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.
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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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.
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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
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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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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.
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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.)
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