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Use of molecular imaging to quantify response to IKK-2 inhibitor treatment in murine arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 1, January 2007, pp 117–128
DOI 10.1002/art.22303
© 2007, American College of Rheumatology
Use of Molecular Imaging to Quantify Response to
IKK-2 Inhibitor Treatment in Murine Arthritis
Elena S. Izmailova,1 Nancy Paz,1 Herlen Alencar,2 Miyoung Chun,1 Lisa Schopf,1
Michael Hepperle,1 Joan H. Lane,1 Geraldine Harriman,1 Yajun Xu,1 Timothy Ocain,1
Ralph Weissleder,2 Umar Mahmood,2 Aileen M. Healy,1 and Bruce Jaffee1
Objective. The NF-␬B signaling pathway promotes the immune response in rheumatoid arthritis
(RA) and in rodent models of RA. NF-␬B activity is
regulated by the IKK-2 kinase during inflammatory
responses. To elucidate how IKK-2 inhibition suppresses disease development, we used a combination of
in vivo imaging, transcription profiling, and histopathology technologies to study mice with antibodyinduced arthritis.
Methods. ML120B, a potent, small molecule inhibitor of IKK-2, was administered to arthritic animals,
and disease activity was monitored. NF-␬B activity in
diseased joints was quantified by in vivo imaging.
Quantitative reverse transcriptase–polymerase chain
reaction was used to evaluate gene expression in joints.
Protease-activated near-infrared fluorescence (NIRF)
in vivo imaging was applied to assess the amounts of
active proteases in the joints.
Results. Oral administration of ML120B suppressed both clinical and histopathologic manifestations of disease. In vivo imaging demonstrated that
NF-␬B activity in inflamed arthritic paws was inhibited
by ML120B, resulting in significant suppression of
multiple genes in the NF-␬B pathway, i.e., KC, epithelial
neutrophil–activating peptide 78, JE, intercellular adhesion molecule 1, CD3, CD68, tumor necrosis factor ␣,
interleukin-1␤, interleukin-6, inducible nitric oxide synthase, cyclooxygenase 2, matrix metalloproteinase 3,
cathepsin B, and cathepsin K. NIRF in vivo imaging
demonstrated that ML120B treatment dramatically reduced the amount of active proteases in the joints.
Conclusion. Our data demonstrate that IKK-2
inhibition in the murine model of antibody-induced
arthritis suppresses both inflammation and joint destruction. In addition, this study highlights how gene
expression profiling can facilitate the identification of
surrogate biomarkers of disease activity and treatment
response in an experimental model of arthritis.
NF-␬B signaling is thought to play an important
role in the inflammatory response underlying the pathogenesis of autoimmune diseases, including rheumatoid
arthritis (RA). NF-␬B activity is regulated by the IKK
complex. This complex consists of 2 kinases, IKK-1 and
IKK-2, and a regulatory subunit, IKK␥/NEMO. Recent
data indicate that IKK-2, rather than IKK-1, participates
in the pathway by which proinflammatory stimuli induce
NF-␬B function (1).
NF-␬B activity is implicated in promoting both
inflammation and tissue remodeling, by activating the
transcription of many key genes (2). Recent data from
both clinical RA studies and studies in rodent models of
arthritis suggest that inflammation and destruction of
articular structures occur independent of one another
(3,4). It is well established that NF-␬B is involved in the
regulation of multiple proinflammatory mechanisms.
Activation of NF-␬B is necessary and sufficient for
transcriptional activation of intercellular adhesion mol-
1
Elena S. Izmailova, PhD, Nancy Paz, MS (current address:
Merrimack Pharmaceuticals, Cambridge, Massachusetts), Miyoung
Chun, PhD (current address: University of California, Santa Barbara),
Lisa Schopf, PhD, Michael Hepperle, PhD (current address: Phenomix
Corporation, San Diego, California), Joan H. Lane, DVM, DACVP,
Geraldine Harriman, PhD, Yajun Xu, PhD, Timothy Ocain, PhD,
Aileen M. Healy, PhD, Bruce Jaffee, PhD: Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts; 2Herlen Alencar, MD, Ralph
Weissleder, MD, PhD, Umar Mahmood, MD, PhD: Massachusetts
General Hospital, Charlestown.
Drs. Izmailova, Schopf, Lane, Harriman, Ocain, and Jaffee
have or have had stock or stock options in Millennium Pharmaceuticals, Inc. Drs. Weissleder and Mahmood have stock or stock options in
Visen Medical. Dr. Mahmood holds patents on activatable optical
agents.
Address correspondence and reprint requests to Elena S.
Izmailova, PhD, Millennium Pharmaceuticals, Inc., 35 Lansdowne
Street, Cambridge, MA 02139. E-mail: Elena.Izmailova@mpi.com.
Submitted for publication March 10, 2006; accepted in revised
form September 22, 2006.
117
118
ecule 1 (ICAM-1) and the chemokines monocyte chemotactic protein 1 and interleukin-8 (IL-8). These molecules facilitate infiltration of inflammatory cells into
the diseased joint. NF-␬B also regulates transcription of
tumor necrosis factor ␣ (TNF␣), IL-1␤, IL-6, inducible
nitric oxide synthase (iNOS), and cyclooxygenase 2
(COX-2) (5), all of which are required for the initiation,
amplification, and maintenance of chronic inflammation. The role of NF-␬B in the mechanisms regulating
tissue remodeling is less well defined.
Several lines of evidence indicate that heightened
protease activity is a contributing factor in the tissue
remodeling associated with RA (6,7). Proteases play a
role in pannus growth and degradation of the articular
structure. Both matrix metalloproteinases (MMPs) and
cysteine proteases are implicated in the development of
RA (8–11). NF-␬B regulates the production of some
MMPs, such as MMP-3 and MMP-13 (12), but its effect
on cysteine proteases remains unclear.
One of the major challenges in assessing the
efficacy of antirheumatic drugs is the inability to estimate protease activity in the joints directly and noninvasively in vivo. Radiographic assessment of the joint
does not generally take place until late in the course of
the disease, which limits its usefulness. The recent
development of near-infrared fluorescence (NIRF) imaging probes allows for the localization, visualization,
and quantification of signal in areas with increased
proteolytic activity in vivo (13). Such molecular reporters may serve as sensitive indicators for use in objective
assessment of treatment response.
ML120B is a potent and selective small molecule
inhibitor of IKK-2 (14) that suppresses the clinical and
histologic disease manifestations of antibody-induced
arthritis in mice. IKK-2 inhibition also results in significant suppression of multiple genes in the NF-␬B pathway that are involved in the development of murine
antibody-induced arthritis.
In the present study, we combined molecular
imaging with gene expression profiling and histopathologic examination to examine the effects of IKK-2
inhibition in murine antibody-induced arthritis. In vivo
imaging demonstrated direct inhibition of NF-␬B activity by ML120B in inflamed paws. We found that the
IKK-2 inhibitor suppressed expression, in the joints, of
several proinflammatory genes, including the adhesion
molecule ICAM-1, the chemokines JE, KC, and epithelial neutrophil–activating peptide 78 (ENA-78), the inflammation mediators TNF␣, IL-1␤, IL-6, COX-2, and
iNOS, as well as 2 markers of mononuclear cell infiltra-
IZMAILOVA ET AL
tion, CD3 and CD68. Of note, ML120B treatment also
inhibited the expression of the tissue remodeling genes
MMP-3 and the cysteine proteases cathepsin B and
cathepsin K. Furthermore, IKK-2 suppression dramatically reduced the level of destructive enzymes in diseased joints as demonstrated by noninvasive NIRF
imaging, and these findings were concordant with histologic evidence of cartilage damage and bone resorption.
Thus, our data demonstrate that IKK-2 inhibition of
antibody-induced arthritis in mice is likely the result
of suppression of both inflammation and degradation of
bone and cartilage.
MATERIALS AND METHODS
Induction of arthritis in mice. Female BALB/c mice
were purchased from Charles River (Wilmington, MA). All
mice were housed at Millennium Pharmaceuticals with free
access to standard rodent chow diet and water and were
studied at 8–9 weeks of age. Animal studies were performed
according to Institutional Animal Care and Use Committee
standards. For induction of arthritis, mice were injected intravenously with 2 mg anti–type II collagen (anti-CII) antibody,
according to the protocol recommended by the manufacturer
(Chemicon, Temecula, CA). Three days later, animals received
an intraperitoneal injection of 12.5 ␮g lipopolysaccharide
(LPS) (Escherichia coli O111:B4).
Administration of the IKK-2 inhibitor ML120B. The
IKK-2 inhibitor ML120B was administered orally at 10 mg/kg,
30 mg/kg, or 60 mg/kg in 0.5% hydroxypropyl-methylcellulose/
0.2% Tween twice daily starting on day 0 after anti-CII
antibody injection. Untreated animals and animals injected
with LPS only were used as nonarthritic controls.
Clinical disease scoring. The severity of arthritis was
graded visually by assessing the level of swelling in each paw,
including the tarsus (ankle) and carpus (wrist) joints. Scores
were assessed consistently 1 hour after morning dosing, at the
following time points: day 0 (before administration of anti-CII
antibody), day 3 (before disease boost with the LPS injection),
day 5 or 6 (when clinical symptoms became clearly visible), and
days 7 and 9 (peak of disease activity). The following scoring
system was used: 0.5 ⫽ slight redness, 1.0 ⫽ 1 or more digits
swollen, 1.5 ⫽ 1 or more digits swollen and red/swollen tarsus,
2.0 ⫽ moderate swelling, 2.5 ⫽ moderate swelling and swollen
digits, 3.0 ⫽ severe swelling, 3.5 ⫽ severe swelling and
moderate ankylosis, and 4.0 ⫽ severe swelling and complete
ankylosis (maximum possible total score per animal 16).
Histopathology. Hind paws from one side of each
mouse were fixed for 4 days in 10% buffered formalin,
decalcified for 2 weeks in 5% formic acid, and processed for
paraffin embedding. Eight-micrometer sections were stained
with toluidine blue and scored, under blinded conditions, by a
veterinary pathologist (at BoulderPath, Boulder, CO) for
inflammation, pannus formation/cartilage loss, and bone erosion. Severity was scored on a 0–5 scale.
In vivo NF-␬B imaging. For NF-␬B imaging studies
(15), antibody-induced arthritis was introduced into transgenic
RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE
female BALB/c mice as described above. ML120B was administered orally at 60 mg/kg twice daily. Imaging experiments
were performed 1 hour after compound administration (the
time of maximum concentration in peripheral blood) on days
0, 3, 7, and 9. For the imaging procedure, mice were anesthetized with 2% isoflurane in oxygen, and luciferin (150 mg/kg)
was injected intraperitoneally. Ten minutes after luciferin
injection, the animals were imaged in an IVIS200 system
(Xenogen, Alameda, CA), with a 1-minute bioluminescence
exposure. Signal quantification was based on region of interest
analysis.
RNA extraction and polymerase chain reaction (PCR)
analysis. Total RNA was prepared from each animal separately using left rear paw whole-joint homogenate. RNA was
extracted by the single-step method using RNA Stat-60 (TelTest, Friendswood, TX). After treatment with DNase I (Qiagen, Valencia, CA), complementary DNA (cDNA) was synthesized using the MultiScribe Reverse Transcription kit
(Applied Biosystems, Foster City, CA). Gene expression was
measured by TaqMan real-time PCR according to the protocol
recommended by the manufacturer (Applied Biosystems).
Target gene probes were labeled with 6-FAM, and the
internal reference probe, rodent GAPDH, was labeled with
VIC. PCRs were performed with the forward and reverse
primers (200 nM) and the probe (100 nM) for GAPDH and the
forward and reverse primers (600 nM) and probe (200 nM) for
the gene of interest. The experiments were performed on an
ABI Prism 7700 Sequence Detection System (Applied Biosystems) under the following conditions: 2 minutes at 50°C and 10
minutes at 95°C, followed by 2-step PCR for 40 cycles of 95°C
for 15 seconds followed by 60°C for 1 minute. The number of
PCR cycles needed for FAM and VIC fluorescence to cross a
threshold of a statistically significant increase in fluorescence
(threshold cycle [Ct]) was measured using Applied Biosystems
software. Relative target gene expression was determined
using the following formula: relative expression ⫽ 2-⌬⌬Ct,
where ⌬⌬Ct ⫽ (Ct target gene – Ct reference gene in experimental
cDNA sample) ⫺ (Ct target gene – Ct reference gene in mock
reverse-transcribed RNA sample).
In vivo protease optical imaging and image analysis.
Fluorescence reflectance imaging was performed using an
epifluorescence system (bonSAI; Siemens, Erlangen, Germany) that is capable of near simultaneous data acquisition in
multiple channels, including a broad-spectrum visible white
light channel providing anatomic detail and an NIR channel
providing molecular imaging information. The system consists
of a 150W halogen excitation light source connected to the
acquisition box through an optical waveguide. The built-in
filter wheel is set to deliver light at 400–745 nm for white light
images and at 645–675 nm for NIR images. On the detection
side, a second filter wheel uses a 4-step neutral optical density
filter for white light and a 720–750–nm filter for activated
Prosense (Visen Medical, Woburn, MA) NIR fluorescence
detection. A charge coupled device camera with a matrix size
of 1,360 ⫻ 1,024 pixels and a resolution of 0.116 mm/pixel was
used for image acquisition. Anesthesia was maintained by
mask inhalation of isoflurane vaporized at concentrations of
up to 4% in the induction phase, and 1.5% during imaging.
The isoflurane was delivered along with 100% oxygen at a flow
119
rate of 2 liters/minute. During imaging, body temperature was
kept constant at 37°C.
Previous experiments aimed at optimizing imaging
time indicated that statistically significant differences in signal
intensity between control and diseased animals can be detected as early as day 6. Fluorescence signal intensity reaches
its maximum at the peak of disease activity, between days 7 and
10. We performed imaging on days 6 and 9 after anti-CII
antibody injection, to detect early disease response to treatment and to assess the effect of the added compound when
disease activity is maximal. The imaging procedure was carried
out 24 hours after intravenous injection of the proteaseactivated probe Prosense, which contained a total of 2 nmoles
of quenched NIRF dye. This dose and timing were based on
doses that had previously been used in tumor model systems.
Signal intensity of this class of activated probe is linear with
respect to enzyme concentration. Exposure time was 0.3
seconds for all fluorescence images, and the data were stored
in DICOM (Digital Imaging and Communications in Medicine) format for subsequent analysis. System control and data
storage were performed with a PC using Syngo software
(Siemens). Using custom software (CMIRImage; Center for
Molecular Imaging Research, Massachusetts General Hospital), signal intensities from all fluorescence images were determined using a circular region of interest placed over ankle
joints, skin (snout), and a reference standard. The fluorescence
signal from the mouse paws, expressed as relative fluorescence
intensity, was calibrated to the standard, subtracting the skin
value as background. Immediately after the last imaging session, animals were killed and paws were obtained for RNA
expression analysis and histologic study.
Statistical analysis. Summary statistics are reported as
the mean ⫾ SEM for each treatment group. Data were tested
for normal distribution. One-way analysis of variance with
Dunnett’s multiple comparison test was used to identify significant differences between experimental and vehicle-treated
control groups. Pearson’s correlation coefficient was used to
express the correlation between fluorescence intensity and the
means of total clinical scores. All statistics were generated
using GraphPad Prism software (GraphPad, San Diego, CA).
P values less than 0.05 were considered significant.
RESULTS
Increase in NF-␬B activity in vivo in murine
arthritis. To assess NF-␬B activity during arthritis, we
induced antibody-induced arthritis in transgenic mice
expressing the luciferase gene, under control of the
NF-␬B–inducible promoter (15). We visualized NF-␬B
activity in diseased joints by in vivo bioluminescence
imaging, captured images, and quantified the luminescence signal intensity in nonarthritic controls and arthritic paws during disease progression. On day 7 after
injection of anti-CII antibodies, signal intensity was
6.5-fold higher in arthritic animals than in the control
120
IZMAILOVA ET AL
Figure 1. Up-regulation of NF-␬B activity in vivo in the murine arthritis model. The model was generated in transgenic mice expressing the
luciferase gene under the control of the NF-␬B–inducible promoter, as described in Materials and Methods. A, In vivo bioluminescence imaging.
Shown are overlays of photographic and color-coded bioluminescence images of the front and hind paws of nonarthritic control mice (day 0) and
arthritic mice on days 3, 7, and 9 after the injection of anti–type II collagen antibodies. B, Quantitative analysis of total bioluminescence signal
intensity in front and hind paws. Values are the mean ⫾ SEM signal intensity in nonarthritic controls (day 0) and in arthritic mice on days 3, 7, and
9, and are representative of 3 independent experiments. Each group of mice consisted of 6–8 animals. ⴱ ⫽ P ⬍ 0.05 versus controls.
group. On day 9, signal intensity was 9.4-fold higher in
the arthritic animals compared with controls (Figure 1).
Inhibition of the development of antibodyinduced arthritis by ML120B treatment. Clinical disease symptoms. To determine if inhibition of the
NF-␬B pathway affects antibody-induced arthritis development, we used ML120B, a potent and selective inhibitor of IKK-2 kinase (14). The antibody-induced
arthritis model was initiated by injection of anti-CII
antibodies, and animals were treated orally twice daily
with various concentrations of ML120B or vehicle
alone, as described above. Vehicle-treated animals developed severe arthritis, with clinical disease symptoms
(redness, swelling, ankylosis) appearing on day 5 and
peaking between days 7 and 9 (Figure 2A). Clinical
disease symptoms were almost completely inhibited
with 60 mg/kg of ML120B. A small number of animals
(2 of 6) developed scores of 0.5 in 1 paw on days 7 and
10 with ML120B at this dose level. Mice treated with
ML120B exhibited a dose-dependent decrease in clinical
symptoms compared with vehicle-treated animals (Figure 2A). Based on these data, further imaging studies
characterizing the effects of IKK-2 inhibition were performed using the maximally efficacious dose evaluated,
i.e., 60 mg/kg.
Histopathologic assessment. We compared nonarthritic, vehicle-treated, and ML120B-treated animals on
days 6 and 9 after disease initiation. Histologic staining
RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE
121
Figure 2. Suppression of disease development in the murine antibody-induced arthritis model by administration of the IKK-2 inhibitor ML120B.
A, Mean ⫾ SEM clinical scores on days 0, 3, 5, 7, and 10 in mice administered anti–type II collagen (anti-CII) antibodies and subsequently treated
with vehicle or with ML120B twice daily at the doses shown. Each group of mice consisted of 6–8 animals. Values shown are representative of 5
independent experiments. ⴱⴱ ⫽ P ⬍ 0.01 versus vehicle-treated animals. B, Histopathologic assessment, on days 6 and 9 after administration of
anti-CII, of joint sections from representative mice treated with vehicle or with ML120B at 60 mg/kg (toluidine blue–stained; original
magnification ⫻ 10). C, Total pathology scores in the paws of nonarthritic control, vehicle-treated, and ML120B-treated mice on days 6 and 9 after
administration of anti-CII. Values are the mean and SEM.
122
IZMAILOVA ET AL
Figure 3. Suppression of NF-␬B activity in vivo in arthritic mice treated with the IKK-2 inhibitor ML120B. A, Overlays of photographic and
color-coded bioluminescence images of the front and hind paws of nonarthritic control mice, vehicle-treated arthritic mice, and 60 mg/kg
ML120B–treated mice on days 7 and 9. B, Quantitative analysis of total bioluminescence signal intensity. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle-treated
animals. Embedded graph shows the total clinical scores of the animals used in the imaging experiments. Values are the mean ⫾ SEM.
of paw sections (Figure 2B) indicated that as early as day
6, joints from the vehicle-treated group had moderate
cellular infiltration, edema, and minimal growth of pannus into the cartilage and subchondral zone. Cellular
infiltrates were composed predominantly of neutrophils,
accompanied by fibroblast-like cells and smaller numbers of lymphocytes and macrophages. The joints displayed minimal-to-mild chondrocyte loss or collagen
disruption, while a few animals (2 of 8) had small areas
of bone resorption and rare osteoclasts in affected joints
at this time point. On day 9, animals in the vehicletreated group showed marked cellular infiltrates and
edema, pronounced pannus formation, mild-to-marked
chondrocyte and collagen loss, larger areas of bone
resorption, and more frequent osteoclasts compared
with day 6. In contrast, joints from ML120B-treated
animals were mostly disease free on both day 6 and day
9. A small number of animals (2 of 8 on day 6) showed
some cell infiltration, mild edema, minimal pannus
formation, and cartilage loss (Figure 2C). Thus, animals
treated with 60 mg/kg of ML120B twice daily exhibited
suppressed disease development as judged by histopathologic as well as clinical features.
Inhibition of NF-␬B activity in arthritic paws by
ML120B treatment. To determine whether ML120B
treatment directly inhibits NF-␬B activity in vivo, we
measured NF-␬B activity by quantifying the luminescence signal intensity in paws of compound-treated
animals and compared the findings with those in the
vehicle-treated group. Luminescence signal intensity
in ML120B-treated animals was significantly lower than
that in the vehicle-treated animals and similar to the
levels detected in nonarthritic controls (Figure 3A).
Moreover, luminescence signal intensity correlated
RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE
123
Figure 4. Suppressed expression of disease-mediating genes in arthritic mice treated with the IKK-2 inhibitor ML120B. Gene expression in the paws
of nonarthritic control mice, vehicle-treated arthritic mice, and 60 mg/kg ML120B–treated mice on days 6 and 9 was measured by quantitative reverse
transcriptase–polymerase chain reaction. Values are the mean ⫾ SEM relative expression (RE) of each gene. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽
P ⬍ 0.001, versus vehicle-treated animals. ICAM-1 ⫽ intercellular adhesion molecule 1; ENA-78 ⫽ epithelial neutrophil–activating peptide 78;
TNFa ⫽ tumor necrosis factor ␣; IL-1b ⫽ interleukin-1␤; iNOS ⫽ inducible nitric oxide synthase; COX-2 ⫽ cyclooxygenase 2; MMP-3 ⫽ matrix
metalloproteinase 3.
124
with clinical scores during disease progression (r ⫽ 0.6,
P ⬍ 0.001). Thus, these results demonstrate a correlation between the inhibition of NF-␬B activity in
arthritic paws in vivo and clinical disease activity
(Figure 3B).
Suppression of the expression of diseasemediating genes by ML120B treatment. The development of RA is associated with chronic active inflammation in the synovial tissue and damage to
articular surfaces. Antibody-induced arthritis exhibits
both periarticular inflammation and cartilage destruction in a subacute to chronic-active setting. To gain
insight into the pathogenic processes blocked by
ML120B, we examined gene expression in whole-joint
homogenates from nonarthritic control, vehicle-treated,
and 60 mg/kg ML120B–treated mice, by quantitative
reverse transcriptase–PCR.
First, we examined molecules involved in the
development of inflammation. We found an 11–13-fold
increased induction of ICAM-1 on days 6 and 9 in the
paws of vehicle-treated animals compared with the
control group. A similar expression profile was observed for ENA-78, KC, and JE, neutrophil and monocyte chemoattractants induced by NF-␬B (Figure 4).
Compared with vehicle-treated animals, ML120Btreated mice exhibited significantly suppressed ICAM-1
levels on days 6 (61%) and 9 (86%). Moreover, on
both day 6 and day 9, ML120B-treated animals displayed significantly inhibited gene expression for chemokines KC (88% and 110%), ENA-78 (98% and 99%),
and JE (77% and 58%) compared with vehicle-treated
animals.
We estimated modulation of the composition of
cellular infiltrate in joints via examination of CD3 and
CD68 expression, for T cells and macrophages, respectively. The expression of CD3 was increased 3.3-fold and
CD68 expression was increased 7.2-fold on day 9 in the
vehicle-treated animals compared with the control
group, indicating increased infiltration of both T cells
and macrophages. Levels of CD3 and CD68 expression
in the joints of ML120B-treated animals were comparable with levels in the joints of the nonarthritic controls;
this was corroborated histologically by observation of
decreased cellular infiltrate.
With regard to expression of the inflammation
mediators TNF␣, IL-1␤, IL-6, iNOS, and COX-2, we
detected a modest but significant increase in expression
of TNF␣ (3-fold; P ⫽ 0.008) and COX-2 (2.5-fold; P ⫽
0.0004) in the vehicle-treated mice, and these animals
exhibited a dramatic increase in levels of messenger
IZMAILOVA ET AL
RNA (mRNA) for IL-1␤ (11-fold), IL-6 (76-fold), and
iNOS (10-fold). ML120B treatment caused a profound
inhibition of all proinflammatory markers on both day 6
and day 9 (TNF␣ 48% and 96%, respectively, IL-1␤ 91%
and 98%, respectively, IL-6 94% and 100%, respectively,
iNOS 80% and 66%, respectively, and COX-2 128% and
116%, respectively) (Figure 4).
We next investigated whether IKK-2 inhibition
ameliorates joint damage via matrix remodeling, by
quantifying the expression of matrix-degrading enzymes.
Expression of MMP-3 (stromelysin) was elevated up to
7.3-fold in the vehicle-treated group. ML120B administration suppressed MMP-3 expression to levels comparable with those in control mice. We also measured
expression of genes for cysteine proteases and found
that cathepsin B expression was elevated 3.2-fold on
day 6 and 6-fold on day 9 in vehicle-treated mice.
Expression of cathepsin K was significantly increased
only on day 9 (12-fold). ML120B administration significantly inhibited the expression of cathepsin B on both
day 6 and day 9 (73% and 66%, respectively). Cathepsin
K expression was inhibited by 70% on day 9 (Figure 4).
The gene expression profiling results are summarized in
Table 1.
Reduction of levels of destructive proteases in the
joint by ML120B treatment. Since we detected increased
mRNA levels for cathepsins B and K in arthritic joints,
which were inhibited in the ML120B-treated group, we
selected an optical probe activated by cathepsin proteases to monitor protease activity in vivo. The Prosense
“smart” probe is an enzyme-cleavable fluorescent probe
specific for a defined subset of proteases, including
cathepsins B and K. Probe fluorescence remains
quenched when the probe is administered intravenously,
until activation by enzyme cleavage in the target tissue
(16). The Prosense probe was injected into control,
vehicle-treated, and 60 mg/kg ML120B–treated animals,
and an in vivo image was captured and fluorescence
intensity in paws quantified (Figure 5A). The fluorescence signal was increased 4.2-fold in the paws of
vehicle-treated mice on day 6 after the injection of
anti-CII antibodies and 8-fold on day 9 (Figure 5B),
which indicates augmented probe enzymatic cleavage in
arthritic paws, resulting in fluorescence activation. The
increased fluorescence signal during disease progression
correlated with clinical scores (r ⫽ 0.95, P ⬍ 0.001)
(Figure 5C). The signal intensity was significantly lower
in the animals receiving ML120B and was similar to the
levels found in the control group (Figures 5A and B).
RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE
125
Table 1. Regulation of gene expression in the antibody-induced arthritis model and suppression by the IKK-2 inhibitor
ML120B*
Day 6
Day 9
Gene
Mean fold induction
over nonarthritic
controls
Mean %
inhibition with
ML120B
CD3
CD68
ICAM-1
JE
KC
ENA-78
TNF␣
IL-6
IL-1␤
iNOS
COX-2
MMP-3
Cathepsin B
Cathepsin K
2.2
3
13
40
8.8
22
3
76
11
10
2.5
7.3
3.2
1.7
75
40
61
77
88
98
48
94
91
80
128
97
73
⫺14
P†
Mean fold induction
over nonarthritic
controls
Mean %
inhibition with
ML120B
P†
⬍0.05
⬍0.01
⬍0.01
⬍0.01
⬍0.001
⬍0.001
⬍0.05
⬍0.001
⬍0.001
⬍0.001
⬍0.001
⬍0.001
⬍0.001
NS
3.3
7.2
11
29
5.2
37
3.4
72
10
8.4
3.2
3.5
6
12
89
87
86
58
110
99
96
100
98
66
116
119
66
70
NS
⬍0.001
⬍0.05
NS
⬍0.001
⬍0.001
⬍0.05
⬍0.001
⬍0.001
⬍0.05
⬍0.001
⬍0.05
⬍0.01
⬍0.01
* NS ⫽ not significant; ICAM-1 ⫽ intercellular adhesion molecule 1; ENA-78 ⫽ epithelial neutrophil–activating peptide 78;
TNF␣ ⫽ tumor necrosis factor ␣; IL-6 ⫽ interleukin-6; iNOS ⫽ inducible nitric oxide synthase; COX-2 ⫽ cyclooxygenase 2;
MMP-3 ⫽ matrix metalloproteinase 3.
† Significance of the percent inhibition with ML120B.
DISCUSSION
Our results demonstrate that the combination of
molecular imaging methods, gene expression profiling,
and histopathologic analysis is a powerful approach to
understanding the mechanism of action of small molecule inhibitors in complex models of disease. We assessed the effects of the IKK-2 inhibitor ML120B in a
murine arthritis model and demonstrated that, at the
efficacious dose, this compound suppressed NF-␬B activity and inhibited mediators of inflammation and joint
destruction.
There are some differences between the pathogenesis of murine antibody-induced arthritis and that of
RA in humans, i.e., the antibody-induced arthritis is
subacute while RA is generally chronic with periodic
flares. Nevertheless, the known shared features of the
characteristic inflammatory response to injury render
the model informative with respect to the pathways of
interest for potential therapeutic intervention.
Several studies have assessed in vitro NF-␬B–
DNA binding activity in synovial tissue both from humans and from animals with experimental disease. NF␬B–DNA binding was significantly greater in human RA
tissue compared with that from patients with osteoarthritis, and moreover, was localized to the sites of
maximum tissue destruction (17). Increased NF-␬B–
DNA binding activity has been demonstrated in synovial
extracts from rats and mice following disease development (18,19). The inhibition of NF-␬B binding after
IKK-2 inhibitor treatment has also been demonstrated
in the mouse collagen-induced arthritis model (19) and
the rat adjuvant-induced arthritis model (18). In vivo
imaging enabled us to visualize and quantify target
activity in the inflamed joints of mice. Our data extend
previous observations by providing in vivo evidence of
NF-␬B activity during disease development and its suppression in arthritic joints via the administration of the
IKK-2 inhibitor ML120B.
NF-␬B regulates the expression of multiple
genes involved in arthritis pathogenesis. This regulation
may be direct, via activation of gene transcription, or
indirect, via the secondary downstream effects of
NF-␬B–regulated gene products. For example, TNF␣
production is regulated by NF-␬B directly via the
TNF␣ promoter, which contains an NF-␬B binding site
(20,21). NF-␬B also regulates the expression of cell
adhesion molecules and inflammatory cell chemoattractants (5), and therefore, indirectly regulates cell
migration. Our mRNA expression analyses provide evidence of decreased cell infiltration into the joints of
ML120B-treated animals, which was confirmed by histopathologic analyses. One potential mechanism ex-
126
IZMAILOVA ET AL
Figure 5. Reduced levels of destructive proteases in the joints of arthritic mice treated with the IKK-2 inhibitor ML120B. Protease-activated probe
was injected intravenously 24 hours prior to the imaging experiment. A, Superimposition of white light and near-infrared fluorescence images of the
paws of nonarthritic control, vehicle-treated, and 60 mg/kg ML120B–treated mice on days 0, 6, and 9. B, Time course of fluorescence intensity.
Embedded graph represents the means of total clinical scores of the animals used in the imaging experiments. Values are the mean ⫾ SEM.
ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle-treated animals. C, Correlation between mean total clinical scores and relative fluorescence intensity in arthritic hind
paws 24 hours after injection of Prosense probe injection. Fluorescence intensities in arthritic animals correlated strongly with clinical scores
(r ⫽ 0.95, P ⬍ 0.001).
RESPONSE TO IKK-2 INHIBITION IN ARTHRITIC MICE
plaining this observation may be inhibition of chemotactic chemokines and/or leukocyte adhesion molecules
in the joint.
Previous reports on the effects of IKK-2 inhibitors in animal models were limited to genes directly
regulated by NF-␬B (19,22). We investigated the impact of IKK-2 inhibition on genes that are both directly
and indirectly regulated by NF-␬B in the development
of experimental arthritis. As expected, we observed the
inhibition of several genes that are directly regulated
by NF-␬B and involved in cell migration, inflammation,
and matrix degradation, including ICAM-1, TNF␣,
IL-1␤, IL-6, KC, ENA-78, JE, iNOS, COX-2, and
MMP-3, in the diseased paws of animals treated with the
IKK-2 inhibitor. Reports concerning the role of NF-␬B
in cathepsin expression are controversial, and it is not
clear whether IKK-2 inhibition has a direct or indirect
effect (23,24). However, in vivo IKK-2 inhibition by
ML120B suppressed the transcription of the cathepsin B
and cathepsin K genes. Overall, our data indicate that
IKK-2 inhibition has effects on multiple genes involved
in the inflammatory and destructive components of
disease.
We observed increased expression of mRNA for
several proteases, including cathepsins B and K, in
arthritic animals. Since reduced expression of these
genes was found in the ML120B-treated group, we
hypothesized that ML120B therapy decreases the levels
of destructive enzymes in joints. To test this, we used
NIRF imaging with a protease-specific probe. We selected a probe that is activated by the cysteine protease
cathepsin, because the results of previous studies demonstrated that cysteine proteases are up-regulated in RA
synovial tissue and fluid (25). Increased cysteine protease expression is predominantly restricted to the synovium at sites of joint damage and can be detected as
early as 2 weeks after the onset of disease symptoms
(26). These enzymes directly degrade cartilage and bone
matrices. In addition to their destructive activities, they
can activate MMPs (27). Consistent with these findings,
we observed a dramatic increase in fluorescence signal
intensity in the joints of vehicle-treated animals, which
indicated increased amounts of active proteases. In
contrast, signal intensity in the ML120B-treated group
was similar to that in the nonarthritic control animals.
The reduction of protease-activated fluorescence
signal in response to methotrexate treatment has been
observed in the murine collagen-induced arthritis model
(16). However, that study did not include analysis of the
correlation between the decrease in protease-activated
127
fluorescence signal and clinical disease indices such as
redness, swelling, and ankylosis. In the present study we
demonstrated that protease-activated NIRF imaging
probes can be used as sensitive biomarkers of subacute
to chronic active joint disease activity and treatment
efficacy in a murine antibody-induced arthritis model.
We observed complete disease suppression with the
IKK-2 inhibitor at the efficacious concentration used in
this study. Current efforts are aimed at testing the
prognostic utility of the Prosense probe for predicting
clinical and histopathologic disease amelioration.
In conclusion, we have demonstrated, using in
vivo imaging and gene expression profiling, that the
ML120B compound offers protection from inflammation and joint destruction in subacute to chronic active
murine antibody-induced arthritis. Moreover, we
showed a direct correlation between the drug’s diseasemodifying effects and biochemical target activity. These
studies highlight how gene expression profiling can be
implemented to identify surrogate biomarkers of disease
activity and treatment response in experimental models
of arthritis.
ACKNOWLEDGMENTS
The authors would like to thank K. Anderson and E.
Grant for technical assistance, and A. Parker and C. Fraser for
critical review of the manuscript.
AUTHOR CONTRIBUTIONS
Dr. Izmailova had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Drs. Izmailova, Alencar, Chun, Schopf, Xu, Ocain,
Weissleder, Mahmood, Healy, and Jaffee.
Acquisition of data. Dr. Izmailova, Ms Paz, and Drs. Alencar, Mahmood, and Jaffee.
Analysis and interpretation of data. Drs. Izmailova, Alencar, Schopf,
Lane, Xu, Ocain, Mahmood, Healy, and Jaffee.
Manuscript preparation. Dr. Izmailova, Ms Paz, and Drs. Alencar,
Schopf, Lane, Ocain, Mahmood, Healy, and Jaffee.
Statistical analysis. Dr. Izmailova, Ms Paz, and Dr. Jaffee.
Medicinal chemistry. Dr. Hepperle.
Invention of chemical matter, progression of key compound. Dr.
Harriman.
Project leadership. Dr. Xu.
Manuscript editing. Dr. Weissleder.
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