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Triggering of proteinase-activated receptor 4 leads to joint pain and inflammation in mice.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 3, March 2009, pp 728–737
DOI 10.1002/art.24300
© 2009, American College of Rheumatology
Triggering of Proteinase-Activated Receptor 4 Leads to
Joint Pain and Inflammation in Mice
Jason J. McDougall,1 Chunfen Zhang,1 Laurie Cellars,1 Eva Joubert,1
Chantelle M. Dixon,1 and Nathalie Vergnolle2
Objective. To investigate the role of proteinaseactivated receptor 4 (PAR-4) in mediating joint inflammation and pain in mice.
Methods. Knee joint blood flow, edema, and pain
sensitivity (as induced by thermal and mechanical stimuli) were assessed in C57BL/6 mice following intraarticular injection of either the selective PAR-4 agonist
AYPGKF-NH2 or the inactive control peptide YAPGKFNH2. The mechanism of action of AYPGKF-NH2 was
examined by pretreatment of each mouse with either the
PAR-4 antagonist pepducin P4pal-10 or the bradykinin
antagonist HOE 140. Finally, the role of PAR-4 in mediating joint inflammation was tested by pretreating mice
with acutely inflamed knees with pepducin P4pal-10.
Results. PAR-4 activation caused a long-lasting
increase in joint blood flow and edema formation, which
was not seen following injection of the control peptide.
The PAR-4–activating peptide was also found to be
pronociceptive in the joint, where it enhanced sensitivity
to a noxious thermal stimulus and caused mechanical
allodynia and hyperalgesia. The proinflammatory and
pronociceptive effects of AYPGKF-NH2 could be inhib-
ited by pepducin P4pal-10 and HOE 140. Finally, pepducin P4pal-10 ameliorated the clinical and physiologic
signs of acute joint inflammation.
Conclusion. This study demonstrates that local
activation of PAR-4 leads to proinflammatory changes
in the knee joint that are dependent on the kallikrein–
kinin system. We also show for the first time that PARs
are involved in the modulation of joint pain, with PAR-4
being pronociceptive in this tissue. Thus, blockade of
articular PAR-4 may be a useful means of controlling
joint inflammation and pain.
Serine proteinases are a group of proteolytic
enzymes whose unique catalytic-reactive domains can
hydrolyze specific peptide bonds. In mammals, serine
proteinases are involved in wound healing, hemostasis,
and degradation of neuropeptides following neurogenic
inflammation. The levels of proteinases such as tryptase
are known to be elevated in synovial fluid extracted from
patients with arthritis (1,2), in whom the enzymatic
activity of proteinases is believed to lead to articular
tissue destruction. In addition to their typical degradative effects, proteinases are able to regulate cell signaling
via unique receptors called proteinase-activated receptors (PARs). The PARs are a superfamily of G protein–
coupled receptors with 7 transmembrane-spanning domains. They are triggered by a novel mechanism in
which the proteinase initially hydrolyzes a specific arginine cleavage site located on the extracellular
N-terminal loop of the receptor (3). This hydrolytic
event unmasks a new N-terminal sequence that then
binds to a docking domain on the same receptor while
remaining tethered at the other end. One of the major
advances in PAR research in recent years has been the
development of short synthetic peptides whose sequence
mimics the tethered ligand and can therefore act as
selective receptor agonists. These PAR-activating peptides are highly potent and resistant to aminopeptidases,
Dr. McDougall’s work was supported by the Canadian Institutes of Health Research; he is an Alberta Heritage Foundation for
Medical Research Senior Scholar and an Arthritis Society of Canada
Investigator. Dr. Vergnolle’s work was supported by the Canadian
Institutes of Health Research, the INSERM-Avenir program, the
Foundation Bettencourt-Schueller, and the Foundation UPSADouleur; she is an Alberta Heritage Foundation for Medical Research
Scholar and a Canadian Institute for Health Research Investigator.
1
Jason J. McDougall, PhD, Chunfen Zhang, MD, Laurie
Cellars, BSc, Eva Joubert, MSc, Chantelle M. Dixon, BSc (Hons):
University of Calgary, Calgary, Alberta, Canada; 2Nathalie Vergnolle,
PhD: University of Calgary, Calgary, Alberta, Canada, INSERM,
U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse,
France, and Université Toulouse III Paul Sabatier, Toulouse, France.
Address correspondence and reprint requests to Jason J.
McDougall, PhD, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1,
Canada. E-mail: mcdougaj@ucalgary.ca.
Submitted for publication May 23, 2008; accepted in revised
form November 7, 2008.
728
PAR-4 AND ARTHRITIS
making them useful pharmacologic tools for the in vivo
study of PARs.
To date, 4 PARs have been cloned: PAR-1,
PAR-2, PAR-3, and PAR-4. Different proteinases, ranging from thrombin, trypsin, cathepsins, kallikrein, or
even microbial proteinases, are able to activate these
receptors, inducing intracellular signaling (4). PAR-4,
the most recently defined member of this family of
receptors (5), can be activated by thrombin, trypsin,
cathepsin G, or activated factor X of the coagulation
cascade (4). In addition to its role in thrombin-induced
platelet aggregation, PAR-4 activation is thought to be
involved in inflammatory mechanisms, as revealed by
studies that have demonstrated the effects of PAR-4
agonists on leukocyte recruitment and plasma extravasation (6–8). Although these studies have demonstrated
a proinflammatory role for PAR-4 agonists, no study has
yet investigated the role of PAR-4 in models of organ
pathology. Studying the role of PAR-4 in debilitating
and painful conditions such as arthritis could reveal
novel signaling pathways related to joint pathology and
symptom development. As such, we tested the hypothesis that PAR-4 activation modulates the progression of
joint inflammation and alters pain sensitivity in an
animal model. Further experiments were carried out to
investigate the potential mechanisms of action of PAR-4
in joint inflammation and the potential role of PAR-4 in
inflammatory joint pain.
MATERIALS AND METHODS
Animals. C57BL/6 mice (6–8 weeks old) were purchased from Charles River Canada (Montreal, Quebec, Canada). All mice were housed under constant humidity and
temperature, under a 12-hour light–12-hour dark cycle. Institutional animal care committees approved all procedures,
which were in compliance with the Canadian Council for
Animal Care. At the end of the experiments, the mice were
humanely killed using sodium pentobarbital (200 mg/kg intraperitoneally), followed by cervical dislocation.
Chemicals. The PAR-4–activating peptide AYPGKFNH2, the PAR-4–inactive control peptide YAPGKF-NH2, and
the palmitoylated PAR-4 antagonist pepducin P4pal-10 (Npalmitoyl-SGRRYGHALR-NH2) were obtained from the
peptide synthesis facility at the University of Calgary. The
composition and purity of the peptides were confirmed by
high-performance liquid chromatography analysis. All peptides were dissolved in 0.9% NaCl.
Experimental design. Joint blood flow, joint diameter,
and thermal and mechanical nociception were determined
before (basal) and after intraarticular injections of the PAR4–activating peptide AYPGKF-NH2 (100-␮g intraarticular injection; 5-␮l bolus), the control inactive PAR-4 peptide
YAPGKF-NH2 (100-␮g intraarticular injection; 5-␮l bolus), or
their vehicle (0.9% NaCl; 5-␮l bolus). In order to elucidate the
729
mechanism of PAR-4 action in the joint, experiments with
AYPGKF-NH2 were repeated in the presence of either the
selective PAR-4 antagonist pepducin P4pal-10 (100 ␮g intraperitoneally) or the bradykinin antagonist HOE 140 (50 ␮g/kg
intraperitoneally). Antagonists were administered 1 hour prior
to the PAR-4–activating peptide. All experimental protocols
were carried out on separate groups of mice.
The role of PAR-4 in acute inflammatory hyperemia
and edema was examined in the following series of experiments. Under urethane anesthesia, the right knee joint was
shaved, swabbed with 100% ethanol, and injected with 2%
kaolin (10-␮l bolus). The joint was then extended and flexed
for 10 minutes to allow cartilage debridement and synovial
irritation. Mice then received a single intraarticular injection of
2% carrageenan (10-␮l bolus). Ten mice with inflamed tissue
were randomly assigned to 2 groups, with 1 cohort of mice
being treated with pepducin P4pal-10 (100 ␮g intraperitoneally) immediately following the induction of inflammation. The
control group of mice received a sterile saline injection (0.9%
NaCl intraperitoneally). Basal blood flow and joint edema
were then measured in both groups of mice over the succeeding 3–4 hours. Six mice (3 mice randomly assigned from each
treatment group) were then prepared for histopathologic
analysis (see below).
Assessment of joint blood flow. A total of 8 mice were
used in the studies assessing blood flow. Under deep urethane
anesthesia (50 mg intraperitoneally), the right carotid artery
was cannulated and attached to a pressure transducer (Stoelting, Wood Dale, IL) and then a blood pressure monitor (BP-1;
World Precision Instruments, Sarasota, FL) to record continuous mean arterial pressure. A small ellipse of skin was
removed from the anterior aspect of the right stifle (knee)
joint, and all underlying fascia was excised. Hydration of the
exposed joint capsule was attained by regular superfusion of
0.9% saline at 37°C. Each mouse was then placed supine on a
thermoregulated heating pad (TR-200; Fine Science Tools,
North Vancouver, British Columbia, Canada), which maintained core body temperature at 37°C as recorded by a rectal
thermometer.
Knee joint blood flow was measured noninvasively by
laser Doppler perfusion imaging, as previously described
(9,10). The imaging system (moorLDI2; Moor Instruments,
Axminster, UK) involves directing a low-power (2-mW) red
(␭ ⫽ 633 nm) laser beam onto the surface of the exposed knee
joint. The scanner head was positioned 15 cm above the
mouse, and the gain settings were optimized for mouse joint
blood flow measurement (DC gain ⫽ 0, flux gain ⫽ 2,
concentration gain ⫽ 2, background threshold ⫽ 100). By
means of a pivoted mirror, the laser beam scans in a continuous raster pattern over the surface of the joint, and a blood
perfusion measurement is made at multiple discrete loci.
Circulating erythrocytes in the articular microvasculature
cause a pulsed Doppler shift in the frequency of the laser light
that is proportional to the velocity of the moving blood cells
(flux component). A photodetection system captures the
Doppler-shifted laser light, and a central processor calculates
tissue blood flow, which is displayed as a color-coded map.
Joint scans were obtained before (control) and following
intraarticular injection of the test compounds (5-␮l bolus total
volume). Blood flow images were captured every 10 minutes
for up to 2 hours following drug administration.
730
MCDOUGALL ET AL
At the end of the recording period in all blood flow
experiments, the mouse was killed by anesthetic overdose
(sodium pentobarbital, 80-mg intracardiac injection), and a
scan of the dead mouse was obtained. This “biologic zero”
(which accounts for tissue optical noise and Brownian motion)
was subtracted from all captured images prior to data analysis.
All perfusion images were analyzed using proprietary
software (LDI Processing Software; Moor Instruments), and
the mean blood flow to the anterior joint capsule was calculated. To obviate any influence due to blood pressure fluctuations, vasomotor changes were expressed as vascular conductance, which was calculated as follows: conductance ⫽ blood
flow divided by mean arterial pressure. The effects of test
agents on joint vascular conductance were expressed as the percentage change in conductance between control and test images.
Joint edema measurement. The knee joint diameter
was measured before and at different time points after intraarticular knee injection in 8 mice and was used as an index of
edema. Measurements were performed by an investigator who
was blinded with regard to the experimental treatments, using
a digital caliper with a resolution of 10 ␮m. The calipers were
oriented in a mediolateral plane across the joint line, with
minimal or no compression being exerted onto the joint at the
time of measurement.
Pain assessment. Pain assessment was carried out in a
separate cohort of 8 mice. The latency of paw withdrawal to
radiant heat stimuli was measured using a Harvard plantar test
apparatus (Harvard Apparatus, South Natick, MA), essentially
as described by Hargreaves et al (11). Thermal stimulation was
applied to the plantar surface of the hind paw of mice that had
received intraarticular injections into the knee. Thermal hyperalgesia was defined as a significant decrease in the withdrawal latency compared with basal measurements at different
time points after the intraarticular injection.
Mechanical nociception was measured as follows. Mice
were placed into individual plastic cages. Von Frey hair
filaments with bending forces of 3.61g, 3.84g, and 4.08g were
pressed perpendicularly against the plantar skin of the ipsilateral hind paw and held for 5 seconds. This stimulation was
repeated 3 times in each hind paw tested, at intervals of several
seconds. The responses to these stimuli were ranked as follows:
0 ⫽ no response, 1 ⫽ move away from the filament, and 2 ⫽
immediate flinching or licking of the hind paw. The nociception score was calculated as follows:
Nociception score 共%兲 ⫽
⌺ 共average score of each animal)
⫻100
2 ⫻ no. of animals tested.
The nociception score was measured before and at different
time points after the intraarticular injection of test agents (5-␮l
bolus total volume).
Joint histomorphology. Following blood flow measurement, 6 of the mice with acute inflammation were prepared for
histopathologic analysis. Three of the mice had been treated
with pepducin P4pal-10 (100 ␮g intraperitoneally) immediately
following inflammation induction, while the remaining 3 mice
served as saline-injected controls (0.9% NaCl injected intraperitoneally). The ipsilateral hind limbs were removed and
fixed in 10% neutral buffered formalin for 5 days. Tissues were
then rinsed in distilled water, and excess muscle was removed.
Limbs were then slowly decalcified in 10% formic acid for ⬃10
days and embedded in paraffin. Sagittal sections of the whole
joint were cut at a thickness of 15 ␮m, mounted on glass
microscope slides, and dried overnight at 50°C. Sections were
deparaffinized in xylene and then dehydrated in serial dilutions
of ethanol before being rehydrated in water for 1 minute.
Sections were then stained with Weigert’s hematoxylin and
eosin and dehydrated in ethanol, coverslipped, and finally left
to dry at room temperature. Sections were viewed under brightfield microscopy and graded by an observer who was blinded with
regard to the degree of joint inflammation (i.e., the degree of
cellular infiltration and level of pannus formation). Wholenumber scoring was used for each parameter, which ranged from
0 (normal) to 3 (severe), for a possible total maximum score of 6.
A total of 30 sections from the saline-treated mice and 48 sections
from the pepducin P4pal-10 group were scored.
Indirect immunofluorescence. Three mice were anesthetized with 2% isoflurane prior to perfusion through the left
ventricle with 10 ml 0.9% saline and then 10 ml 4% paraformaldehyde (PFA). Knee joints were removed immediately and
post-fixed in 4% PFA overnight prior to decalcification with
Cal-Ex (Fisher Scientific, Fairlawn, NJ). Tissues were cryopreserved in 3 changes of 30% sucrose/phosphate buffered saline
(PBS), embedded in OCT compound (Sakura Finetek USA,
Torrance, CA), and sectioned to 10 ␮m on a Microm HM 500
cryostat (Microm, Waldorf, Germany). Sections were washed
once for 5 minutes in PBS (pH 7.2) and blocked for 60 minutes
in 10% normal donkey serum (Sigma-Aldrich, St. Louis, MO),
followed by incubation in a humidity chamber overnight at 4oC
with goat polyclonal anti–PAR-4 (1:50 dilution; Santa Cruz
Biotechnology, Santa Cruz, CA). Slides were washed 3 times
for 5 minutes in PBS, and specific staining was detected using
Cy3-labeled donkey anti-goat IgG (Jackson ImmunoResearch,
West Grove, PA) or anti–PAR-4 antibody together with the
blocking peptide (Santa Cruz Biotechnology). Slides were
washed for a final time with PBS and coverslipped with
FluorSave (Calbiochem-Novabiochem, San Diego, CA). Imaging was analyzed using OpenLab 3.0 software (Quorum Technologies, Guelph, Ontario, Canada) on a Leica DM6000B
(Leica, Wetzlar, Germany). The following controls were carried out to test the specificity of the primary antibody: 1)
immunohistochemistry was performed in the absence of the
primary antibody, and 2) PAR-4–blocking peptide (1:100
dilution; Santa Cruz Biotechnology) was used together with
the primary antibody. No immunofluorescence was detected in
either of these control experiments. Positive immunostaining
with PAR-4 was confirmed in sections of rat colon, as previously described (12) (data not shown).
Statistical analysis. All data conformed to a Gaussian
distribution and were analyzed using parametric statistics, i.e.,
Student’s t-test, one-way analysis of variance (ANOVA), and
two-way ANOVA. A Bonferroni post hoc test was used to
determine differences at individual time points. P values less
than 0.05 were considered significant. All data points are
expressed as the mean ⫾ SEM.
RESULTS
Immunohistochemical localization of PAR-4 in
mouse knee joints. Immunopositive staining for PAR-4
was detected in chondrocytes and subchondral bone
PAR-4 AND ARTHRITIS
731
Figure 1. Immunolocalization of proteinase-activated receptor 4 (PAR-4). A and C, Polarized light photomicrographs. B and
D, Higher-magnification views of the boxed areas in A and C. PAR-4 was detected in chondrocytes of the femur as well as in
some regions of the subchondral bone and meniscus (B). PAR-4 was also detected in the synovium (D), where it appeared to
be associated with nerve fiber bundles (arrows). F ⫽ femur; T ⫽ tibia; M ⫽ meniscus; S ⫽ synovium.
associated with the femur and tibia of the mouse knee
(Figure 1B). PAR-4 was also detected in the menisci of
the normal knee joint, with positive staining appearing
throughout the depth of the tissue (Figure 1B). Finally,
pronounced PAR-4 staining was also observed in the
synovium (Figure 1D). No staining for PAR-4 was
observed in slices exposed to both anti–PAR-4 antibody
and the blocking peptide (results not shown).
Joint inflammation caused by PAR-4 activation.
Intraarticular injection of the selective PAR-4 agonist
AYPGKF-NH2 caused a gradual increase in knee joint
blood flow, which reached a maximal response ⬃2 hours
after administration (Figure 2A). This hyperemic response to the PAR-4 agonist was statistically significantly different from the response to vehicle control and
the PAR-4–inactive peptide YAPGKF-NH2 (P ⬍ 0.0001
by two-way ANOVA; n ⫽ 8). Similarly, the intraarticular
injection of AYPGKF-NH2 caused an increase in joint
diameter, characteristic of edema (Figure 2B). This
increase was maximal 2 hours after the injection, and at
⬃4 hours was still significantly higher than the effect of
the saline injection or injection of the control peptide
YAPGKF-NH2 (P ⬍ 0.05 by two-way ANOVA; n ⫽ 8)
(Figure 2B).
The AYPGKF-NH2–mediated increase in joint
vascular conductance and edema in the mice was significantly attenuated by treatment with the selective
PAR-4 antagonist pepducin P4pal-10 (Figures 2C and
D), suggesting that PAR-4 activation is responsible for
the AYPGKF-NH2–induced inflammatory response.
Pepducin P4pal-10 given alone had no effect on joint
edema.
MCDOUGALL ET AL
732
Figure 2. Time course of changes in joint vascular conductance (A and C) and joint diameter (B and D) following intraarticular
injection of the proteinase-activated receptor 4 agonist AYPGKF-NH2, the inactive peptide YAPGKF-NH2, or saline vehicle
control, in naive mice (A and B) or mice treated with the PAR-4 antagonist pepducin P4pal-10, the bradykinin B2 antagonist
HOE 140, or vehicle (C and D). Values are the mean and SEM results from 8 mice per group. ⴱ ⫽ P ⬍ 0.05 versus
YAPGKF-NH2 (A and B) or versus vehicle (C and D).
Joint pain caused by PAR-4 activation. Intraarticular injection of the selective PAR-4 agonist
AYPGKF-NH2 caused a significant decrease in paw
withdrawal latency in response to a plantar thermal
stimulus, compared with the effects of the control peptide YAPGKF-NH2 or vehicle (saline) (Figure 3A). This
decreased withdrawal latency is characteristic of thermal
hyperalgesia. In response to plantar mechanical stimulation with a small-size von Frey hair filament, mice that
had received a knee joint injection of saline or the
control peptide YAPGKF-NH2 did not show a nociceptive response (Figure 3B). In contrast, mice that had
received an intraarticular injection of the PAR-4–
activating peptide AYPGKF-NH2 showed a strong no-
ciceptive response to this small-size von Frey filament
(Figure 3B). Such a response is characteristic of allodynia. When stimulated with larger-size von Frey filaments, mice injected with saline or control peptide
YAPGKF-NH2 showed a nociceptive response proportional to the size of the filament, and this response did
not vary significantly by time after injection (Figures 3C
and D). However, mice injected with the PAR-4–
activating peptide AYPGKF-NH2 showed a significant
increase in nociceptive response to middle-size (3.84g)
(Figure 3C) and large-size (4.08g) filaments (Figure 3D)
compared with basal values. This increased nociceptive
response is characteristic of mechanical hyperalgesia.
AYPGKF-NH2–induced thermal hyperalgesia
PAR-4 AND ARTHRITIS
733
Figure 3. Time course of withdrawal latency in response to a plantar thermal stimulus (A) and of the nociception score in
response to plantar mechanical stimulation with von Frey filaments at 3.61g (B), 3.84g (C), or 4.08g (D) following intraarticular
injection of the proteinase-activated receptor 4 agonist AYPGKF-NH2, the inactive peptide YAPGKF-NH2, or saline vehicle
control. Plantar stimulation was performed on the paw that received the intraarticular injection. Values are the mean and
SEM results from 8 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, versus basal (time 0).
(decrease in withdrawal latency) was not observed in
mice that received systemic treatment with the PAR-4
antagonist pepducin P4pal-10 (Figure 4A). Similarly,
AYPGKF-NH2–induced mechanical allodynia (increased nociception score in response to small-size von
Frey filament) and hyperalgesia (increased nociception
score in response to medium- and large-size von Frey
filaments) were not observed in mice treated with pepducin P4pal-10 (Figures 4B–D). When given alone,
pepducin P4pal-10 had no effect on thermal sensitivity
or mechanosensitivity (see Supplementary Figure 1,
available on the Arthritis & Rheumatism Web Site at
http://www3.interscience.wiley.com/journal/76509746/
home). Taken together, these results show that PAR-4
activation is responsible for AYPGKF-NH2–induced
allodynia and hyperalgesia.
Involvement of a bradykinin B 2 receptor–
mediated mechanism in PAR-4–induced joint inflammation and pain. An AYPGKF-NH2–mediated increase
in joint vascular conductance and edema was completely
inhibited by treatment of mice with the bradykinin B2
receptor antagonist HOE 140 (Figures 2C and D).
Similarly, thermal and mechanical hyperalgesia, characterized by decreased withdrawal latency and an increased nociception score, respectively, were fully inhibited in mice treated systemically with HOE 140 (Figure
MCDOUGALL ET AL
734
Figure 4. Time course of withdrawal latency in response to a plantar thermal stimulus (A) and of the nociception score in
response to plantar mechanical stimulation with von Frey filaments at 3.61g (B), 3.84g (C), or 4.08g (D) following
intraarticular injection of the proteinase-activated receptor 4 (PAR-4) agonist AYPGKF-NH2 in mice treated with the
PAR-4 antagonist pepducin P4pal-10, the bradykinin B2 antagonist HOE 140, or vehicle. Plantar stimulation was performed
on the paw that received the intraarticular injection. Values are the mean and SEM results from 8 mice per group. ⴱ ⫽ P ⬍
0.05; ⴱⴱ ⫽ P ⬍0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, versus basal (time 0).
4). HOE 140 by itself had no significant effect on joint
edema, thermal sensitivity, or mechanosensitivity in
these normal mice.
Pivotal role of PAR-4 activation in joint inflammation. Three hours after intraarticular injection of
kaolin/carrageenan, there was a pronounced inflammatory reaction in the joint, characterized by synovial
hyperplasia and an influx of inflammatory cells (see
Supplementary Figure 2, available on the Arthritis &
Rheumatism Web site at http://www3.interscience.
wiley.com/journal/76509764/home). In contrast, pretreatment of mice with acute tissue inflammation with
pepducin P4pal-10 ameliorated the severity of tissue
inflammation. Histologic scoring of whole joint sections
under blinded conditions revealed that the levels of
cellular infiltration and synovial hyperplasia were significantly reduced (P ⬍ 0.0001 by Student’s unpaired t-test;
n ⫽ 30–48) in mice pretreated with the PAR-4 antagonist (Table 1).
Table 1. Histologic scores of acutely inflamed knee joints in mice
treated with either saline or pepducin P4pal-10
Pannus formation
Cellular infiltrate
Total score
Saline
Pepducin P4pal-10
2.5
2.2
4.7
1.3*
1.4*
2.7*
* P ⬍ 0.0001 versus saline treatment, by Student’s unpaired t-test.
PAR-4 AND ARTHRITIS
735
DISCUSSION
Figure 5. Progression of joint hyperemia (A) and edema formation
(B) in the acutely inflamed knee joints of mice treated with either
saline or the proteinase-activated receptor 4 antagonist pepducin
P4pal-10. Both synovial blood flow and joint swelling were attenuated
by pepducin treatment. Values are the mean and SEM results from
6–10 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus saline
treatment.
Blood flow studies showed that treatment of
kaolin/carrageenan-injected knees with the PAR-4 antagonist pepducin P4pal-10 reduced the hyperemia associated with acute joint inflammation (Figure 5A).
Joint edema was also significantly reduced at 2 hours
and 3 hours after kaolin/carrageenan injection in mice
treated with pepducin P4pal-10 (Figure 5B).
One of the pronounced hallmarks of arthritis is
the extensive destruction and overall abnormal remodeling of joint tissues. High levels of proteinases released
into diseased joints are likely the primary driving force
responsible for these structural changes. In addition to
the enzymatic activity of proteinases, it has been eloquently demonstrated that these agents can act as signaling molecules by triggering a specialized family of
receptors called PARs (for review, see ref. 3). This study
provides the first evidence that PAR activation modulates pain sensitivity in joints and reveals a vital role of
PAR-4 in promoting joint inflammation.
The results of the immunolocalization experiments presented here clearly show for the first time that
PAR-4 is expressed extensively throughout the mouse
knee joint in tissues such as the cartilage, subchondral
bone, menisci, and synovium. Articular activation of
PAR-4 with the selective peptide agonist AYPGKF-NH2
caused a gradual rise in synovial blood flow with concomitant edema formation, which was maximal ⬃2
hours after intraarticular injection. These inflammatory
reactions to the peptide could be blocked by pretreatment with the PAR-4 antagonist pepducin P4pal-10.
These results are consistent with observations in the
hind paw and the mesenteric circulation, in which
PAR-4 activation leads to edema and granulocyte infiltration (6–8). Considering the strong expression of
PAR-4 in endothelial cells (8), it could be hypothesized
that in the joint, PAR-4 activation–induced edema is
attributable to endothelial cell activation and subsequent increased vascular permeability; however, the
expression of PAR-4 in the various cell types described
here indicates that there are multiple targets in which
PAR-4 agonists could induce inflammation.
Other PARs have similarly been implicated in the
pathogenesis of arthritis. PAR-4, for example, has been
detected in the synovial tissue of patients with rheumatoid arthritis and patients with osteoarthritis (13), and
thrombin-induced PAR-1 activation led to increased
inflammatory chemokine expression by human synovial
fibroblasts (14). In other studies, antigen-induced arthritis was found to be less severe in PAR-1–knockout mice
compared with wild-type controls (15). Similarly, PAR-2
has been implicated in joint disease, as evidenced by its
isolation from the joints of patients with arthritis (16,17)
and by the observation that PAR-2 activation leads to
proinflammatory changes in the knee joints in animal
models (18,19). Thus, the data presented here extend
MCDOUGALL ET AL
736
the role of PARs in arthritis by showing that PAR-4 is
also proinflammatory in joints.
PAR activation is known to modulate pain in
animal models (20–22); however, the role of PARs in
joint pain has not previously been explored. In this study,
it was demonstrated that intraarticular injection of a
PAR-4 agonist caused a heightened pain response to
both thermal and mechanical stimuli in mice. This
allodynic and hyperalgesic effect of AYPGKF-NH2 was
inhibited by treating the mice with the selective PAR-4
antagonist pepducin P4pal-10. These pain-causing effects of PAR-4 activation are in contrast to the previously described antinociceptive effects of AYPGKFNH2 in the rat hind paw (20). In addition to the obvious
differences in species and injection sites between the
studies, the most likely explanation for these divergent
results is that in the present study, a higher dose of the
PAR-4 agonist was used. Thus, it appears that low-dose
AYPGKF-NH2 has the ability to reduce nociceptive
pain, whereas higher concentrations of the peptide cause
hyperalgesia and allodynia.
The mechanism of action of PAR-4 in causing
joint inflammation and pain appears to involve the
kallikrein–kinin system, because the bradykinin antagonist HOE 140 attenuated edema, hyperemia, and nocifensive responses to AYPGKF-NH2. This observation is
consistent with findings of a previous study showing that
PAR-4–mediated paw edema could be inhibited by
pretreatment with a bradykinin B2 antagonist (7). Because PAR-4 has been detected on the surface of
leukocytes, endothelial cells, and vascular smooth muscle cells (8), it is hypothesized that PAR-4–activating
peptides stimulate these cells to release kallikreins,
which then cleave kininogens to produce active kinins,
which ultimately bind to bradykinin receptors, resulting
in vasodilatation and increased vascular permeability.
Indeed, bradykinin is known to be vasoactive in knee
joints, where it causes synovial vasodilatation and protein extravasation (23–25). The role of the kallikrein–
kinin system in promoting joint pain is also corroborated
by other studies showing that bradykinin causes peripheral sensitization of knee joint afferent nerves, leading
to enhanced pain sensation (26,27). Other PARs such as
PAR-1 and PAR-2 produce pain and inflammation by
causing the secondary release of proinflammatory neuropeptides from sensory neurones and by activating
connective tissue mast cells (28–31). In contrast, however, the inflammatory actions of PAR-4 are not neurogenically driven, nor do they involve mast cell degranulation (6).
Experiments were also undertaken to determine
whether blockade of PAR-4 with a selective antagonist
could inhibit disease severity in a model of acute synovitis. Pepducin P4pal-10 treatment of mice with kaolin/
carrageenan–induced inflammation significantly ameliorated the severity of joint inflammation, as evidenced by
a reduction in pannus formation and inflammatory cell
infiltration. The antiarthritic effects of PAR-4 antagonism were consistent throughout the whole depth of the
joint as well as in both lateral and medial compartments.
In other experiments, it was shown that pretreating
acutely inflamed knees with pepducin P4pal-10 attenuated the hyperemia and edema associated with this
model. These results clearly indicate that blockade of
PAR-4 has a beneficial effect on the pathogenesis of
acute synovitis.
In summary, this study has shown that the triggering of PAR-4 caused a proinflammatory and painful
reaction in the mouse knee joint. Blockade of bradykinin
receptors attenuated the physiologic activity of
AYPGKF-NH2, suggesting that the kallikrein–kinin system is involved in mediating the nocifensive and inflammatory responses to PAR-4 activation. Finally, blockade
of PAR-4 with a selective antagonist ameliorated pannus
formation and leukocyte infiltration in response to an
acute inflammatory insult, implicating PAR-4 as one of
the major proinflammatory signals in this model. As
such, PAR-4 may be a useful target for the treatment of
inflammatory joint disease and associated pain.
AUTHOR CONTRIBUTIONS
Dr. McDougall 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. McDougall, Vergnolle.
Acquisition of data. McDougall, Zhang, Cellars, Joubert, Dixon.
Analysis and interpretation of data. McDougall, Zhang, Joubert,
Vergnolle.
Manuscript preparation. McDougall, Dixon, Vergnolle.
Statistical analysis. McDougall, Zhang, Joubert.
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