Protease-activated receptor 2 has pivotal roles in cellular mechanisms involved in experimental periodontitis.
ABSTRACT The tissue destruction seen in chronic periodontitis is commonly accepted to involve extensive upregulation of the host inflammatory response. Protease-activated receptor 2 (PAR-2)-null mice infected with Porphyromonas gingivalis did not display periodontal bone resorption in contrast to wild-type-infected and PAR-1-null-infected mice. Histological examination of tissues confirmed the lowered bone resorption in PAR-2-null mice and identified a substantial decrease in mast cells infiltrating the periodontal tissues of these mice. T cells from P. gingivalis-infected or immunized PAR-2-null mice proliferated less in response to antigen than those from wild-type animals. CD90 (Thy1.2) expression on CD4(+) and CD8(+) T-cell-receptor beta (TCRbeta) T cells was significantly (P < 0.001) decreased in antigen-immunized PAR-2-null mice compared to sham-immunized PAR-2-null mice; this was not observed in wild-type controls. T cells from infected or antigen-immunized PAR-2-null mice had a significantly different Th1/inflammatory cytokine profile from wild-type cells: in particular, gamma interferon, interleukins (interleukin-2, -3, and -17), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha demonstrated lower expression than wild-type controls. The absence of PAR-2 therefore appears to substantially decrease T-cell activation and the Th1/inflammatory response. Regulation of such proinflammatory mechanisms in T cells and mast cells by PAR-2 suggests a pivotal role in the pathogenesis of the disease.
- SourceAvailable from: jimmunol.org[show abstract] [hide abstract]
ABSTRACT: Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.The Journal of Immunology 07/2003; 170(11):5690-6. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Proteinase 3 (PR3), a 29-kDa serine proteinase secreted from activated neutrophils, also exists in a membrane-bound form, and is suggested to actively contribute to inflammatory processes. The present study focused on the mechanism by which PR3 activates human oral epithelial cells. PR3 activated the epithelial cells in culture to produce IL-8 and monocyte chemoattractant protein-1 and to express ICAM-1 in a dose- and time-dependent manner. Incubation of the epithelial cells for 24 h with PR3 resulted in a significant increase in the adhesion to neutrophils, which was reduced to baseline levels in the presence of anti-ICAM-1 mAb. Activation of the epithelial cells by PR3 was inhibited by serine proteinase inhibitors and serum. The epithelial cells strongly express protease-activated receptor (PAR)-1 and PAR-2 mRNA and weakly express PAR-3 mRNA. The expression of PAR-2 on the cell surface was promoted by PR3, and inhibited by cytochalasin B, but not by cycloheximide. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization, and rendered cells refractory to subsequent stimulation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein-coupled PAR-2 and actively participates in the process of inflammation such as periodontitis.The Journal of Immunology 11/2002; 169(8):4594-603. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Periodontal diseases are chronic inflammatory diseases that can result in resorption of the alveolar bone of the jaw. We have developed a murine model in which alveolar bone loss is induced by oral infection with Porphyromonas gingivalis, an oral anaerobic bacterium associated with periodontal disease in humans. Here we compared a strain of immunocompetent mice (C57BL/6J) to the same strain of mice made T cell deficient by genetic deletion of the alpha chain of their T cell receptors (C57BL/6J-Tcra). T cell deficiency did not affect the ability of P. gingivalis to implant in the oral cavity. The two strains of mice had equal percentages of P. gingivalis among their cultivable anaerobes 7 weeks after infection. The same bacterial load led to much less bone resorption in the T cell deficient mice than in the immune normal mice, measured as either the number of sites with significant loss, or as the total amount of bone resorbed. T cell deficient mice lost bone at only three out of 14 measurement sites, compared with eight out of 14 sites in the wild-type mice. The total amount of bone lost was 70% less in the T cell deficient mice. T cell deficient mice had lower titers of P. gingivalis-specific IgG than the wild-type mice after oral infection did, but the same titers of specific IgA. Lower titers did not correlate with greater bone loss. Antigen-activated T lymphocytes are known to induce osteoclastogenesis; here we demonstrate that T cell deletion decreases the amount of alveolar bone loss induced by infection of the murine oral cavity.FEMS Immunology & Medical Microbiology 10/2002; 34(1):45-50. · 2.68 Impact Factor
INFECTION AND IMMUNITY, Feb. 2010, p. 629–638
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 2
Protease-Activated Receptor 2 Has Pivotal Roles in Cellular
Mechanisms Involved in Experimental Periodontitis?
David M. Wong,1,3,4Vivian Tam,1,2Roselind Lam,1,2Katrina A. Walsh,1,2Liliana Tatarczuch,1,3
Charles N. Pagel,1,3Eric C. Reynolds,1,2Neil M. O’Brien-Simpson,1,2†
Eleanor J. Mackie,1,3† and Robert N. Pike1,4*†
CRC for Oral Health Sciences1and Melbourne Dental School,2University of Melbourne, Carlton, Victoria 3010, Australia;
Faculty of Veterinary Science, University of Melbourne, Parkville, Victoria 3010, Australia3; and Department of
Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia4
Received 7 September 2009/Returned for modification 11 October 2009/Accepted 15 November 2009
The tissue destruction seen in chronic periodontitis is commonly accepted to involve extensive upregulation
of the host inflammatory response. Protease-activated receptor 2 (PAR-2)-null mice infected with Porphyromo-
nas gingivalis did not display periodontal bone resorption in contrast to wild-type-infected and PAR-1-null-
infected mice. Histological examination of tissues confirmed the lowered bone resorption in PAR-2-null mice
and identified a substantial decrease in mast cells infiltrating the periodontal tissues of these mice. T cells from
P. gingivalis-infected or immunized PAR-2-null mice proliferated less in response to antigen than those from
wild-type animals. CD90 (Thy1.2) expression on CD4?and CD8?T-cell-receptor ? (TCR?) T cells was
significantly (P < 0.001) decreased in antigen-immunized PAR-2-null mice compared to sham-immunized
PAR-2-null mice; this was not observed in wild-type controls. T cells from infected or antigen-immunized
PAR-2-null mice had a significantly different Th1/inflammatory cytokine profile from wild-type cells: in
particular, gamma interferon, interleukins (interleukin-2, -3, and -17), granulocyte-macrophage colony-stim-
ulating factor, and tumor necrosis factor alpha demonstrated lower expression than wild-type controls. The
absence of PAR-2 therefore appears to substantially decrease T-cell activation and the Th1/inflammatory
response. Regulation of such proinflammatory mechanisms in T cells and mast cells by PAR-2 suggests a
pivotal role in the pathogenesis of the disease.
Protease-activated receptors (PARs) are G-protein-coupled,
seven-transmembrane proteins present on many cell types, in-
cluding epithelial, endothelial, and neuronal cells; T cells; and
osteoblasts (1, 37, 39, 48, 57). Rather than being activated
solely by ligand occupancy, these receptors are activated by
proteolysis of their N terminus to reveal a tethered ligand that
interacts with the extracellular loops of the receptor (42).
There are four known PARs; PAR-1, -3, and -4 have throm-
bin as their usual physiological activator, whereas PAR-2 is
activated by a variety of proteases, including trypsin, mast cell
tryptase, and neutrophil protease 3 (57). PAR-2 is thought to
play pivotal roles in stimulating inflammation, with involve-
ment in increased leukocyte rolling (38), increased epithelial
cell permeability (32) and a possible role(s) in chronic arthritis
PAR-2 has been implicated in periodontitis, which is a
chronic inflammatory disease associated with the destruction
of the periodontal tissues, including bone, leading to tooth loss
(23, 28, 47). Chronic periodontitis has also been linked with
certain systemic diseases, such as diabetes and cardiovascular
diseases. Porphyromonas gingivalis, which has been strongly
associated with chronic periodontitis (7, 27, 46), produces cys-
teine proteases known as gingipains (31, 49, 50). The arginine-
specific gingipains, RgpA and RgpB, are known to activate
PAR-1 and PAR-2, with studies showing that PAR-2 activation
by RgpB in oral epithelial cells leads to an increased release of
interleukin-6 (IL-6), a known stimulator of osteoclasts, the
cells responsible for bone resorption (39). In addition, activa-
tion of PAR-2 on gingival epithelial cells by gingipains induces
the production of the human ?-defensin antimicrobial peptide
(14), and RgpB upregulates proinflammatory neuropeptides in
dental pulp cells in a PAR-2-dependent manner (56). Other
studies have shown that activation of PAR-2 on oral epithelial
cells by neutrophil proteases (58) and by human leukocyte
elastase and cathepsin G in gingival fibroblasts upregulates
IL-8 production (57). IL-8 is a chemokine that activates neu-
trophils, as well as a chemoattractant that leads neutrophils
and T cells to sites of inflammation (5).
The in vitro studies mentioned above suggest a proinflam-
matory role of PAR-2 in periodontitis. Recently, an in vivo
study showed that oral challenge of rats with a PAR-2 agonist
peptide induced many symptoms of periodontitis, such as in-
creased alveolar bone loss and gingival granulocyte infiltration,
as well as overexpression of matrix metalloproteinase 2
(MMP-2) and MMP-9 and cyclooxygenase 1 (COX-1) and
COX-2 (29). That study suggested that a cascade of events
starting with PAR-2 activation induces cytokine release, lead-
ing to bone resorption and tissue degradation. Confirmation of
the involvement of PAR-2 in experimental periodontitis was
provided by a study where PAR-2?/?mice orally challenged
with P. gingivalis exhibited less alveolar bone loss than did
PAR-2?/?challenged mice (28). We have show here that
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Biology, Monash University, Clayton, Victoria
3800, Australia. Phone: 61-3-99029300. Fax: 61-3-99029500. E-mail:
† N.M.O.-S., E.J.M., and R.N.P. contributed equally to this study.
?Published ahead of print on 23 November 2009.
PAR-2 is involved in a number of the cellular mechanisms that
underlie the pathogenesis of experimental periodontitis.
MATERIALS AND METHODS
Animals and materials. Mice in which the PAR-1gene had been disrupted by
homologous recombination (16) were kindly provided by S. Coughlin (University
of California, San Francisco). These mice have been extensively backcrossed on
the C57BL/6J background and were bred at Monash University. Age- and sex-
matched C57BL/6J mice were used as wild-type controls. PAR-2-null mice were
generated on a 129Sv background by J. Morrison and M. Stevens (52). For
experiments utilizing PAR-2?/?mice, PAR-2?/?littermates were used as con-
trols. The mouse colony was maintained by PAR-2?/?? PAR-2?/?matings, and
all mice were genotyped by using a PCR-based approach. Experiments making
use of animal tissues were approved by the Animal Care and Use Committee of
the Department of Biochemistry and Molecular Biology, Monash University
(Ethics approval, BAM/B/23/2003). All primers for PCR and quantitative PCR
were synthesized by Geneworks (Adelaide, Australia). PCR reagents, such as
deoxynucleoside triphosphates, 50-bp DNA ladder, PCR buffer, 25 mM MgCl2,
and GoTaq DNA polymerase were purchased from Promega (Madison, WI). P.
gingivalis strain W50 was grown and harvested as described previously (44, 45)
and used as a live inoculum for the mouse periodontitis model. For immuniza-
tion and T-cell assays P. gingivalis cultures were harvested, and the cell pellets
were resuspended in 40 ml of 0.5% (vol/vol) formalin overnight at room tem-
perature. Fresh formalin was added the next day, after which the bacteria (for-
malin-killed W50 [FK-W50]) were ready for use.
Mouse periodontitis model. The mouse periodontitis model was performed as
described previously (49) and involved orally inoculating mice with live P. gin-
givalis cells (strain W50). Overall, 24 wild-type and 24 PAR-1 or PAR-2-null mice
at 8 weeks of age were used for the experiment. The wild-type and receptor-null
mice groups were equally subdivided into groups that were infected with P.
gingivalis or sham infected. The infection regimen for the periodontitis model
was as follows: four doses of 1010bacterial cells 2 days apart, followed by a 10-day
break before another four doses of 1010bacterial cells 2 days apart. Mice were
killed 4 weeks after the last inoculation, and then the maxillae were removed and
dissected into left-half and right-half maxillae for analyses as previously de-
Histomorphometric analysis of maxillae. For one-half maxilla from each an-
imal, a digital image of the buccal aspect was captured with an Olympus DP12
digital camera mounted on a dissecting microscope, using OLYSIA BioReport
software version 3.2 (Olympus Australia Pty, Ltd., New South Wales, Australia)
to assess horizontal bone loss, defined as loss occurring in a horizontal plane,
perpendicular to the alveolar bone crest (ABC) that results in a reduction of the
crest height. Each half-maxilla was aligned so that the molar buccal and lingual
cusps of each image were superimposed, and the image was captured with a
micrometer scale in frame, so that measurements could be standardized for each
image. The area of exposed root surface, between the cementoenamel junction
and the ABC for each molar tooth was measured using the software. These
measurements were determined twice by a single blinded examiner using a
randomized and coded protocol.
The other half maxillae from three mice of each group were processed for
histomorphometry as previously described (2). Briefly, the specimens were fixed,
decalcified, and embedded in Spurr’s resin. Semithin sections (2 to 5 ?m) were
stained in 0.5% (wt/vol) methylene blue. Mast cell counts and analysis of alveolar
bone resorption (eroded surface) were conducted on images of sections captured
with a digital camera (Spot; Diagnostic Instruments, Inc.) linked to an Olympus
BX60 microscope. Measurements were made with Image-Pro Plus image analysis
software (Media Cybernetics, Silver Spring, MD).
Mast cells were identified as large cells with unilobed nuclei containing nu-
merous intensely metachromatically stained granules in their cytoplasm (Fig. 1b,
inset) (4). Mast cells were counted in a specific field located at the base of the
gingival sulcus (buccal and lingual aspects) and in between the cementum and
alveolar bone (illustrated in Fig. 1a and b). The eroded surface (ES) of the
alveolar bone on the buccal aspect was measured in a region extending 450 ?m
from the ABC toward the apex of the tooth (illustrated in Fig. 1c) and expressed
as a percentage of bone surface (BS), that is, ES/BS ? 100. For the mast cell
counts and the percentage of alveolar surface erosion, results from three sections
were averaged to give the result for each animal. The results for mast cell counts
and alveolar surface erosion were obtained from three animals per genotype and
treatment group. All histomorphometric analyses were performed by a single
blinded examiner using a randomized and coded protocol.
Flow cytometric analysis. Mice were immunized (25 ?g/mouse with formalin-
killed P. gingivalis W50 in incomplete Freund adjuvant administered subcutane-
ously to the hind paw), and 7 days later the popliteal and inguinal lymph nodes
were removed and the lymph nodes were pooled into their respective groups.
Lymphocytes were isolated from the four treatment groups (PAR-2?/?sham
immunized, PAR-2?/?immunized, PAR-2?/?sham immunized, and PAR-2?/?
immunized) after processing the lymph nodes through a sieve, following which
the separated tissue was collected and gently layered onto 5 ml of Lympholyte M
and centrifuged (800 ? g, 20 min). The lymphocyte layer was collected and
washed twice with Dulbecco modified Eagle medium (DMEM) containing 10%
(vol/vol) fetal calf serum (FCS), penicillin (100 U/ml), streptomycin sulfate (100
?g/ml) and glutamate. The lymphocytes were counted by using a Coulter particle
counter (Beckman Coulter, Fullerton, CA), after which 106cells were added per
well for each experimental group on a 96-well plate. The plate was centrifuged
at 800 ? g for 5 min, and the supernatant was removed. For each treatment
group, four wells were used, with two wells for antibody mixture 1 (anti-CD90/
Thy-1.2-fluorescein isothiocyanate [FITC], anti-CD8-phycoerythrin [PE] and
anti-TCR?-allophycocyanin [APC]; BD Biosciences, New South Wales, Austra-
lia), and two wells for antibody mixture 2 (anti-CD90/Thy-1.2-FITC, anti-CD4-
PE, and anti-TCR?-APC). Both antibody mixtures containing 1% (vol/vol) FC
blocker (BD Biosciences) were incubated with shaking for 20 min at room
temperature in the dark. Cells alone (PAR-2?/?nonimmunized) and single-
color controls (where PAR-2?/?nonimmunized lymphocytes were incubated
with one fluorescent antibody) were used as background controls. A Cytomics
FC500 flow cytometer (Beckman-Coulter, New South Wales, Australia) was used
to detect the stained lymphocytes.
T-cell proliferation and ELISPOT assays. The remaining isolated lymphocytes
from the immunized and sham-immunized mice were further purified by using
mouse PAN T-cell MAC beads and an Automac bead cell sorter (Miltenyi
Biotech, New South Wales, Australia). Spleens were also removed from the
sham-immunized mice and pooled into PAR-2?/?and PAR-2?/?groups.
Spleens were processed through a sieve, the single-cell suspension was washed
twice (800 ? g) in DMEM, and the red blood cells were lysed using red cell lysis
buffer (Sigma-Aldrich, New South Wales, Australia). After three washes (800 ?
g) in DMEM, the splenic cells were irradiated (2,200 rads) and used as a source
of syngeneic antigen-presenting cells in the T-cell and enzyme-linked immuno-
spot (ELISPOT) assays.
A serial dilution of P. gingivalis FK-W50 cells (starting from 25 ?g of protein/
100 ml) was added to a 96-well tissue culture plate. T cells from the four
experimental treatment groups were added in triplicate to the wells containing P.
gingivalis FK-W50 at a concentration of 105cells/well. PAR-2?/?sham-immu-
nized antigen presenting cells at a concentration of 105cells were added to the
wells containing T cells from PAR-2?/?sham-immunized and immunized mice.
PAR-2?/?sham-immunized antigen presenting cells were added to the wells
containing T cells from PAR-2?/?sham-immunized and immunized mice. For
each group, a negative control was used where wells only contained T cells and
antigen-presenting cells. Serially diluted concanavalin A was used as a positive
control by adding it to wells that contained T cells and antigen-presenting cells.
The plates were incubated at 37°C in 5% CO2for 3 days, after which 1 ?Ci of
[3H]thymidine (Amersham Biosciences, Buckinghamshire, United Kingdom)
was added to the cells, followed by incubation for a further 18 h. After incuba-
tion, 40 ?l of mammalian cell lysis buffer (Sigma-Aldrich) was added to the wells
for 20 min. Cells were subsequently harvested onto glass fiber filter mats using
the MACHIII cell harvester (Tomtec, Hamden, CT), and the filter was air dried
and sealed into sample bags containing 5 ml of scintillant fluid. A Wallac Mi-
crobeta ?-scintillation counter instrument (Perkin-Elmer, New South Wales,
Australia) was used to count the radioactivity emitted from the glass fiber filter
mats, and the counts per minute (cpm) in each well were determined. The mean
cpm values for either FK-W50-stimulated or control cells were calculated by
averaging the counts in the respective triplicate wells. The stimulatory index (SI)
was calculated by dividing the mean cpm obtained after antigen stimulation by
the mean cpm detected in control, unstimulated wells.
For the ELISPOT assays, Millipore Multiscreen 96-well filtration plates
(MAHAS450; Millipore, New South Wales, Australia) were coated with anti-
mouse cytokine capture antibodies (eBiosciences, San Diego, CA), specific for
IL-4 and gamma interferon (IFN-?), at a concentration of 4 ?g/ml in 0.1 M
sodium bicarbonate buffer (pH 9.5), followed by incubation overnight at 4°C. The
ELISPOT plates were washed with Dulbecco phosphate-buffered saline (PBS)
and blocked with enriched DMEM for 1 h at 37°C. Lymph node T cells from each
group and spleens were prepared as described above. Lymph node cells (105/
well) from FK-W50-immunized PAR-2?/?or PAR-2?/?mice were incubated
with gamma-irradiated (2,200 rads) syngeneic spleen cells as a source of antigen-
presenting cells (PAR-2?/?or PAR-2?/?, respectively, 105cells/well) and P.
gingivalis FK-W50 cells (1.0 ?g/ml). Plates were incubated at 37°C in an atmo-
sphere of 5% CO2in air for 48 h in a humidified incubator, after which they were
630WONG ET AL.INFECT. IMMUN.
washed with PBS containing 0.05% (vol/vol) Tween 20 (PBST) three times
and once with deionized water. Cytokine-specific biotinylated antibodies
(eBiosciences, San Diego, CA) specific for IL-4 and IFN-? were added at a
concentration of 2 ?g/ml in Dulbecco PBS-enriched DMEM (1:1 [vol/vol]) and
incubated at room temperature for 2 h. Plates were washed six times with PBST
and streptavidin-alkaline phosphatase conjugate (Roche, Castle Hill, New South
Wales, Australia) was added to the plates at a 1:1,000 dilution in Dulbecco
PBS-enriched DMEM (1:1 [vol/vol]) and incubated for 1 h at room temperature.
The plates were washed with PBST and PBS, after which substrate (5-bromo-4-
chloro-3-indolyl phosphate/nitroblue tetrazolium [BCIP/NBT]; Sigma-Aldrich,
St. Louis, MO) was added to allow spots to develop for 20 to 30 min, which were
counted by using an EliSpot Reader Lite (version 2.9; Autoimmun Diagnostika
GmbH, Strassberg, Germany). As described above, ELISPOT studies were also
undertaken to analyze the T-cell response in the mouse periodontitis model,
where T cells were isolated from the submandibular lymph nodes, which are the
gingival tissue draining lymph nodes. Statistical analysis of the ELISPOT data
was carried out on the data represented as spot-forming cells (SFC)/million,
SFC/million minus control, and also as a ratio (test/control variable), where
appropriate. All analyses gave the same results, but only data for SFC/million
and SFC/million minus control are shown.
Bio-Plex cytokine array. For the simultaneous quantitation of multiple se-
creted cytokines, undiluted supernatants were collected from three separate
T-cell proliferation assays where T cells obtained from FK-W50-immunized
PAR-2?/?and PAR-2?/?mice were exposed to 0.78 ?g of P. gingivalis FK-
W50/ml (see Fig. 6) and were analyzed by using a mouse cytokine kit containing
IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-13, IL-17, granulocyte-
macrophage colony-stimulating factor (GM-CSF), IFN-?, and tumor necrosis
factor alpha (TNF-?) beads on the Bio-Plex suspension array system (Bio-Rad,
Hercules, CA) according to the manufacturer’s instructions. Standard curves
were prepared, and samples were analyzed by using the Bio-Plex Manager
software on the Bio-Plex 2200 instrument. The concentrations of these cytokines
in the supernatants from each T-cell assay were analyzed in triplicate, and the
results were averaged.
Statistical analysis. Bone loss (mm2) data were statistically analyzed by using
a one-way analysis of variance, Dunnett’s 3T test, and Cohen’s effect size (d).
Cohen’s effect sizes (15) were calculated using an effect size calculator provided
online (http://cem.dur.ac.uk/ebeuk/researc/effectsize/). According to Cohen (15),
a small effect size (d) is ?0.2 and ?0.5, a moderate effect size is ?0.5 and ?0.8,
and a large effect size is ?0.8. All other data were statistically analyzed by using
the Student t test and Cohen’s effect size, and the results are expressed as the
means ? the standard errors of the mean (SEM). The significance of differences
between experimental groups was determined with the Student t test, assuming
unequal variances. P values of ?0.05 were considered significant.
Role(s) of PAR-1 and PAR-2 in P. gingivalis-induced alveo-
lar bone resorption. To determine whether PAR-1 or PAR-2
have a role in P. gingivalis-induced periodontal bone loss,
PAR-1?/?and PAR-1?/?mice and PAR-2?/?and PAR-2?/?
littermates were orally infected with P. gingivalis strain W50,
and the alveolar bone loss induced was analyzed. The maxillae
of PAR-1?/?, PAR-1?/?, and PAR-2?/?mice orally infected
with P. gingivalis W50 had a significantly (P ? 0.05) greater
area of exposed root surface than the sham-infected groups,
indicating that the infection had resulted in alveolar bone loss
(Fig. 2). There was no significant difference between PAR-
FIG. 1. Regions used for histomorphometry in sections of mouse
maxillae. (a) Section of a maxilla from a PAR-2?/?mouse after chal-
lenge with P. gingivalis. The arrows indicate the fields in which the mast
cells were counted. E, gingival epithelium; T, tooth; A, alveolar bone.
(b) Section showing two mast cells (arrows); insert shows mast cells at
higher magnification. (c) Section of a maxilla showing the field taken
for measuring the eroded surface of the alveolar bone. The bold line
indicates the region of interest, which is 450 ?m in length. C, alveolar
bone crest; T, tooth.
VOL. 78, 2010ROLE OF PAR-2 IN PERIODONTITIS 631
1?/?and PAR-1?/?infected mice in terms of the exposed root
surface (Fig. 2c). However, the PAR-2?/?mice orally infected
with P. gingivalis W50 were found to have significantly (P ?
0.01) less exposed root surface than their respective PAR-2?/?
counterparts (Fig. 2d). Moreover, there was no significant dif-
ference in the values between the PAR-2?/?mice that were
orally infected with P. gingivalis and the sham-infected PAR-
Histological examination of mast cells in periodontal tissue.
In the histological analysis (Fig. 1), mast cells were found in
higher numbers in the periodontal tissue of PAR-2?/?mice
orally infected with P. gingivalis compared to sham-infected
PAR-2?/?mice. In contrast, P. gingivalis oral infection had no
effect on mast cell numbers in PAR-2?/?mice. It is noteworthy
that sham-infected PAR-2?/?mice were also found to have
significantly fewer mast cells in their periodontal tissue than
PAR-2?/?unchallenged mice (Fig. 3).
Eroded surface, as a histomorphometric parameter of recent
bone resorption, was greater in alveolar bone from PAR-2?/?
mice orally infected with P. gingivalis than in alveolar bone
from their sham-infected counterparts (Fig. 4). There was no
difference in the alveolar bone eroded surface between P.
gingivalis-infected and sham-infected PAR-2?/?mice.
FIG. 2. Quantification of periodontal bone loss, in PAR-1 (?/?
and ?/?) and PAR-2 (?/? and ?/?) mice in experimental periodon-
titis. Half maxillae from PAR-2?/?(a) and PAR-2?/?(b) mice orally
of exposed root surface (outlined by white lines in panel b) was measured
and counted blind by one evaluator. The results for the comparison
between infected and sham-infected PAR-1?/?and PAR-1?/?(c) and
PAR-2?/?and PAR-2?/?(d) mice are shown as means ? SEM (n ? 12;
*, P ? 0.05;**, P ? 0.01) .
FIG. 3. Quantification of the number of mast cells in the periodon-
tal tissue of mice orally challenged with P. gingivalis W50. The results
are presented as the mean ? the SEM (n ? 3).*, P ? 0.05;**, P ?
0.01 (for comparisons indicated by lines above the bars). S-Inf, sham-
infected mice; Inf, infected mice.
FIG. 4. Eroded bone surface measurements in sections of mouse
maxillae (see Fig. 1c). The results are presented as means ? the SEM
(n ? 3;*, P ? 0.05 for comparison between infected and sham-
infected mice). S-Inf, sham-infected mice; Inf, infected mice.
632 WONG ET AL.INFECT. IMMUN.
PAR-2?/?mice display impaired T-cell immune responses.
Initially, T cells from mice that had been immunized with P.
gingivalis FK-W50 cells were isolated by using CD90 (Thy1.2)
microbeads to positively select for T cells. However, we con-
sistently obtained poor yields (?105total cells) of T cells from
the PAR-2?/?immunized mice, whereas for the PAR-2?/?
immunized mice we obtained a typical yield ranging from 3 ?
107to 7 ? 107T cells. Preliminary phenotyping of the lympho-
cyte populations from the PAR-2?/?and PAR-2?/?mice in-
dicated that there was no significant difference in the numbers
of TCR??, CD4?, or CD8?T cells between the strains of mice
(data not shown).
Thus, T cells were isolated from both strains of mice by
negative sorting using PAN T-cell microbeads (depleting all
lymphocytes except T cells), and we obtained cell numbers in
the typical (higher) yield range above for both genotypes using
this strategy. Phenotyping analysis of the lymphocyte popula-
tions from P. gingivalis FK-W50-immunized and sham-immu-
nized (PBS) PAR-2?/?and PAR-2?/?mice by flow cytometry
indicated differing expression levels of CD90/Thy-1.2 (Fig. 5).
In the PAR-2?/?mice there was no significant difference be-
tween the nonimmunized and immunized animals in CD90/
Thy-1.2 expression in TCR??CD4?T cells or TCR??CD8?
T cells (Fig. 5a and b). However, there was a significant (P ?
0.001) decrease in CD90/Thy-1.2 expression in both TCR??
CD4?T cells and TCR??CD8?T cells from immunized
compared to sham-immunized PAR-2?/?mice (Fig. 5c and d).
Furthermore, in PAR-2?/?immunized mice, 59% of TCR??
CD4?T-cell populations and 55% of TCR??CD8?T-cell
populations were determined to have no CD90/Thy-1.2 expres-
sion (data not shown). In comparing the CD90/Thy-1.2 expres-
sion in sham-immunized mice, the PAR-2?/?mice had signif-
icantly (P ? 0.01) less CD90/Thy-1.2?in CD4?and CD8?T
cells than their PAR-2?/?counterparts. Interestingly, in the
PAR-2?/?mice, although there was no significant difference in
the CD4?or CD8?T-cell populations positive for CD90/Thy-
1.2, there was an observed decrease in CD90/Thy-1.2 expres-
sion (as determined by the mean fluorescence intensity) in
both T-cell populations in the immunized groups compared to
the sham-immunized groups.
To further examine differences in the T-cell populations,
cells isolated (using PAN-T microbeads) from PAR-2?/?and
PAR-2?/?mice immunized with P. gingivalis FK-W50 were
subjected to proliferation and cytokine analysis in response to
P. gingivalis W50 cells. Figure 6 shows that the P. gingivalis
FK-W50-primed T cells from PAR-2?/?mice had significantly
FIG. 5. Expression of CD90 on T cells of PAR-2?/?and PAR-2?/?mice immunized with P. gingivalis FK-W50. Lymphocytes were isolated from
inguinal and popliteal lymph nodes of mice 7 days after immunization. Expression of molecules on T-cell subsets was detected by FACS analysis
with relevant antibodies. The data, expressed as cytometry histograms, are representative of three independent experiments. CD90 expression of
T-cell (TCR??) subsets (CD4?and CD8?) is represented by a solid line for PBS-immunized mice (control) and a dashed line for antigen (P.
gingivalis FK-W50)-immunized mice for the PAR-2?/?isolated lymphocytes (a and b) and for the PAR-2?/?isolated lymphocytes (c and d). A total
of 10,000 events were measured.
VOL. 78, 2010ROLE OF PAR-2 IN PERIODONTITIS 633
(P ? 0.05) lower maximal proliferation in response to P. gin-
givalis than their PAR-2?/?counterparts. Furthermore, the
numbers of IL-4- and IFN-?-secreting T cells from P. gingivalis
FK-W50-immunized PAR-2?/?mice were significantly (P ?
0.05) less than their PAR-2?/?counterparts (Fig. 7a), with a
large difference in the IFN-? response (d ? 7.49, 95% confi-
dence interval [CI] ? 12.88 to 3.22) compared to the IL-4
response (d ? 5.25, 95% CI ? 7.42 to 1.46). On comparing the
FIG. 6. T-cell proliferation assay. T cells isolated from the lymph nodes of mice 7 days after immunization were incubated with serially diluted
P. gingivalis FK-W50 antigen in the presence of gamma-irradiated syngeneic antigen presenting cells in vitro. The T-cell proliferation was measured
by determining the [3H]thymidine incorporation, with the results presented as the stimulatory index (SI), calculated by dividing the mean cpm
obtained after antigen stimulation by the mean cpm detected in control, unstimulated wells. This graph is a representation of four independent
experiments with similar results. S-Im, sham-immunized mice; Im, immunized mice.
FIG. 7. Cytokine responses of P. gingivalis FK-W50-primed T cells from PAR-2?/?and PAR-2?/?mice. T cells isolated from inguinal and
popliteal lymph nodes of mice 7 days after immunization (a) and T cells isolated from submandibular lymph nodes (b) from the mouse periodontitis
model were stimulated with P. gingivalis FK-W50 (1 ?g/ml) in the presence of gamma-irradiated syngeneic antigen-presenting cells in vitro. After
2 days, the assay was stopped and developed. The data are expressed as SFC/million ? the SD minus the background and are the averages of
triplicate assays. S-Im, sham-immunized mice; Im, immunized mice; S-Inf, sham-infected mice; Inf, infected mice.
634 WONG ET AL.INFECT. IMMUN.
IL-4 and IFN-? response in each strain, the PAR-2?/?mice
had a significantly (P ? 0.05) higher IFN-? response compared
to the IL-4 response. In contrast, the number of IFN-?-secret-
ing T cells was not significantly higher than the number of
IL-4-secreting T cells in PAR-2?/?mice; in fact, the mean
number of IL-4-secreting T cells was slightly higher (d ? 0.91,
95% CI ? ?0.86 to 2.46).
In the mouse experimental periodontitis model, T cells were
also isolated and used in an ELISPOT assay to determine the
numbers of IL-4- and IFN-?-secreting T cells in response to P.
gingivalis stimulation. No significant difference was found in
the numbers of IL-4-secreting T cells between PAR-2?/?and
PAR-2?/?mice orally infected with P. gingivalis (Fig. 7b).
However, there was a significantly higher number of IFN-?-
secreting PAR-2?/?T cells compared to PAR-2?/?T cells
(P ? 0.05, d ? 19.58). Furthermore, in PAR-2?/?mice orally
infected with P. gingivalis there were significantly higher num-
bers of IFN-?-secreting T cells than IL-4-secreting T cells (P ?
0.05, days ? 8.34). However, in the PAR-2?/?mice orally
infected with P. gingivalis, there was no significant difference
between the numbers of IL-4-secreting T cells and IFN-?-
secreting T cells; in fact, the mean number of IL-4-secreting T
cells was slightly higher (d ? 0.97, 95% CI ? ?0.77 to 2.59).
To further characterize the T-cell cytokine response, the
supernatants from the T-cell proliferation assays were ana-
lyzed by Bioplex cytokine array for the presence of the follow-
ing cytokines: IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12
(p70), IL-13, IL-17, GM-CSF, IFN-?, and TNF-?. T cells from
P. gingivalis-immunized PAR-2?/?mice secreted significantly
(P ? 0.05) higher amounts of IL-2, IL-3, IL-12 (p70), IL-17,
GM-CSF, IFN-?, and TNF-? than their PAR-2?/?counter-
parts in response to P. gingivalis FK-W50 (Fig. 8). The stron-
gest response (fourfold higher than other cytokines) in both
PAR-2?/?and PAR-2?/?T cells, was the level of IL-17
(145.8 ? 69.4 pg/ml and 14.4 ? 4.9 pg/ml for PAR-2?/?and
PAR-2?/?, respectively). Although there was no significant
difference in the levels of the other cytokines analyzed, there
were higher numbers (as determined by effect size) of PAR-
2?/?T cells secreting IL-5, IL-6, IL-9, IL-10, and IL-13, but
not IL-4, than PAR-2?/?T cells (d ? 0.87, 1.24, 0.67, 0.89, and
0.97, respectively). Furthermore, although analysis of the levels
of secreted cytokines showed that no significant difference was
observed (Student t test), PAR-2?/?T cells did secrete higher
concentrations of IL-5, IL-6, IL-9, IL-10, and IL-13 than were
secreted by PAR-2?/?T cells (d ? 0.87, 1.24, 0.67, 0.89, and
0.97, respectively, as analyzed by Cohen’s effect size). Overall,
Fig. 6, 7, and 8 show that the proliferation and cytokine secre-
tion responses to antigen of T cells from PAR-2?/?animals
were significantly lower compared to T cells from PAR-2?/?
PAR-2 can be activated by several proteases, including tryp-
sin, mast cell tryptase and the gingipains from P. gingivalis (28,
48). Here we have shown that PAR-2 apparently plays a pivotal
role in the progression of the inflammatory events that under-
pin the pathogenesis of experimental periodontitis. Chronic
periodontitis is commonly viewed as an inflammatory condi-
tion involving a host response to bacterial components that
have diffused into the subjacent gingival tissue from the sub-
gingival plaque biofilm. The inability of the host immune sys-
tem to remove the biofilm, which is external to the tissue and
accreted on a nonshedding tooth root surface, results in con-
tinual external stimulation, leading to a chronic inflammatory
state. This chronic inflammation leads to periodontal tissue
damage, including bone resorption caused by the cells and
molecules of the host system response (23, 46, 47).
P. gingivalis, a major causative agent of chronic periodontitis,
was orally inoculated into PAR-1?/?, PAR-1?/?, PAR-2?/?,
and PAR-2?/?mice to induce disease. Essentially, no differ-
ence was found between PAR-1?/?and PAR-1?/?mice in the
model, indicating that this receptor does not play a pivotal role
in the progression of experimental periodontitis. However, it
was clearly shown that less alveolar bone resorption occurred
in mice lacking the PAR-2 gene, confirming the bone loss
findings of Holzhausen et al. (28). In the present study, further
histological, cellular, and molecular analysis was carried out to
investigate the immunological mechanisms involved in the re-
duction of bone loss in the PAR-2-null mice in response to oral
infection with P. gingivalis.
Histological analyses indicated that the increased numbers
of mast cells seen in the maxillary tissue of infected PAR-2?/?
mice were not seen in infected PAR-2?/?mice. It must be
FIG. 8. Cytokine secretion responses of T cells from PAR-2?/?and
PAR-2?/?mice to stimulation with P. gingivalis FK-W50 antigen. T
cells isolated from inguinal and popliteal lymph nodes of mice (PAR-
2?/?and PAR-2?/?) 7 days after immunization in the hind limb were
stimulated with P. gingivalis FK-W50 in the presence of gamma-irra-
diated syngeneic antigen-presenting cells in vitro. Supernatants from
the T-cell proliferation assays (Fig. 6) were collected, and supernatants
corresponding to maximal T-cell proliferation in response to P. gingi-
valis FK-W50 antigen (0.78 ?g/ml, Fig. 6) were analyzed for IL-2, IL-3,
IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, GM-CSF, IFN-?,
and TNF-? by using the Bio-Plex cytokine array system. The data are
expressed in pg/ml minus the no-antigen control and are representative
of three independent T-cell proliferation assays. Sample size, n ? 3.
VOL. 78, 2010ROLE OF PAR-2 IN PERIODONTITIS635
noted that in sham-infected animals, there were also signifi-
cantly fewer mast cells in the tissue of PAR-2?/?mice com-
pared to the PAR-2?/?mice, indicating that the lack of PAR-2
may also have a role in mast cell differentiation or infiltration
into tissues. Thus, activation of PAR-2 expressed by mast cells
(19, 55) may participate in the recruitment of these cells. Mast
cells can be activated by bacterial antigens (33, 35, 53), leading
to the release of inflammatory mediators that are pivotal to the
innate immune response (20, 22, 54). These cells may therefore
play a pivotal role in the early inflammatory response to P.
gingivalis in chronic periodontitis. This may result from activa-
tion of PAR-2 on their surface by the arginine-specific gingi-
pains or other tissue proteases, as well as other mechanisms of
activation. It has been shown that activation of PAR-2 on mast
cells leads to degranulation by these cells, causing the release
of proinflammatory compounds that kill pathogens and up-
regulate the immune response (3). Mast cell tryptase, released
from the granules of these cells upon activation, activates
PAR-2 (17), and therefore these cells could play a primary role
in periodontitis by causing the activation of the receptor on
other cells in the periodontal tissues.
These results indicate that PAR-2 may have a role in the
innate immune response to P. gingivalis, so the next question
was whether PAR-2 was involved in the adaptive immune
response. T cells obtained from the lymph nodes of PAR-2?/?
mice that were immunized with formalin-killed P. gingivalis
W50 proliferated significantly less in response to antigen than
T cells from PAR-2?/?immunized mice. This is consistent
with previous observations (43), noting a marked difference in
proliferation between T cells from PAR-2?/?and PAR-2?/?
myelin oligodendrocyte glycoprotein-immunized mice. The de-
creased proliferation of T cells is another plausible reason for
the reduction in bone loss seen in the PAR-2?/?mice in the
mouse periodontitis model, since impairment in T-cell activa-
tion would lead to a less effective immune response, thereby
also decreasing the tissue damage associated with the inflam-
matory response. Studies have also shown that major histo-
compatibility complex class II-null and CD4-null mice have
less periodontal bone loss when challenged with P. gingivalis
compared to wild-type mice (6, 8, 9).
It was noted in both the periodontitis model and the immu-
nization experiments that use of the pan-T-cell marker, CD90/
Thy-1, for isolation of T cells led to poor recovery of cells from
PAR-2?/?mice, indicating a decreased expression of CD90/
Thy-1 compared to wild-type controls. fluorescence-activated
cell sorting (FACS) analysis of whole lymph node lymphocyte
isolates verified this hypothesis since expression of CD90/
Thy-1 was markedly downregulated after immunization in
PAR-2?/?mice, with the majority of CD4?and CD8?T cells
being CD90/Thy-1 negative. CD90/Thy-1 has important roles
in T-cell activation and maturation (25), possibly due to its
action as coreceptor for the T-cell receptor, which may act as
a cis ligand for Thy-1 (10, 24). Thy-1 also regulates cell adhe-
sion and migration (51), thus lower levels of this receptor
would have a detrimental effect on T-cell migration to sites of
infection and thereby decrease their contribution to inflamma-
tion. Thus, the marked downregulation of expression of CD90/
Thy-1 in T cells from immunized PAR-2?/?mice might con-
tribute to the reduced proliferation of the T cells from PAR-
2?/?mice noted here and previously (43). It is also possible
that the reduction of expression of Thy-1 might influence the
profile of cytokines expressed by T cells from the PAR-2?/?
ELISPOT analysis of T cells from both immunized and in-
fected wild-type mice showed strongly increased IFN-?-posi-
tive T-cell colonies compared to IL-4-positive colonies upon
stimulation by the bacterium. The pronounced increase in
IFN-?-positive colonies upon immunization or infection was
not seen for the T cells from PAR-2?/?mice. The increase in
IFN-?-secreting T cells from wild-type mice would be expected
to induce a strong T-cell helper 1 (Th1) type of immune re-
sponse, which would likely contribute strongly to the tissue
destruction observed in periodontitis. In the PAR-2?/?mice,
most T cells were Thy-1 negative after antigen stimulation,
which may affect the T-cell activation state and thus the pro-
duction of cytokines. Interestingly, naturally occurring Thy-1
negative CD4?T-cell populations only secrete IL-4 and not
IFN-? (13). Our data suggest that the antigen-induced down-
regulation of Thy-1 resulting in CD4?and CD8?Thy-1 neg-
ative T cells results in a “deactivated” T-cell population. The
findings from the present study therefore suggest that antigen
stimulation of PAR-2?/?T cells results in a downregulation of
CD90/Thy-1, leading to deactivated cells that are unable to
contribute to an inflammatory response and thus induction of
tissue destruction. This is consistent with our results showing a
pronounced decrease in bone resorption in the receptor-null
mice. These findings also point to the potential use of a PAR-2
antagonist to restrict the contribution of T cells to inflamma-
tion, as has been shown in mouse models of rheumatoid ar-
thritis (34), a disease known to be modulated by T cells.
Supernatants from the T-cell proliferation experiments were
tested for the levels of a range of cytokines to further under-
stand the role of PAR-2 in these cells. The levels of IL-2, which
plays a vital role in T-cell proliferative responses, were mark-
edly downregulated in the supernatants of T cells from PAR-
2?/?mice, which provides an explanation for the lack of pro-
liferation by T cells from these mice. The levels of other
cytokines were also affected, including the reduction of IFN-
? expression, a finding consistent with the results of the
ELISPOT assays. IL-17 protein levels were highly upregulated
in the supernatants of T cells from PAR-2?/?mice and were
?10-fold higher than in supernatants of T cells from PAR-
2?/?immunized mice. Notably, there was no difference be-
tween the supernatants from PAR-2?/?versus PAR-2?/?de-
rived T cells in the sham-immunized mice, suggesting that the
differences reflected events occurring during an immune re-
sponse to antigen. The cells responsible for the expression of
IL-17 have been identified as a subset of T cells (Th17) (26).
IL-17 is a potent inducer of inflammation and studies have
shown it to be involved in autoimmune diseases such as rheu-
matoid arthritis (40, 41). Recent studies have reported that the
IL-17 levels in gingival samples are higher in periodontitis
patients than healthy patients, indicating that Th17 cells may
play a significant role in the inflammatory response associated
with chronic periodontitis (12, 30, 59). Since PAR-2 activation
in a variety of cells causes upregulation of IL-6, a potent
inducer of Th17cell production (11), this pathway could be
involved in the upregulation of IL-17 levels seen in the T-cell
supernatants from PAR-2?/?mice and thus the lower levels of
636 WONG ET AL.INFECT. IMMUN.
IL-17 seen in the PAR2?/?mice may be a consequence of the
lower levels of IL-6 observed.
In addition to the highly altered cytokine levels noted above,
it is worth noting that almost every cytokine tested showed a
difference between the PAR-2?/?and the PAR-2?/?derived
cells, with the levels of IL-3, IL-12p70, GM-CSF, and TNF-?
all significantly decreased in the samples from PAR-2?/?mice.
These data suggest that although the CD4 and CD8 T-cell
population numbers in PAR-2?/?mice are similar to those in
PAR-2?/?mice, upon antigen activation, potential inflamma-
tory T cells become less active, resulting in reduced prolifera-
tion, cytokine secretion, and downregulation of CD90/Thy-1.
The reduction in the inflammatory T-cell response in PAR-
2?/?mice fits with the receptor’s role in stimulating inflam-
mation. Proliferation of T cells from the PAR-2?/?mice was
not completely prevented; this may be due to a response by
CD90/Thy-1-negative T cells, a small population of cells that
secrete IL-4 in response to antigen, but not IFN-? (13). We
found a low level of proliferation of T cells in PAR-2?/?mice
despite there being similar or higher numbers of IL-4-secreting
T cells in the population compared to PAR-2?/?mice. This
indicates that Th2 and/or CD90/Thy-1-negative cells may be
stimulated by antigen in the PAR-2?/?mice, resulting in pro-
liferation and IL-4 secretion that is not regulated by the in-
flammatory/Th1 T cells that would normally be the major re-
sponsive population to bacterial infection. Notably, the
absence of Thy-1 from T cells has been shown to profoundly
affect T-cell signaling through the T-cell receptor and CD3
(36); thus, the downregulation of this receptor on PAR-2-
negative T cells may have caused a widespread effect on the
ability of these cells to be activated by antigen. The molecular
basis for the effect of PAR-2 on Thy-1 levels will be an inter-
esting avenue for future research.
The role of PAR-2 in periodontal disease progression needs
to be considered at several levels. First, P. gingivalis releases
arginine-specific gingipains, which may penetrate gingival tis-
sue and activate PAR-2 on epithelial, endothelial, and connec-
tive tissue cells, thus causing an inflammatory response. One of
the possible outcomes of such activation of PAR-2 is the pro-
duction of IL-6 by gingival epithelial cells. IL-6 is a proinflam-
matory cytokine that can directly promote the resorption of
bone through the induction of osteoclast formation, and it is
possible that this pathway operates to cause bone loss in peri-
odontal disease (39). Second, mast cells attracted to the site of
inflammation could also recognize P. gingivalis, causing them
to release TNF-?, which would recruit neutrophils to the site
of infection, and mast cell tryptase, which would activate
PAR-2 on surrounding cells. Third, the recruitment of neutro-
phils would add more potential activators of PAR-2 as neutro-
phils produce proteinase-3, which is another known activator
of PAR-2 (18). The activation of PAR-2 by the gingipains,
tryptase, and proteinase 3 may all lead to activation and pro-
liferation of T cells, strongly upregulating a tissue destructive
immune response. Due to the multiple potential levels of ac-
tivation of the receptor during the inflammatory process, it is
therefore highly possible that in its absence, the inflammatory
response to the bacterium is markedly reduced, thereby de-
creasing host tissue degradation and bone resorption. Our
findings strongly indicate the potential for antagonists of
PAR-2 as a treatment for periodontitis and potentially other
chronic inflammatory diseases.
We have no conflicting financial interests.
We acknowledge funding from the Cooperative Research Centre for
Oral Health Sciences and the National Health and Medical Research
Council of Australia.
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Editor: A. J. Ba ¨umler
638WONG ET AL.INFECT. IMMUN.