IKK signalsome and
initiate the NF-␬B cascade (6). IKKs then phosphorylate
I␬B␣ at serine 32 and serine 36 (or other I␬B isoforms
at homologous amino acid residues), which targets the
inhibitor for ubiquitination and degradation by the 26S
Gary S. Firestein, MD: University of California, San Diego
School of Medicine.
Address correspondence and reprint requests to Gary S.
Firestein, MD, Division of Rheumatology, Allergy, and Immunology,
University of California, San Diego School of Medicine, 9500 Gilman
Drive, La Jolla, CA 92093-0656. E-mail:
Submitted for publication April 5, 2004; accepted in revised
form May 12, 2004.
This process, initiated within minutes of surface
receptor ligation, releases NF-␬B and leads to its nuclear
translocation, followed by initiation of gene transcription. The specific genes that are activated depend on the
various NF-␬B–binding sequences in promoter regions
as well as the components of the NF-␬B dimers. IKK-2
is an absolute requirement for this pathway in many
circumstances, especially those related to innate immunity, while the absence of IKK-1 in knockout mice
typically has little effect on NF-␬B translocation. However, IKK-1 is an integral part of signaling through
RANK, the receptor activator of NF-␬B (7). Defective
IKK-1 activity has little effect on innate immune responses but is required for lymphoid development and T
cell–dependent antibody responses (8). Other kinases,
such as NF-␬B–inducing kinase, can be activated by
lymphotoxin and signals via an alternative pathway that
involves only IKK-1, with subsequent processing of
NF-␬B1 to produce p52 dimers (9).
The rate of I␬B degradation and resynthesis
depends on a variety of factors, including the particular
isoforms of I␬B expressed by each cell lineage. I␬B␣,
which is commonly found in synovial macrophages and
fibroblast-like synoviocytes, is rapidly degraded and then
resynthesized within 1–2 hours. Resynthesis of I␬B␤ in
other cells, however, is often delayed and leads to
prolonged NF-␬B translocation (10). Degradation of
I␬B⑀ is relatively slow, although this protein can be
quickly resynthesized. The binding affinities of different
I␬B isoforms for NF-␬B components also determine
gene regulation. For instance, I␬B␣ and I␬B␤ bind to
p65, but not to ␣ c-Rel; however, I␬B⑀ can associate with
both (11). The p50/p65 heterodimer binds to I␬B␤ with
greater avidity than do p50/RelB or p50/c-Rel dimers.
These differences are clearly relevant to approaches
designed to enhance I␬B expression, such as the TatsrI␬B␣ construct, when the variability in NF-␬B dimer
components are observed.
NF-␬B activation has been implicated in the
pathogenesis of RA. As demonstrated by electromobility shift assays, NF-␬B binding is significantly higher in
RA synovium compared with osteoarthritis synovium
(12,13). Immunohistochemistry studies identify nuclear
p50 and p65 translocation in the synovial lining and
mononuclear cells of the sublining. In vitro studies
confirm a role of NF-␬B in the production of cytokines
by macrophages, as well as elevated constitutive production of IL-6 by RA synoviocytes (14). Animal models of
RA also support the notion that NF-␬B participates in
synovitis. Time-course studies in both murine collageninduced arthritis and rat adjuvant-induced arthritis dem-
Figure 1. Potential therapeutic interventions designed to suppress
nuclear factor ␬B (NF-␬B) in arthritis. MAPKKK ⫽ mitogen-activated
protein kinase kinase kinase; NAK ⫽ NF-␬B–activating kinase; I␬B ⫽
inhibitor of ␬B; IKK ⫽ I␬B kinase; P ⫽ phosphate; U ⫽ ubiquitin.
onstrate NF-␬B activation prior to the appearance of
clinical disease (15). Selective activation of IKK-2 by
intraarticular gene transfer leads to arthritis in rats, thus
confirming that IKK activation is sufficient to initiate
synovitis (16).
Several commonly used therapeutic agents, most
notably, corticosteroids, can suppress NF-␬B, but more
selective inhibitors are clearly desirable in order to
minimize NF-␬B–independent toxicity. Blockade of the
IKK signal complex has attracted considerable attention
based on its pivotal role in NF-␬B signaling and potential inhibition by small molecules (Figure 1). IKK-2 is an
especially attractive target for therapy because it regu-
lates cytokine production in many cell types, including
cultured synoviocytes. For example, a dominantnegative IKK-2 adenovirus construct almost completely
abrogates cytokine-induced IL-6, IL-8, and ICAM-1
expression (17). Intraarticular gene therapy with
dominant-negative IKK-2 in rats with adjuvant-induced
arthritis decreases NF-␬B nuclear translocation and
suppresses joint swelling.
Although gene transfer has some advantages
when considering local therapy, the development of
small-molecule inhibitors of IKK-2 has contributed additional proof that this kinase is a potential target.
Published data on 2 selective IKK-2 inhibitors demonstrate remarkable clinical efficacy in rat adjuvantinduced arthritis and murine collagen-induced arthritis
(18,19). In addition to decreased inflammation, bone
and cartilage destruction is also significantly lower in the
animals treated with the IKK-2 inhibitors. Gene-transfer
studies suggest that cytokine production by cultured
rheumatoid synovial tissue cells is largely dependent on
IKK-2 (20). However, production of some cytokines,
such as TNF␣, is not blocked by dominant-negative
IKK-2, indicating a substantial degree of complexity that
was not anticipated. Information on the role of IKK-1 in
arthritis is less robust, and this kinase plays little role in
cytokine production by fibroblast-like synoviocytes. Endogenous peroxisome proliferator–activated receptor ␥
agonists, such as 5-deoxy-⌬12,14-prostaglandin J2, can
also decrease inflammatory responses through inhibition
of IKK and could serve as a normal counterregulatory
response (21).
While the early focus in arthritis has been on
IKK-2, other signaling pathways that feed into NF-␬B
also might be targeted. Upstream MAPKs, such as
MEKK-1 or NAK, have potential, as do the IKK-related
kinases inducible IKK (IKK-i) and TANK-binding kinase 1 (TBK-1) (22). The role of the latter two molecules in inflammation is only partially understood at the
present time. Although IKK-i can recognize I␬B as a
substrate, the process is inefficient, because only 1 of the
2 serines required for rapid degradation is phosphorylated. In contrast, the IKK-related kinases also participate in other pathways relevant to RA, including TLR
signal transduction and expression of IL-6 and
interferon-␤ (23). Little or no information is available on
the pharmacology of IKK-related kinases in inflammation.
Moving downstream from IKK, methods for stabilizing I␬B have been evaluated extensively in cell
culture. Typically, genetic constructs that overexpress
I␬B or an engineered protein that lacks the sites for
phosphorylation (I␬B super-repressor) have been used.
These proteins, although very effective in vitro, present
major technical hurdles in vivo because they must
enter the cells to be effective. In practice, this usually
requires gene transfer using viral or nonviral vectors.
Blackwell et al have added a very interesting twist to
I␬B therapy by using a novel construct that allows the
therapeutic protein to traverse cell membranes in
order to reach the cytoplasm. In a rat model of
pleurisy, they demonstrated that this fusion protein
does, indeed, penetrate cells and forms a stable
complex with NF-␬B that blocks activation. Even
more striking, systemic administration inhibits leukocyte migration into the pleural space after injection
with a proinflammatory stimulus. The mechanism
appears to be related to enhanced apoptosis, probably
due to loss of NF-␬B–mediated protection from cell
death. In addition, cytokine and chemokine production in the pleural cavity are decreased and probably
contribute to the antiinflammatory effect.
The authors also noted that local administration
of the protein has much less benefit than systemic
exposure. One possible explanation is that circulating
leukocytes are exposed to the construct after intravenous injection, thereby inducing apoptosis and preventing extrapleural cell activation that is required for cell
migration. Alternatively, the primary effect could be on
the vascular endothelium, where induction of adhesion
molecules, such as VCAM-1, ICAM-1, and E-selectin,
requires NF-␬B (24). Regardless of the mechanism, the
results suggest that in this model of acute inflammation,
systemic inhibition is necessary for the full benefit. The
observation is consistent with the clinical efficacy of
selective IKK-2 inhibitors in arthritis, which may be
greater with systemic administration than with intraarticular gene therapy techniques. However, it is important to recognize the significant differences between
acute inflammation, which is highly dependent on transient influx of neutrophils, and chronic disease, where
the prominent infiltration of the synovium with longerlived mononuclear cells dominates. Furthermore, the
pharmacokinetics of the Tat-srI␬B␣ protein after local
injection still needs to be determined, and reformulation
could improve efficacy.
The Tat-srI␬B␣ approach is a technical tour de
force and is unquestionably valuable as a proof-ofconcept experiment, but some questions should be answered to determine the utility of the construct as a
therapeutic agent. For example, the dosing and frequency of administration need to be assessed for longterm administration. Although it is clear that the con-
struct enters leukocytes, the distribution in other tissues
and the toxicity are not defined. Some concerns about
the role of NF-␬B inhibition in hepatocyte apoptosis
need to be addressed, along with effects on other host
responses to pathogens. These questions are not unique
to Tat-srI␬B␣, but should be considered with any longterm therapy that inhibits NF-␬B.
In addition to enhancing intracellular levels of
I␬B by providing exogenous protein, efforts to prevent
degradation of endogenous I␬B have been explored. For
instance, inhibitors of E2 ubiquitin–conjugating enzymes could interfere with I␬B ubiquitination. Alternatively, one could target the proteasome to prevent
degradation of I␬B after ubiquitination. One such compound, MG132, increases intracellular I␬B levels and,
like Tat-srI␬B␣ and other NF-␬B–targeted approaches,
induces synovial apoptosis in arthritis (25). One concern
with this method is the multitude of proteins that are
processed by ubiquitin and the proteasome, and toxicity
could be a major hurdle. Interfering with NF-␬B binding
and transactivating effects represents another post-I␬B
targeting alternative. For instance, decoy oligonucleotides that contain an NF-␬B-binding site can be delivered into cells to prevent gene transcription (26). This
approach is effective in rat streptococcal cell wall–
induced arthritis, and the beneficial effects again appear
to be related to induction of synovial apoptosis. Strategies to decrease the expression of specific NF-␬B proteins using antisense or RNA interference could selectively alter the gene expression patterns by changing the
composition of NF-␬B dimers.
Despite the abundant data indicating a key role
of NF-␬B in inflammatory diseases, significant safety
concerns about inhibition of this transcription factor
remain. NF-␬B sits at the crossroads of host defense,
homeostasis, cell survival, and response to stress, and
there are major issues related to systemic inhibition of
this primordial protective mechanism. Surely, a price
will be paid for blocking the innate immune responses
that begin with engagement of TLRs and result in
NF-␬B–driven gene expression. Some anticipated defects in host defense have already been identified, such
as increased susceptibility to infections such as Listeria
monocytogenes (27).
Equally important, the induction of apoptosis
due to NF-␬B inhibition has significant potential for
collateral damage to normal or physiologically stressed
tissues. The most prominent example is florid hepatic
apoptosis, which occurs in IKK-2– or RelA-knockout
mice (28). This problem can be abrogated by suppressing TNF responses, as shown in TNF and RelA–double-
knockout mice (29). TNF-dependent apoptosis is obviously relevant to diseases marked by increased
production of TNF␣, such as RA, where the risks of
NF-␬B blockade might be amplified. A similar doubleedged sword is apparent in mice lacking IKK-2 in
gastrointestinal enterocytes. Although these animals are
protected from systemic inflammatory responses after
gut ischemia-reperfusion, the local mucosa is damaged
by extensive apoptosis related to the lack of NF-␬B.
Because IKK-2 plays an essential role in B cell development (30), inhibitors could have a significant impact on
antibody production, germinal center formation, and
lymphoid tissue maturation. The role of NF-␬B proteins
in other normal immune responses and development
also raises concerns. Abnormal antibody production is
observed in p50-knockout mice, and absence of RelB
interferes with the development of dendritic cells. In
addition to B cell defects, osteoclast development has
also been noted in p50 and p52–double-knockout mice
The use of small-molecule inhibitors of various
kinases and regulatory proteins in the NF-␬B pathway
also suffers from potential issues related to the specificity of the compound. It is difficult to design truly
selective inhibitors because many bind to ATP-binding
sites with significant homology across the universe of
kinases. Screening for specificity against all possible
targets, as well as other ATP- or substrate-binding sites,
is problematic at best. Despite this, significant strides
have been made in the chemistry of kinase inhibitors,
and at least some specificity appears feasible. These
issues are, of course, less critical with biologic agents
such as Tat-srI␬B␣.
How are we to approach NF-␬B under such
circumstances? One goal could be to modulate, rather
than completely ablate, NF-␬B function. Global inactivation of NF-␬B, such as with proteasome inhibitors,
enhancement of cellular I␬B levels, or decoy oligonucleotides, could have the greatest potential for toxicity,
although some selectivity based on cell-specific I␬B
isoforms is possible. IKK-2 blockade suffers from many
constraints as well, but there are clearly alternative
pathways that permit some activation of NF-␬B. For
example, one selective IKK-2 inhibitor, SC-514, suppresses and delays I␬B degradation but does not entirely
block it (32). Even if IKK-2 were inhibited, IKK-1– and
IKK-related kinase-mediated I␬B phosphorylation
would remain intact, thereby permitting basal or stressinduced NF-␬B activation in some tissues. Other selective kinase-targeted approaches could potentially block
pathogenic NF-␬B activation and inhibit macrophage
cytokine production while permitting basal or normal
responses in other cell lineages. The use of biologics
such as Tat-srI␬B␣ could achieve site and event specificity based on the method of delivery, the NF-␬B
heterodimers and homodimers expressed, and the relative ability of the construct to enter different cell lineages.
The jury is still out on the relative balance
between the benefits and risks of NF-␬B blockade; we
do not know if NF-␬B is the Holy Grail or a tantalizing
and dangerous temptation. The recognition that RA is a
serious disease with considerable morbidity and mortality helps pave the way for aggressive therapy, and we
should not shy away from interventions that might
provide a major positive impact on our patients’ lives.
One recalls that worries about the dangers of anticytokine therapy contributed to considerable trepidation
over a decade ago; it turns out that humans are remarkably resilient and adapt more readily than our murine
brethren. Proof-of-concept in RA awaits while the multitude of NF-␬B–directed approaches move inexorably
toward the clinic.
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