ARTHRITIS & RHEUMATISM

Vol. 54, No. 4, April 2006, pp 11841197

DOI 10.1002/art.21771
2006, American College of Rheumatology

Intraarticular Induction of Interleukin-1 Expression in the

Adult Mouse, With Resultant Temporomandibular Joint
Pathologic Changes, Dysfunction, and Pain
Yu-Ching Lai, Solomon S. Shaftel, Jen-nie H. Miller, Ross H. Tallents, Yoon Chang,
Carl A. Pinkert, John A. Olschowka, Ian M. Dickerson, J. Edward Puzas,
M. Kerry OBanion, and Stephanos Kyrkanides
Objective. To examine the effects of intraarticular
induction of interleukin-1 (IL-1) expression in adult
mice.
Methods. We used somatic mosaic analysis in a
novel transgenic mouse with an inducible IL-1 transcription unit. Transgene activation was induced by Cre
recombinase in the temporomandibular joints (TMJs)
of adult transgenic mice (conditional knockin model).
The effects of intraarticular IL-1 induction were subsequently evaluated at the cellular, histopathologic, and
behavioral levels.
Results. We developed transgenic mice capable of
germline transmission of a dormant transcription unit
consisting of the mature form of human IL-1 as well as
the reporter gene -galactosidase driven by the rat
procollagen 1A1 promoter. Transgene activation by a
feline immunodeficiency virus Cre vector resulted in
histopathologic changes, including articular surface fibrillations, cartilage remodeling, and chondrocyte clon-

ing. We also demonstrated up-regulation of genes implicated in arthritis (cyclooxygenase 2, IL-6, matrix
metalloproteinase 9). There was a lack of inflammatory
cells in these joints. Behavioral changes, including
increased orofacial grooming and decreased resistance
to mouth opening, were used as measures of nociception
and joint dysfunction, respectively. The significant increase in expression of the pain-related neurotransmitter calcitonin gene-related peptide (CGRP) in the sensory ganglia as well as the auxiliary protein CGRP
receptor component protein of the calcitonin-like receptor in the brainstem further substantiated the induction
of pain.
Conclusion. Induction of IL-1 expression in the
TMJs of adult mice led to pathologic development,
dysfunction, and related pain in the joints. The somatic
mosaic model presented herein may prove useful in the
preclinical evaluation of existing and new treatments for
the management of joint pathologic changes and pain,
such as in osteoarthritis.

Dr. Lais work was supported by the William H. Bowen

International Fellowship. Dr. Shaftels work was supported by the
University of Rochester Medical Scientist Training Program
(GM07356). Dr. Dickersons work was supported by an NIH grant
(DK-52328). Dr. OBanions work was supported by an NIH grant
(NS-048522). Dr. Kyrkanides work was supported by funds from the
Department of Dentistry, University of Rochester School of Medicine
& Dentistry.
Yu-Ching Lai, DDS, Solomon S. Shaftel, BS, Jen-nie H.
Miller, MS, Ross H. Tallents, DDS, Yoon Chang, DDS, Carl A.
Pinkert, PhD, John A. Olschowka, PhD, Ian M. Dickerson, PhD, J.
Edward Puzas, PhD, M. Kerry OBanion, MD, PhD, Stephanos
Kyrkanides, DDS, PhD: University of Rochester School of Medicine &
Dentistry, Rochester, New York.
Drs. OBanion and Kyrkanides have applied for a patent for
the nucleic acids (patent pending PCT/US2005/042058).
Address correspondence and reprint requests to Stephanos
Kyrkanides, DDS, PhD, University of Rochester Eastman Dental
Center, 625 Elmwood Avenue, Rochester, NY 14620. E-mail:
stephanos_kyrkanides@urmc.rochester.edu.
Submitted for publication September 1, 2005; accepted in
revised form January 17, 2006.

Osteoarthritis (OA) manifests as a slowly progressing debilitating disease that affects one or more
joints of the body. Clinical symptoms include pain,
dysfunction, and swelling and enlargement of the joints.
The primary pathologic features of OA are fibrillation
and loss of articular cartilage, accompanied by remodeling of subchondral bone. OA seems to be a node of
convergence for a number of potentially independent
pathologic processes that, ultimately, can lead to joint
dysfunction and pain (1). Although the role of inflammation in OA has been long debated (2), recent evidence now confirms proinflammatory cytokines as mediators in this disease (3). For example, the catabolism
of OA cartilage is thought to involve the action of
proinflammatory cytokines such as interleukin-1 (IL-1)
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INDUCTION OF IL-1 EXPRESSION AND CHANGES IN TMJs OF ADULT MICE

and tumor necrosis factor (TNF) (35). In fact, it has

been suggested that inflammation and cartilage destruction may be 2 separate pathogenic events: IL-1 seems
more potent in its ability to cause cartilage degradation,
whereas TNF seems to be responsible for inflammatory events (6).
IL-1 limits the synthesis of extracellular matrix
(ECM) by up-regulating the synthesis of metalloproteinase (7). Furthermore, IL-1 receptor antagonist (IL-1Ra)
has been shown to be an effective therapeutic agent in
animal models of arthritis and in humans with rheumatoid arthritis (RA) (8). The 2 isoforms of IL-1 ( and )
show similar potency, but IL-1 seems to be active in
early synovial inflammation, whereas IL-1 is found to
be up-regulated in advanced disease states (9). Studies
involving various animal models have revealed a role of
IL-1 in the pathogenesis of arthritis, leading primarily
to the development of RA (1012). For example, intraarticular injection of methylated bovine serum albumin
into the mouse knee, together with subcutaneous injection of recombinant human IL-1, led to the development
of synovitis and pannus tissue (10,11). Autografts of
retrovirally infected synoviocytes expressing human
IL-1 in rabbit knees resulted in severe monarthritis
similar to chronic RA in humans (12). Injection of IL-1
into the knee joints of mice resulted in profoundly
accelerated proteoglycan breakdown and enhanced expression of gelatinolytic activities (6). The aforementioned models, however, are primarily based on the
acute effects of IL-1 in joints, and do not replicate the
long-term process underlying the development of OA in
humans.
The purpose of this study was to examine the
effects of IL-1 expression in the adult mouse joint. To
this end, we used a somatic mosaic analysis method in a
transgenic mouse. This Cre/loxP-based molecular genetic method utilizes a germline-transmitted recombinational substrate containing a dormant transcription unit
coupled to somatic gene transfer of Cre recombinase to
activate the gene of interest (13). Using this method, we
evaluated the effects of intraarticular induction of IL-1
at the cellular, histopathologic, and behavioral levels in
the mouse temporomandibular joint (TMJ).
MATERIALS AND METHODS
Vector and transgene construction. The ssIL-1 gene
(539 bp) codes for the signal sequence of human IL-1Ra that
is fused to the complementary DNA (cDNA) of the mature
form of the human IL-1 structural gene (Figure 1A). The
construct was created as follows.
Total RNA was extracted from primary human mono-

1185

cytes and was reversed transcribed with reverse transcriptase

derived from Moloney murine leukemia virus (Superscript II;
Invitrogen, Carlsbad, CA) using oligo(dT) primers, as previously described (14). The human IL-1 cDNA (464 bp) was
amplified by polymerase chain reaction (PCR) using the 17-kd
upper primer (5-GCA-CCT-GTA-CGA-TCA-CTG-AACTGC-3) and the 17-kd lower primer (5-CTT-TAG-GAAGAC-ACA-AAT-TGC-ATG-G-3) (30 cycles at an annealing
temperature (TA) of 58C). The human IL-1Ra signal sequence was added upstream by a series of 3 consecutive PCR
amplifications of the 464-bp human IL-1 cDNA using the
same 17-kd lower primer and each of the following signal
sequence upper primers (ssUP): ssUP-1 (5-TTC-CAT-TCAGAG-ACG-ATC-TGC-GCA-AAT-GTA-CGA-TCA-3 at
TA 59C), ssUP-2 (5-TCA-CCT-AAT-CAC-TCT-CCTCCT-CTT-CCT-GTT-CCA-TTC-AGA-GAC-G-3 at TA
59C), and ssUP-3 (5-ATG-GAA-ATC-TGC-AGA-GGCCTC-CGC-AGT-CAC-CTA-ATC-ACT-CTC-C-3 at TA
59C).
The final 539-bp PCR product was gel purified and
subsequently cloned into the pCRII-TOPO vector (Invitrogen)
according to the manufacturers instructions. The insert size,
sequence, and orientation were confirmed by multiple restriction enzyme digestions and direct DNA sequencing using the
M13 and T7 primers. A segment containing the ssIL-1
construct was then cloned into the Eco RI site of pBS KS
plasmid (Stratagene, La Jolla, CA) leaving the Eco RI site
intact.
The cytomegalovirus (CMV) promoter was inserted
into the Pac I site of the pBigT vector (15), which contains a
loxP-flanked (floxed) transcription termination cassette as
follows. The CMV promoter sequence was amplified from the
pRc/CMV vector (Invitrogen) using primers that included the
Pac I restriction enzyme cutting sites (5-AAT-ATC-TTAATT-AAA-TCT-CTA-GAT-GCT-TCG-CGA-TGT-ACGGGC-3, 5-TAG-TCA-TAT-ATG-ATC-TTA-ATT-AAAAGC-TTG-GGT-CTC-CC-3). This PCR product was
digested with Pac I, gel purified, and subsequently cloned into
the Pac I site of the pBigT vector upstream of the floxed
transcriptional termination cassette. A DNA sequence consisting of internal ribosome entry site (IRES)lacZpoly(A) was
cloned from the vector pBS-IRES-LacZ-poly(A) (16) into
pBigT/CMV using the unique Xho I and Not I sites within each
of these vectors. Subsequently, the Bam HI sites of the
constructs in pBSII KS and the Nhe I site of pBigT/CMV
were blunt ended using T4 DNA polymerase (Invitrogen)
according to the manufacturers instructions. Next, both plasmids were digested with Sal I, agarose gel purified, and ligated.
The predicted final vector (CMV-IL-1XAT) was confirmed
via Eco RI digestion.
The rat Col1a1 (Col1) promoter (17) was kindly donated to us by Dr. Barbara Kream (University of Connecticut,
Farmington, CT) in the pUC12 plasmid. The 3.6-kb promoter
sequence was excised by Xba I digestion, gel purified, and
subcloned into the following plasmid containing a custommade cloning site. This cloning site was prepared by direct
DNA oligo synthesis through the commercially available Gibco
BRL (Gaithersburg, MD) service, and contained an Xba I site
flanked by Pac I sites: 5-ATT-AAT-TAA-TCG-ATG-CGGCCG-CTC-TAG-ATT-AAT-TAA-TA-3 and 5-TAA-TTAATT-AGC-TAC-GCC-GGC-GAG-ATC-TAT-TTA-ATT-

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LAI ET AL

Figure 1. Construction of an excisionally activated IL-1XAT transgene. A, A bicistronic gene consisting of the cDNA of mature human interleukin-1
(IL-1) fused to the signal sequence (ss) of human IL-1 receptor antagonist and the reporter gene lacZ was driven by the cytomegalovirus (CMV)
promoter. A loxP-flanked (or, floxed) transcription termination cassette (STOP) that included the neomycin resistance gene was inserted upstream of the
first open-reading frame (ORF). Translation of the second ORF was accomplished by an internal ribosome entry sequence (IRES). IL-1XAT activation
is accomplished after loxP-directed STOP excision by Cre recombinase. The 293GLVP/CrePr cell line that carries an RU-486inducible Cre system was used
to further characterize CMV-IL-1XAT. Addition of RU-486 (107M) to the media of CMVIL-1XATtransfected 293GLVP/CrePr cells yielded B,
loxP-directed DNA recombination, which was detected as a decrease (2.5 kb) in the size of the polymerase chain reaction (PCR) product amplified by
primers flanking the STOP cassette. L 1-kb ladder; cntl control. To this end, we detected C, an induction of -galactosidase expression, as assessed
by X-Gal histochemistry (original magnification 4), D, an induction of human IL-1 transcript, as determined by reverse transcriptionPCR (RT-PCR),
and E, a significant increase in human IL-1 secreted protein, as determined by enzyme-linked immunosorbent assay (ELISA), following Cre-mediated
recombination. Furthermore, the rat procollagen 1 (Col1) promoter was used to target IL-1XAT expression in chondrocytes, bone cells, and fibroblasts
(Col1-IL-1XAT). Values are the mean and SD of 3 tissue culture samples per group. F, NIH3T3 stable cell lines carrying Col1-IL-1XAT were transduced
by Cre recombinase using the lentiviral vector HIV(Cre) containing the human immunodeficiency virus (HIV). Transfection and infection of the stable cell
line with HIV(Cre) resulted in expression of human IL-1 transcript concomitantly with Cre recombinase expression (both determined by RT-PCR). In
contrast, samples treated with plain media lacking HIV(Cre) () did not display any IL-1 or Cre induction. The presence of the IL-1XAT transgene was
confirmed in these samples by PCR amplification of DNA.

INDUCTION OF IL-1 EXPRESSION AND CHANGES IN TMJs OF ADULT MICE

AT-3. The 2 oligos were then hybridized by a single PCR cycle

using Taq polymerase, and subsequently cloned directly into
the pCRII-TOPO vector (Invitrogen) according to the manufacturers instructions. The Xba I site of the pCRII-TOPO
vector was excised by Eco RIApa I digestion, DNA blunted,
and re-ligated using standard molecular biology methods.
Subsequently, the Xba Iexcised Col1 promoter was cloned
into the Xba I site of pCRII-TOPO containing the custommade cloning site, and the desired 533 orientation was
confirmed. Next, the Col1a1 promoter containing the Pac
IPac I sequence was excised by restriction enzyme digestion
(Pac I), gel purified, and cloned into the Pac I site of pBigT in
place of the CMV promoter, resulting in the desired Col1-IL1XAT transgene (pCol1-IL-1XAT vector).
CMV-IL-1XAT and Col1-IL-1XAT induction was
mediated by the administration of Cre recombinase using viral
vectors. The HIV(Cre) vector containing the human immunodeficiency virus (HIV) was developed based on the pLenti6/
V5-D-TOPO vector (Invitrogen) as follows. The fusion gene
containing the nuclear localization sequence (nls) and the
bacterial cre recombinase gene were amplified by PCR from
the pCrePrH vector (18) using the following primers: 5-TCCAAT-TTA-CTG-ACC-GTA-CAC-C-3 and 5-GCA-ACACCA-TTT-TTT-CTG-ACC-3. The subsequent PCR product
was directly cloned into the pLenti6/V5-D-TOPO vector according to the manufacturers instructions. The FIV(Cre)
vector was developed based on the feline immunodeficiency
virus (FIV) system from System Biosciences (Mountain View,
CA). Briefly, the Spe IBpu11021 segment of HIV(Cre) containing the CMV-nlsCreV5-pA gene along with the blasticidin
resistance gene was cloned in place of the lacZ gene between
the Xba I and the Sal I sites of the FIV vector by blunt ligation.
In vitro studies. The regulation of CMV-IL-1XAT was
evaluated in the 293GLVP/CrePr cell line previously developed in
our laboratory (18). Briefly, the 293GLVP/CrePr is a stable cell
line expressing the dually regulated GLVP/CrePr system,
whereby inclusion of RU-486 (106108M) in the culture
medium induces Cre recombinase activity in a temporally
controlled manner. To this end, 293GLVP/CrePr cells were
transfected with CMV-IL-1XAT and treated with RU-486
(108M). Forty-eight hours later, the cultured cells were fixed
with 10% formalin and processed by X-Gal histochemistry as
previously described (19,20). Conditioned media were collected, and the level of human IL-1 protein was assessed in
the conditioned medium by enzyme-linked immunosorbent
assay (ELISA) using the Quantikine human IL-1 kit (R&D
Systems, Minneapolis, MN) according to the manufacturers
instructions. Total RNA was extracted by the TRIzol reagent
(Invitrogen) according to the manufacturers instructions.
IL-1 XAT transcript levels were evaluated by reverse
transcriptionPCR (RT-PCR) as previously described (18),
using the 17-kd upper and lower primers. The data were
normalized against CMV-IL-1XAT DNA levels after DNA
extraction by the TRIzol method according to the manufacturers instructions.
The regulation of Col1-IL-1XAT by Cre recombinase
was tested in a stable cell line developed from the murine
NIH3T3 fibroblast cell line as follows. The Col1-IL-1XAT cell
line was transiently transfected with the HIV(cre) vector using
Lipofectamine 2000 reagent (Invitrogen) according to the
manufacturers instructions. Gene expression was assessed at

1187

the transcript and protein levels by RT-PCR, X-Gal histochemistry, and ELISA. Subsequently, the HIV(Cre) vector was
packaged in the 293FT packaging cell line with the aid of
vectors pLP1, pLP2, and pLP/VSV-G (Invitrogen) and titered
in the HT1080 cell line according to the manufacturers
instructions. The virus was then used to infect the stable
fibroblast cell line carrying the Col1-IL-1XAT gene. The data
were normalized against CMV-IL-1XAT DNA levels after
DNA extraction by the TRIzol method according to the
manufacturers instructions.
Transgenic mice. Col1-IL-1XATtransgenic mice
were generated at the University of Rochester Transgenic
Facility (URTF). A Not INot I linearized fragment from the
pCol1-IL-1XAT was gel purified and prepared following the
established protocols of the URTF. The fragment was
microinjected into inbred C57BL/6 zygotes and subsequently
implanted into pseudopregnant dams as previously described
(21). Founders were bred to C57BL/6 controls to establish
transgenic lineages. Genotyping was accomplished by PCR
amplification of DNA extracted from tail snips using FIX upper primer and 17-kd lower primer as previously described (16).
Transgene expression was evaluated in vivo in
2-month-old Col1-IL-1XAT mice after intraarticular injection
of FIV(Cre) virus (50 l containing a total of 5 105
infectious particles) into the right and left TMJs as previously
described (19). Eight-week-old transgenic and wild-type mice
were anesthetized by ketamine (40 mg/kg), and under surgical
plane of anesthesia, the right and left TMJs were injected with
50 l of an aqueous solution containing a total of 105 FIV(Cre)
infectious particles. The TMJ area was located by palpation
over the zygomatic arch from anterior to posterior and from
the interval between the medial aspect of the zygomatic arch
and the skull. The zygomatic arch is attached posteriorly to the
skull at the temporal bone. At this V-shaped notch, the glenoid
fossa is inferior and posterior. A 271/2-gauge needle was
inserted in a posterior inferior direction, and solutions were
injected into the right and left TMJ areas. After injection, the
mice were returned to their cages.
Additional groups of mice received intraarticular injections of FIV(gfp), a viral vector encoding for the reporter
gene green fluorescent protein. Controls received normal sterile
saline injections. A total of 20 Col1-IL-1XATtransgenic mice
and 16 wild-type littermates were used in our studies.
Eight weeks after injection, the heads of mice were
harvested for histologic analysis, fixed by immersion in 10%
formalin for 10 days, and subsequently processed as described
below. For protein and transcript analyses, tissue blocks (4
4 2 mm) consisting of the TMJ (portion of the temporal
bone and mandibular condyle) were harvested and processed
as described below. In addition, the brainstem and trigeminal
ganglia were harvested and fixed in 10% formalin for 7 days,
followed by immersion in 30% sucrose in phosphate buffered
saline (PBS) for 3 days and then stored at 80C until they
were further processed.
All animal procedures described herein were reviewed
and approved by the Institutional Animal Care and Use
Committee (University Committee on Animal Resources) for
compliance with federal regulations prior to the initiation of
the study (OLAW/PHS Assurance A3292-01). All mice were
maintained in an American Association for Accreditation of
Laboratory Animal Careaccredited specific pathogenfree

1188

barrier facility and were anesthetized by 2,2,2-tribromoethanol

(Avertin; Sigma, St. Louis, MO) injection, or as noted for
specific experiments, during any potentially painful procedure.
Mice were monitored carefully after anesthesia until they
experienced full recovery. Mice were killed by CO2 inhalation
or sedation with 2,2,2-tribromoethanol, followed by cervical
dislocation. All procedures followed the American Veterinary
Medical Association guide, per institutional policy.
Behavioral analyses. Grooming behavior was evaluated by adapting a method previously described (22). Briefly,
mice were placed in a custom-made cage (12 12 12 inches)
with 4 mirrored walls. The cage lacked a roof so that the mice
could be observed and their behavior recorded on video tape.
To minimize stress, each mouse was transferred into the
observation chamber containing bedding from its original cage
and was allowed a 30-minute habituation period (22). Behaviors were recorded on video tape for a period of 60 minutes
using a Sony digital recorder (Digital Handycam/Digital 8;
Sony, Tokyo, Japan) with a Cokin macro digital lens (mode
C043; Sony) added for image enlargement. The mouse was
then returned to its original cage.
Grooming was measured during video playback by
counting the number of seconds a mouse rubbed its face and/or
flinched its head during the session. The mice did not have
access to food or water during the brief testing period. Behavioral
evaluation was performed by an investigator (RHT) who was
blinded to the mouse group assignment. The behavior was
characterized in 3-minute increments over the 60 minutes of
evaluation. These data were entered into FileMaker Pro V7
software (FileMaker, Santa Clara, CA) and exported to Excel
software (Microsoft, Redmond, WA) for analysis.
Resistance to jaw opening was used as a method of
assessing TMJ dysfunction, based on the principles of the
pain-adaptation model (23), which suggests that pain reduces
muscle force. In preparation for the test, mice were anesthetized via intraperitoneal injection of ketamine (40 mg/kg). An
orthodontic hook was attached using dental bonding material
onto the lower incisors, and the mouse was returned to its cage
to recover from the anesthesia for a minimum of 4 hours.
Immediately prior to each test, mice were anesthetized with a
CO2 (60%)/O2 (40%) mixture under constant pressure (25
pounds per square inch), a method that provides 2 minutes
of anesthesia, since CO2 is quickly cleared from the mouse via
exhalation with minimal physiologic changes and is therefore
suitable for the objectives of our experiments. During this
period, each mouse was placed in a plastic (single-use) restraining device that immobilizes the head and the maxilla
while leaving the mandible free. The lower jaw was then
connected via the orthodontic hook to a digital dynamometer
(FGF series; National Instruments, Austin, TX) wired to a
Dell PC (Round Rock, TX) through an A/D conversion card
(NIO16E1; National Instruments, Austin, TX) that recorded
the resistance exhibited by the mouse during an attempt to
displace the mandible vertically by 4 mm.
A total of 10,000 data points over 220 seconds were
collected by the Lab View software package (National Instruments) on a PC computer and plotted over a 5-minute time
period. Within each period, the mandible was lowered 10 times
and held for 2 seconds, with a 20-second interval between
each depression of the mandible. At the end of each session,
the mice were killed.

LAI ET AL

Histologic studies. After fixation in 10% formalin, the

mouse heads were dissected, defleshed, and decalcified by
immersion in an EDTA solution for 7 days at 4C under
constant agitation. The TMJs were then processed on an
RHS-1 microwave tissue processor (Milestone Medical, Shalton, CT) after which the samples were embedded in paraffin,
and 3-mthick sections were cut with a microtome and
collected onto glass slides. The brainstem and trigeminal
ganglia were cut with a cryostat while frozen (18-mthick
sections) and collected onto glass slides.
Overall histopathologic features of the TMJs were evaluated in sections stained with Alcian blueorange G histochemistry. Joint pathologic changes were evaluated using a 04 scale
modified from that described by Wilhelmi and Faust (24). This
scale is defined as follows: 0 no apparent changes; 1
superficial fibrillation, striation of cartilage; 2 injuries limited to
uncalcified cartilage; 3 defects extending into calcified cartilage; and 4 deep defects extending into calcified cartilage.
Immunohistochemical analysis was performed for a
number of antigens using the antibodies described below. In
general, the TMJ sections were deparaffinized in xylene,
rehydrated through graded alcohols, and quenched in 3%
H2O2 for 20 minutes. Antigen retrieval was performed in a
pressure cooker using a 10 mM citrate buffer, pH 6.0. Brainstem and ganglia sections were rehydrated in PBS for 60
minutes, bleached in 3% H2O2 for 15 minutes, and processed
as follows. Tissues were blocked using appropriate primary
serum solution followed by overnight incubation in primary
antibody solution at 4C. The following morning, the TMJ
sections were rinsed with PBS and incubated in an appropriate
biotinylated secondary antibody solution for 30 minutes, followed by a PBS wash and incubation in horseradish
peroxidaseconjugated streptavidin. Aminoethylcarbazole was
used as chromogen, and sections were counterstained with
hematoxylin, followed by a wash in PBS. Brainstem and ganglia
sections were processed in a similar manner except that the
avidinbiotin complex reagent (Vector, Burlingame, CA) was
used in conjunction with nickeldiaminobenzidine as chromogen as previously described (25). The sections were then
dehydrated in alcohols, cleared by xylene, and coverslipped
with permanent mounting media.
The histology sections were evaluated under light
microscopy using an Olympus BX51 microscope (Lake Success, NY). Photomicrographs were obtained with a Spot CCD
digital camera (Diagnostic Imaging, Sterling Heights, MI)
attached to the microscope. The brainstem and ganglia sections were analyzed as follows. The number of immunoreactive
pixels per trigeminal ganglion examined in each microscopic
field (4) was calculated using the National Institutes of
Health Image software (25).
Antibodies used in these experiments included a rat
anti-mouse CD11b (1:100 dilution; Serotec, Raleigh, NC), a
rabbit antitype II collagen (anti-CII) (1:40 dilution, NeoMarkers, Fremont, CA), a rabbit anti-human mature IL-1 (1:100
dilution; Abcam, Cambridge, MA), a rabbit anti-murine cyclooxygenase 2 (COX-2) (1:100 dilution; Cayman Chemical, Ann
Arbor, MI), a goat antimatrix metalloproteinase 9 (anti
MMP-9) (1:100 dilution; Biogenesis; Kingston, NH), a rabbit
antiIL-6 (1:100 dilution; Santa Cruz Biotechnology, Santa
Cruz, CA), a rat anti-V5 (1:500 dilution; Invitrogen), a rabbit
anti-galactosidase (1:1,000 dilution; Sigma), rat anti

INDUCTION OF IL-1 EXPRESSION AND CHANGES IN TMJs OF ADULT MICE

polymorphonuclear neutrophil (anti-PMN) clone 7/4 (1:100

dilution; Serotec), rat anti-mouse I-A/I-E clone M5/114.15.2
(class II major histocompatibility complex) (1:100 dilution;
PharMingen, San Diego, CA), a rat anti-CD3 (1:100 dilution;
Serotec), a rabbit anticaspase 3 (1:500 dilution; Chemicon,
Temecula, CA), and a biotin-conjugated rabbit anti
proliferating cell nuclear antigen (anti-PCNA) (1:100 dilution;
Invitrogen). The mu33 antibody (26) was used to detect
calcitonin gene-related peptide (CGRP; 1:1,000 dilution), and
the 1065 antibody (27,28) was used to detect CGRP receptor
component protein (RCP) (1:500 dilution).
Quantification of messenger RNA (mRNA) using realtime quantitative RT-PCR. Quantification of mRNA levels
was accomplished using an iCycler (Bio-Rad, Richmond, CA)
and real-time quantitative RT-PCR with TaqMan probes
constructed with FAM as the fluorescent marker and Blackhole I quencher (Biosearch Technologies, Novato, CA). Prior
to PCR of the cDNA samples, PCR conditions were optimized
for each mRNA to be analyzed. Standard curve reactions were
performed by varying annealing temperatures, primer concentrations, and TaqMan probe concentrations. Serial dilution of
the starting cDNA template demonstrated linear amplification
over at least 5 orders of magnitude.
PCRs were performed in a volume of 25 l and
contained iQ Supermix (Bio-Rad), 0.625 units of Taq, 0.8 mM
dNTP, 3 mM Mg2, 0.20.6 M concentrations of each primer,
10100 nM probe, and 1 l of cDNA sample. To ensure
consistency, a master mixture was first prepared containing all
reagents except the cDNA sample. Primers were designed
using the Primer Express (Applied Biosystems, Foster City,
CA) and Oligo 6.83 programs (Molecular Biology Insights,
Cascade, CO). The primers used in the real-time quantitative
RT-PCR were as follows. For murine COX-2, the primers
were 5-TGA-CCC-CCA-AGG-CTC-AAA-TA-3 and 5CCC-AGG-TCC-TCG-CTT-ATG-ATC-3, and the probe was
5-CTT-TGC-CCA-GCA-CTT-CAC-CCA-TCA-GTT-3. For
inducible nitric oxide synthase (iNOS), the primers were
5-GGG-CAG-CCT-GTG-AGA-CCT-T-3 and 5-GCATTG-GAA-GTG-AAG-CGT-TTC-3, and the probe was 5TGT-CCG-AAG-CAA-ACA-TCA-CAT-TCA-GAT-CC-3.
In general, PCR conditions were as follows: denaturation at
95C for 3 minutes, followed by 40 cycles of amplification by
denaturing at 95C for 30 seconds, annealing at 60C for 30
seconds, and extension at 72C for 60 seconds.
For each real-time PCR, a standard curve was performed to ensure direct linear correlation between product
yield (expressed as the number of cycles to reach threshold)
and the amount of starting template. The correlation was
always greater than r 0.925. PCR efficiency was determined
for each reaction. To correct for variations in starting RNA
values, the level of ribosomal 18S RNA or GAPDH RNA was
determined for all samples and was used to normalize all
subsequent RNA determinations. Normalized threshold cycle
(Ct) values were then transformed, using the function expression (1 reaction efficiency)Ct, in order to determine the
relative differences in transcript expression.
Statistical analysis. Data were compared by analysis of
variance and Tukeys post hoc tests, as well as by linear
regression to determine correlations using the JMP statistics
program (SAS Institute, Cary, NC). P values less than 0.05
were considered statistically significant.

1189

RESULTS
Activation of the IL-1XAT transgene by Cre recombinase. Regulation of the IL-1XAT transgene by Cre
recombinase (Figure 1) was first evaluated in the
293GLVP/CrePr cell line, which carries an inducible Cre
recombination system that is regulated by RU-486 (18)
in vitro. Exogenous administration of RU-486 (108M) to
cultures of 293GLVP/CrePr cells transfected with CMV-IL1XAT resulted in loxP-directed DNA recombination and
excision of the stop transcription termination sequence
cassette, as determined by PCR using primers that flank
this cassette (Figure 1B). Transgene activation in these
cultures was also evidenced by the induction of the reporter gene lacZ, as assessed by X-Gal histochemistry
(Figure 1C), as well as significant elevation (P 0.05) of
human IL-1 mRNA (Figure 1D) and secreted protein
(Figure 1E).
The tissue-specific Col1-IL-1XAT transgene was
evaluated in a stable cell line that was developed using
the NIH3T3 murine fibroblast cell line (Figure 1F). The
Col1-IL-1XAT stable cell clone was developed in the
NIH3T3 cell line following transfection of the Col1-IL1XAT vector and selection using G418 (800 g/ml) over
a period of 12 days (a neomycin resistance gene was
included in the floxed stop cassette of the pBigT plasmid).
Subsequently, the cells were seeded into a 96-well plate (1
cell per well) and grown in culture media containing G418
(400 g/ml). After selection with G418, 5 cell lines carrying
the transgene (confirmed by PCR) were further expanded
and evaluated by RT-PCR for gene induction by Cre
recombinase following transfection and infection of
HIV(Cre) in vitro. Our data demonstrated activation of
Col1-IL-1XAT by Cre recombinase following transfection,
as well as infection in vitro by a viral vector expressing Cre
recombinase (Figure 1F).
In addition, Cre-mediated Col1-IL-1XAT activation was assessed in transgenic mice in vivo. Fourteen
founder mice were produced; 5 transgenic mice were
identified that harbored the Col1-IL-1XAT transgene,
as determined by PCR of genomic DNA extracted from
tail snips using the FIX upper primer and the 17-kd
lower primer. Two of these pups died at neonatal stages
of development. The remaining 3 founders (A, B, and C)
were then bred with C57BL/6 wild-type mice for germline transgene transmission analysis. Mouse line C demonstrated the highest behavioral changes following
transgene activation and was used in subsequent experimentation; the other 2 transgenic mouse lines (A and B)
were terminated.
Targeted transgene function was evaluated in
vivo following intraarticular injection of FIV(Cre) into

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LAI ET AL

Induction of articular cartilage changes in the

TMJs by Col1-IL-1XAT activation. Eight weeks following FIV(Cre) injection in the TMJs of 2-month-old
transgenic mice, we observed histopathologic changes in
the articular cartilage of the joints, including cellular
disorganization, loss of normal cytoarchitecture, and
chondrocyte cloning, along with superficial erosion and
fibrillations (Figures 3E and F). No synovial membrane
changes were observed in our model. Examination for

Figure 2. Transgene activation and induction of the expression of

mature human interleukin-1 (IL-1) in the temporomandibular
joints (TMJs) of adult Col1-IL-1XAT mice following intraarticular
injection of FIV(Cre) containing the feline immunodeficiency virus
(FIV). Intraarticular injection of FIV(Cre) (50 l containing 5 105
infectious particles) into the right and left TMJs of 2-month-old
Col1-IL-1XATtransgenic mice resulted in transgene activation. A,
Fluorescein isothiocyanateconjugated immunofluorescence of
-galactosidase (arrows) coupled with B, Texas Redconjugated immunofluorescence of Cre recombinase using a V5-specific antibody in
activated Col1-IL-1XATtransgenic mice 8 weeks following FIV injection (arrows). C, Light microscopy image of the histology section
depicting the cellular anatomy of the condylar surface. Arrows indicate
the same cells as in A and B. D, Superimposition of the images in A and
B, demonstrating overlap of -galactosidase and the V5 epitope of our
tagged Cre recombinase (arrows). E, Superimposition of the images in
A, B, and C. F, The expression of mature human IL-1 in transgenic
mice injected with FIV(Cre) was detected by immunohistochemistry
with nickeldiaminobenzidine staining (black) using a commercially
available specific antibody. G, In contrast, transgenic mice injected
with FIV(gfp), a viral vector encoding for the reporter gene green
fluorescent protein, did not show any positive staining.

the TMJs of the Col1-IL-1XATtransgenic mice at 8

weeks of age. Transgene activation was identified primarily
in articular chondrocytes, as evidenced by double immunofluorescence analysis using antibodies against bacterial
-galactosidase and the V5 epitope of Cre recombinase
(Figures 2AE). Expression of mature human IL-1 was
evaluated in these joints by immunohistochemistry using a
commercially available antibody specifically raised against
this antigen (Figures 2F and G). No differences in weight
or locomotive activity (evaluated by Rotorod; Columbus
Instruments, Columbus, OH) were found between transgenic mice receiving intraarticular injections of FIV(Cre),
FIV(gfp), or saline (data not shown).

Figure 3. Pathologic changes in the temporomandibular joints

(TMJs) of Col1-IL-1XATtransgenic mice following intraarticular
injection of FIV(Cre) containing the feline immunodeficiency virus
(FIV). Intraarticular injection of FIV(Cre) (50 l containing 5 105
infectious particles) into the right and left TMJs of 2-month-old
Col1-IL-1XATtransgenic mice resulted in the development of joint
pathologic changes 8 weeks following viral transduction. Alcian blue
orange G histochemistry revealed an overall expansion of the hypertrophic chondrocyte zone, chondrocyte cloning, cellular disorganization, and loss of normal cytoarchitecture in the TMJ articular cartilage.
A, Normal TMJ from a control (transgene plus green fluorescent
protein gene). B, TMJ from an experimental (transgene plus Cre)
mouse. C, Higher-magnification view of A. D, Abnormal cartilage
architecture, cellular disorganization, and chondrocyte hypertrophy
are evident in this Col1-IL-1XATtransgenic mouse following intraarticular FIV(Cre) injection. Brace indicates cartilage erosion. E,
Higher-magnification view of C, showing signs of surface erosions.
Arrows indicate lacunae forming on the surface of the TMJ condyle in
experimental mice (transgene plus Cre). F, Higher-magnification view
of D, showing microscopic fibrillations throughout the surface of the
articular cartilage (arrows). (Original magnifications are indicated on
each figure.)

INDUCTION OF IL-1 EXPRESSION AND CHANGES IN TMJs OF ADULT MICE

1191

Figure 4. Articular changes in Col1-IL-1XATtransgenic mice following intraarticular

injection of FIV(Cre) containing the feline immunodeficiency virus (FIV). A, Changes in
articular cartilage were evaluated in experimental (transgene plus Cre) and control
(transgene plus green fluorescent protein [gfp] gene and transgene plus saline) mice and
scored on a numeric scale of 04, where 0 normal and 4 worse (see Materials and
Methods for details). Experimental mice demonstrated significantly higher articular cartilage pathology scores in the temporomandibular joints (TMJs) as compared with controls at
8 weeks following viral transduction. B, Chondrocyte cloning, another joint pathologic
characteristic, was examined in experimental and control mice. Experimental mice showed
a significantly higher number of chondrocyte clones per microscopic field as compared with
controls. Values in A and B are the mean and SD. P 0.01 versus each control group.
Proteoglycan content was evaluated by SafraninO fast green histochemistry (purple stain
on green background) in C, transgene plus gfp and D, transgene plus Cre experimental mice.
Articular cartilage changes were also evaluated by type II collagen (CII) immunohistochemistry in E, control (transgene plus gfp) and F, experimental (transgene plus Cre) mice.
Overall, we observed an induction of CII staining in the hypertrophic articular zone of the
TMJs in experimental mice. (Original magnifications of CF are indicated on each figure.)

cellular apoptosis or cellular proliferation did not show

any caspase 3positive or PCNA-positive cells in the
TMJs of experimental or control mice at this time point.
Joint pathologic changes were evaluated quantitatively
in all mice and the findings are summarized in Figures
4A and B.

We observed a concomitant loss of proteoglycan

content in the transgenic mice treated with FIV(Cre)
along with an increase in CII expression (Figures 4CF).
We also observed a significant increase in COX-2, IL-6,
and MMP-9 protein expression in experimental mice as
compared with controls by immunohistochemistry ana-

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LAI ET AL

Figure 5. Transgene activation and induction of the expression of mediators of inflammation in the temporomandibular joints (TMJs) of adult
Col1-IL-1XAT mice. Eight weeks after injection of FIV(Cre) containing the feline immunodeficiency virus (FIV) into the TMJs of Col1-IL-1XAT
transgenic mice, the TMJs of control (transgene plus green fluorescent protein [gfp] gene) (A, C, and E) and experimental (transgene plus Cre) (B,
D, and F) mice were harvested and evaluated by immunocytochemistry using antibodies against murine interleukin-6 (IL-6), cyclooxygenase 2
(COX-2), and matrix metalloproteinase 9 (MMP-9). A total of 20 Col1-IL-1XATtransgenic mice and 16 wild-type littermates were used in this
experiment. A and B, Induction of IL-6 in the proliferative zone of the articular surface, and C and D, increased COX-2 expression, as assessed by
immunocytochemistry (red staining), were observed in control and experimental mice. E and F, MMP-9 (gelatinase B) was also increased in the
experimental mice as compared with the controls, as assessed by immunocytochemistry (red staining; hematoxylin nuclear counterstaining). (Original
magnifications of AF are indicated on each figure.) We examined the induction of IL-6, COX-2, and MMP-9, which suggests the presence of
inflammation, in the TMJs of activated Col1-IL-1XATtransgenic mice. G, The numbers of cells staining positive for COX-2, IL-6, and MMP-9 were
counted in immunohistochemistry sections of TMJs from experimental (EXP) and control (CNTL) mice. Experimental mice showed significantly
higher numbers of immunoreactive cells as compared with the controls ( P 0.01). Values are the mean and SD of 4 mice per group. H, COX-2
and inducible nitric oxide synthase (iNOS) transcript levels were also significantly increased in the experimental group as compared with the control
group ( P 0.01), as assessed by quantitative reverse transcriptionpolymerase chain reaction analysis of TMJ total RNA extracts. Values are
the mean and SD of 4 mice per group.

INDUCTION OF IL-1 EXPRESSION AND CHANGES IN TMJs OF ADULT MICE

lysis (Figure 5). Transcript levels of COX-2 were also

significantly increased in the experimental mice, but not
transcript levels of iNOS (Figure 5H). In addition,
examination for inflammatory cells in the joints, including CD11b (monocyte/macrophages), CD3 (lymphocytes), and PMNs, showed no accumulation of these
cells in the TMJs of experimental or control mice.
CD11b, CD3, and PMN cells were identified in
other areas of the sections we examined (data not
shown).
Orofacial pain and dysfunction caused by IL-1
induction in the TMJs. Orofacial grooming was used as
a method of assessing pain (Figure 6A). FIV(Cre)treated transgenic mice displayed increased grooming
activity compared with FIV(gfp)-treated and salineinjected controls (23-fold increase; P 0.05), suggesting the presence of pain in the area of the head and face.
Joint/muscle dysfunction was evaluated in experimental
and control mice by assessing resistance to mouth opening, a method based on the pain-adaptation model (23)
(Figure 6B). FIV(Cre)-treated transgenic mice displayed significantly reduced resistance to vertical mandibular displacement as compared with FIV(gfp)treated (40% reduction; P 0.05) and with salinetreated (60% reduction; P 0.05) control mice. There
was significant induction of protein expression for the
pain-related neurotransmitter CGRP in the trigeminal
ganglia (2-fold increase) as well as RCP, an auxiliary
member of the calcitonin-like receptor (CLR), in the
brainstem (40-fold increase) in experimental mice compared with controls (Figure 6). These results indicate
activation of the trigeminal sensory system that innervates the orofacial region following induction of IL-1 in
the TMJs of adult Col1-IL-1XATtransgenic mice.
DISCUSSION
We present herein a novel model of TMJ pathologic changes, dysfunction, and pain using somatic mosaic analysis in the Col1-IL-1XATtransgenic mouse.
Our data demonstrated pathologic development in the
articular cartilage, accompanied by joint dysfunction and
pain, in the TMJs of adult mice 2 months after the
intraarticular induction of IL-1. Pain was measured by
orofacial grooming, a previously established method for
evaluating pain, accompanied by changes in pain-related
neurotransmission in the trigeminal ganglion and brainstem. TMJ dysfunction was evaluated by resistance to
mouth opening, which can develop from several causes,
including pain, muscle dysfunction, and joint pathologic
changes. In this model, joint pathologic processes evi-

1193

dently included changes in the articular cartilage, but not

in the bone.
Our model is characterized by most, but not all,
of the classic features of OA. Specifically, we observed
articular cartilage pathologic changes, including superficial fibrillations, articular surface erosions, and chondrocyte cloning, accompanied by an apparent loss of proteoglycan content. Moreover, we detected the induction
of mediators of inflammation associated with cartilage
destruction, including IL-6, COX-2, and MMP-9. Mice
with the Col1-IL-1XAT activation displayed increased
levels of orofacial grooming and decreased resistance to
mouth opening, suggesting joint pain and dysfunction,
respectively. Our studies also revealed the absence of
inflammatory cells (monocyte/macrophages, lymphocytes, PMNs), as well as inflamed pannus, in experimental or control joints. The lack of inflammatory cells or
inflamed pannus has also been previously described in
the rat model of iodoacetate-induced OA, where an
inflammatory process that is not associated with cell
infiltration may underlie the development of OA, and
there is pain behavior related to the joints (29). Conversely, previous studies using IL-1 in rodent models of
arthritis have shown RA-like pathologic development in
the joint for up to 1 month following intraarticular
cytokine injection of methylated bovine serum albumin
(10) or ex vivo administration of IL-1transduced synovial cells (30,31), which resolved shortly thereafter. In
contrast to these short-term models, the Col1-IL1XATtransgenic mouse may serve as a model for
long-term studies of arthritis. The transgenic mouse
model described herein does not have the limitation of
potential genetic compensations and adaptations that
are often found in germline knockout or transgenic mice
(32).
Eight weeks following transgene activation in
Col1-IL-1XAT mice, we also observed increased orofacial grooming, which has previously been used as a
measure of pain (22). Although orofacial grooming
resembles face washing, asymmetric and prolonged face
rubbing is not displayed spontaneously by healthy, normal rodents (33). The patterns of rubbing provoked by
local irritation or by noxious stimulation have an organization different from that related to maintenance of
the pelage, thermoregulation, or social signaling. Rubbing activity occurs more specifically at the painful body
area (33).
Resistance to mouth opening as a measure of
joint dysfunction is based on the pain-adaptation model
as proposed by Lund et al (23) and suggests that in the
presence of nociceptive input, there is a decrease in

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LAI ET AL

Figure 6. Col1-IL-1XAT activation in the mouse temporomandibular joint (TMJ) and the
development of pain and dysfunction. A, Pain was evaluated in adult transgenic mice by
assessing orofacial grooming 8 weeks following viral transduction. Transgene (Tg) activation by
intraarticular injection of FIV(Cre) containing the feline immunodeficiency virus (FIV) (n 6)
into the TMJs of mice resulted in increased grooming behavior as compared with Col1-IL1XATtransgenic mice injected with either FIV(gfp) containing the green fluorescent protein
(gfp) (n 5) or saline (n 4). B, Eight weeks after treatment, joint dysfunction was evaluated
by assessing resistance to jaw opening. FIV(Cre)-injected transgenic mice demonstrated
significantly decreased levels of resistance to a 4-mm vertical mandibular displacement. C,
Expression of calcitonin gene-related peptide (CGRP) in the trigeminal ganglia of experimental
and control mice was calculated as the total immunoreactivity in 4 microscopic fields and is
presented as the relative percentage ratio. D, Expression of the CGRP receptor component
protein (RCP) was assessed by immunohistochemistry in brainstem sections and was calculated
as the total immunoreactivity in 20 microscopic fields. Values in AD are the mean and SD,
and P 0.05 versus the other 2 groups. E, Representative section (40 microscopic fields)
of a trigeminal ganglion of a Col1-IL-1XATtransgenic mouse injected with FIV(Cre) and
stained for CGRP. F, Representative section (40 microscopic fields) of RCP immunostaining
in the principal trigeminal nucleus of a Col1-IL-1XATtransgenic mouse injected with
FIV(Cre). (Original magnifications of E and F are indicated on each figure.)

INDUCTION OF IL-1 EXPRESSION AND CHANGES IN TMJs OF ADULT MICE

muscle strength in concentric muscle work (chewing,

clenching), a reduced range of motion, and a slowing of
movement due to antagonistic co-contraction of extensors during eccentric muscle work (jaw opening). Previous studies support this model by demonstrating decreased muscle strength associated with pain. Reduced
grip strength (34), sustained pain on jaw closing (35),
and reduced bite strength (36) have been reported in
patients with pain. The reduction in muscle force exertion associated with TMJ disorders may result from
decreased activity of agonist muscles and increased
activity of antagonist muscles (37). It has been reported
that bite force increases over time as pain decreases in
patients with TMJ disease (38). Indeed, unilateral pain
causes a bilateral reduction in bite force, indicating that
pain has rather widespread effects. Kehl and coworkers
(39) demonstrated that forelimb grip force reduction is a
behavioral index of hyperalgesia in the carrageenan
model of muscle hyperalgesia. Our data demonstrate a
decrease in the resistance to jaw opening in the activated
Col1-IL-1XAT mice (transgene plus Cre) as compared
with the controls (transgene plus gfp and transgene plus
saline), which suggests pain avoidance (Figure 6). This
supports the pain-adaptation hypothesis and other pain
models in which force is decreased (35,36,39).
CGRP has been demonstrated to mediate vasodilation, inflammation, and nociception, either alone or
by modulating other neuropeptides (40). CGRP induction in sensory ganglia has previously been demonstrated in a number of rodent models, including the
injection of capsaicin or Freunds complete adjuvant
into the rat TMJ (41,42), the injection of nerve growth
factor into rat hind paws (43), or the intraarticular
injection of capsaicin into the rat TMJ (44). In the
present studies, transgene induction in the Col1-IL1XAT mouse significantly increased CGRP immunoreactivity in the trigeminal ganglion as compared with the
controls 8 weeks after transgene activation, indicating a
long-term activation of the trigeminal sensory system in
our model. The CGRP system can also be augmented by
increased receptor signaling. The CGRP receptor is a
trimer of proteins, consisting of the CLR and 2 accessory
proteins, receptor activitymodifying protein 1 and RCP
(45). RCP enhances signaling at the CGRP receptor
(26), and in previous studies, expression of RCP correlated with increased CGRP efficacy during gestation
(46). A similar up-regulation of the CGRP receptor
system has been proposed in the rat dorsal root ganglia
and dorsal horn, where RCP expression was shown to be
increased following peripheral inflammation (28,47).
Surprisingly, intraarticular levels of human IL-1
in the experimental mice (transgene plus Cre) were

1195

below the detection threshold of our ELISA-based

method. We calculated the efficacy of our method in
detecting intraarticular cytokine concentrations by injecting 20 ng of recombinant human IL-1 into the
mouse TMJ. A total of 11.45 pg was detected by our
method (range 22,000 pg/ml), indicating a signal to
cytokine concentration ratio of 1:2,000. Based on these
data, we concluded that our method of IL-1 extraction
did not allow for the accurate assessment of this cytokine
in the mouse TMJ. In fact, patients with arthritis of the
TMJ reportedly present with relatively low levels of
IL-1. One study detected an average of 0.02 pg/ml in
healthy volunteers, 0.25 pg/ml in OA patients without
joint effusion, and 0.39 pg/ml in OA patients with joint
effusion (48). These low levels of IL-1 in the TMJs of
humans are consistent with our inability to detect IL-1
in mouse TMJs with our ELISA method.
In conclusion, our data suggest the development
of joint pathologic changes, dysfunction, and pain in
response to increased expression of IL-1 in the TMJs of
adult Col1-IL-1XATtransgenic mice. These findings
may prove important in the development of novel therapies for the management of joint disorders, including
pain. In this model, the pathologic processes in the joint
evidently included changes in the articular cartilage, but
not in the bone, which were accompanied by pain. To
this end, not all patients experiencing joint pain display
degenerative joint disease (OA). Moreover, not all
patients presenting with degenerative joint disease have
pain. From a clinical standpoint, pain is undoubtedly a
chief complaint that often prompts arthritis patients to
seek medical care. Therefore, our model is characterized
by the majority of the classic, although not all, features
of OA and may prove valuable in future studies for
evaluating new and existing treatments for the management of OA.
ACKNOWLEDGMENTS
We would like to thank Ms Barbara F. Stroyer for
valuable technical expertise in the calcified tissue processing
and staining, as well as Dr. Yoon Chang for help in the jaw
resistance evaluation.

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