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RESEARCH REPORTS |
1 Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, 3330 Hospital Drive, NW, Calgary, T2N 4N1, Alberta, Canada; and
2 Department of Oral Pathology, Dental School of Araraquara, State University of São Paulo (UNESP), Araraquara, São Paulo, Brazil;
* corresponding author, nvergnol{at}ucalgary.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: proteinase-activated receptor-2 • alveolar bone loss • inflammation • matrix metalloproteinase • cyclo-oxygenase • periodontitis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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A role for PAR2 during the inflammatory reaction has been suggested by several studies demonstrating that activation of PAR2 can lead to blood vessel relaxation, hypotension, increased vascular permeability, granulocyte infiltration, and leukocyte adhesion and margination (Vergnolle et al., 2001; Coughlin and Camerer, 2003). PAR2 activation also leads to the release of prostanoids and cytokines, including interleukin (IL)-6 and IL-8 in epithelial or non-epithelial cells (Lourbakos et al., 2001; Uehara et al., 2003).
A possible participation of PAR2 in chronic oral inflammation such as periodontitis was indirectly suggested by several studies. PAR2 is expressed in osteoblasts, oral epithelial cells, and human gingival fibroblasts (Abraham et al., 2000; Lourbakos et al., 2001; Uehara et al., 2003). Lourbakos et al.(2001) showed that, in an oral epithelial cell line, PAR2 activation by gingipain induced the secretion of the pro-inflammatory cytokine interleukin-6 (IL-6), which is a potent stimulator of osteoclast differentiation and bone resorption. Uehara et al.(2003) demonstrated that a synthetic PAR2 agonist peptide activates human gingival fibroblasts to produce IL-8, which has the ability to stimulate MMP activity selectively, thereby accounting for collagen destruction within periodontitis lesions. These two studies suggest a role for PAR2 activation in inducing inflammation and bone resorption during periodontitis. In contrast, another study suggests that PAR2 activation could inhibit bone resorption: In that study, the authors showed that the PAR2 agonist SLIGRL inhibits osteoclast differentiation (Smith et al., 2004). These contradictory results reflect the difficulties of using in vitro systems to evaluate which mediators are involved in periodontal diseases. We hypothesized that PAR2 activation may have an important role in inflammatory processes associated with periodontitis. Therefore, we propose to study, in vivo, the effects of PAR2 agonist applied topically to the gingiva, either in healthy control rats or during the course of periodontitis.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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To induce periodontitis, we anesthetized the rats by intramuscular administration of ketamine (0.08 mL/100 g body weight) and xylazine (0.04 mL/100 g body weight), and placed a 3.0 silk ligature around each rat’s right mandibular first molar, as previously described (Holzhausen et al., 2002). Sham-operated rats were anesthetized and treated as ligated rats, except without the ligature.
Treatments with PAR2 Agonist Peptide (SLIGRL-NH2) and Control Peptides
Via a micropipette, rats received (under light anesthesia; halothane 0.5%) a daily topical application of saline (NaCl 0.9%), control peptide LRGILS-NH2 (1 µg/day) (complete reverse sequence of PAR2-tethered ligand, inactive on PAR2), or PAR2-activating peptide SLIGRL-NH2 (1 µg/day), at the mesial gingival sulcus of the right mandibular first molar. These treatments began the same day as the surgical procedure. Rats were randomly separated into the following groups: (i) sham + saline treatment (n = 32); (ii) sham + LRGILS-NH2 treatment (n = 32); (iii) sham + SLIGRL-NH2 treatment (n = 32); (iv) ligature + saline treatment (n = 32); (v) ligature + LRGILS-NH2 treatment (n = 32); and (vi) ligature + SLIGRL-NH2 treatment (n = 32). Eight animals per group were killed at 3, 7, 15, and 30 days after daily treatments began.
Bone Loss Evaluation
The distance between the cemento-enamel junction and the height of alveolar bone were determined for mesial root surfaces of lower right first molars as previously described (Holzhausen et al., 2002). Millimeters of bone loss for each radiograph were measured in a blind fashion 3 times, by the same examiner.
Myeloperoxydase (MPO) Activity Measurement
At the animals’ death, the gingivomucosal tissues encircling the right first mandibular molars were removed and processed for MPO activity, an index of tissue granulocyte infiltration, as previously described (Cenac et al., 2002).
Effect of MMP and COX Inhibition on PAR2 Agonist Peptide-induced Alveolar Bone Loss and Gingival Granulocytic Infiltration
Groups of 8 rats were treated with: indomethacin (daily oral dose of 5 mg/kg/day), a non-selective COX-1/COX-2 inhibitor; doxycycline (daily oral dose of 6 mg/kg/day), a MMP inhibitor; or their vehicle (carboxymethylcellulose, 0.2 mL/day). Then, 1 hr after these treatments, rats received either saline or the PAR2-activating peptide SLIGRL-NH2 (1 µg/day), topically, at the mesial gingival sulcus of the right mandibular first molar, as described earlier. All animals were killed 7 days after the beginning of treatments. MPO and alveolar bone loss were measured as described above.
Western Blot
Gingival tissues collected from animals treated with daily topical application of SLIGRL-NH2 (1 µg/day) or the control peptide LRGILS-NH2 (1 µg/day) at the mesial gingival sulcus of the right mandibular first molars were homogenized and run on a 10% SDS polyacrylamide gel as previously described (Vergnolle et al., 1995). Membranes were incubated with anti-COX-1 (1:500), anti-COX-2 (1:500), anti-MMP2 (5 µg/mL), anti-MMP9 (1:200), anti-MMP13 (1:200), or anti-TIMP1 (2 µg/mL) polyclonal antibody overnight at 4°C. The expressional changes following SLIGRL treatment were analyzed densitometrically by means of the molecular analyst program Quantity One from Bio Rad (Hercules, CA, USA).
Data Analysis
We used one-way analysis of variance (ANOVA) to compare means of alveolar bone loss and MPO activity among groups. In cases of significant differences among the groups, post hoc two-group comparisons were assessed with the Tukey-Kramer test. We used an unpaired t test to analyze the differences in the Western-blot densitometries. A P value < 0.05 was considered statistically significant. Data are expressed as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, 1B). In contrast, topical treatments with SLIGRL-NH2 led to significantly (p < 0.05) increased alveolar bone destruction, in comparison with saline treatments, at all time-points (3, 7, 15, and 30 days) (Figs. 1A, 1C, 1D). At 7 days, a significant increase in MPO activity, an index of tissue granulocytic infiltration, was found after SLIGRL-NH2 treatment, when compared with the control peptide-treated or vehicle-treated groups (Fig. 1B). No differences were found in the MPO activity between saline and control peptide groups at all the observed time-points.
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, 2B). Bone loss in both saline and control-peptide groups progressed significantly between days 3 and 7 (p < 0.05), and between days 7 and 15 (p < 0.01), and no difference between the two groups was observed. The alveolar bone destruction at the mesial surface of the ligated first molar was exacerbated (p < 0.05) by SLIGRL-NH2 daily treatments when compared with treatment with the control peptide LRGILS-NH2 treatment at experimental periods of 7, 15, and 30 days (Figs. 2A, 2C, 2D).
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Effect of MMP and COX Inhibition on PAR2 Agonist Peptide-induced Alveolar Bone Loss and Gingival Granulocytic Infiltration
Seven days of topical treatment with SLIGRL-NH2 caused significant (p < 0.05) increase in alveolar bone destruction and increased granulocytic infiltration when compared with treatment with vehicle alone. Indomethacin and MMP inhibitor treatments both significantly reduced PAR2 agonist-induced alveolar bone loss (Fig. 3A) and granulocytic infiltration (Fig. 3B), while they had no effect on the same parameters in animals that received topical treatment with saline instead of PAR2 agonist.
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, 4B). Tissue expression of MMP-13 and TIMP-1 was unchanged in PAR2-activating peptide-treated rats compared with control peptide-treated rats (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In agreement with results of previous in vitro studies which supported a destructive role for PAR2 (Uehara et al., 2000; Lourbakos et al., 2001), our in vivo approach definitively defines a pro-inflammatory and bone-destructive role for PAR2 activation in periodontal tissues.
Seven days after PAR2-agonist treatment, a peak in MPO activity was observed, which decreased thereafter (see Fig. 1B). Since polymorphonuclear neutrophils, which constitute the front line of the acute host inflammatory response, represent the main source for MPO, it can be concluded that PAR2 agonist treatment led to an acute inflammatory response. Neutrophil-mediated tissue injury and release of inflammatory mediators can promote the initiation of bone metabolism breakdown by stimulating osteoclasts (Dennison and Van Dyke, 1997). Thus, one possible explanation for PAR2 agonist-induced bone loss could be attributed, at least in part, to the induction of an acute inflammatory response.
There is compelling evidence in the literature that pro-inflammatory cytokines not only have a direct effect on periodontal destruction but also can act indirectly by up-regulating COX-2 and MMP expression (Gemmell et al., 1997). It can be suggested, therefore, that periodontal breakdown followed by exposure of gingival tissues to the PAR2 agonist, as shown by our present study, may be due to an initial stimulation of cytokine production by oral epithelial cells, resulting in matrix degradation and alveolar bone resorption through a MMP- and COX-dependent pathway.
The evidence for the involvement of MMPs in PAR2 agonist-induced periodontitis was confirmed by our results showing that doxycycline administration significantly reduced PAR2 agonist-induced alveolar bone loss and granulocyte infiltration at 7 days. Doxycycline is a non-antimicrobial tetracycline that has been shown to reduce connective tissue destruction, including bone resorption, in human and/or animal models of disease (Crout et al., 1996). Inhibition of connective tissue breakdown by doxycycline can occur through different mechanisms: (i) inhibition of active MMPs (collagenases, gelatinases) in the extracellular matrix, (ii) prevention of the conversion of pro-MMPs into active MMPs in the extracellular matrix, and (iii) down-regulation of the gene expression of MMPs in epithelial, endothelial, and bone cells (Lee et al., 2004). In the present study, a significant overexpression of MMP-2 and MMP-9 was found in gingival tissues of rats 7 days after topical treatment with the PAR2 agonist. Considering the fact that MMP-2 and MMP-9 are capable of degrading the organic component of bone (Mansell et al., 1997), it is therefore reasonable to think that doxycycline reduces PAR2-induced periodontitis by inhibiting MMP-2 and MMP-9 overexpression.
The results from our experiments also evidenced the involvement of COXs in PAR2 agonist-induced periodontitis, since non-selective inhibition of COX enzymes (indomethacin treatment) led to a significant decrease in alveolar bone loss and granulocyte infiltration. In addition, SLIGRL treatment induced an overexpression of COXs in gingival tissues. At sites of injury and inflammation, macrophages and fibroblasts express COX-2, subsequently up-regulating the production of prostaglandin E2. Since PGE2 is an important pro-inflammatory mediator in gingivitis and alveolar bone resorption (Lohinai et al., 2001), it is possible that overproduction by PAR2 agonist-induced COX-2 overexpression in gingival tissues may constitute another possible pathway by which SLIGRL induces periodontal disease. It is generally admitted that, while COX-2 expression is up-regulated upon inflammatory stimulation, the expression of COX-1 is constitutive. In our study, we observed that not only COX-2 but also COX-1 expression was up-regulated in gingival tissues (see Fig. 4). A possible explanation is that the increased COX-1 expression is due to the massive periodontal infiltration of granulocytes, which constitutively express COX-1. However, overexpression of COX-1 has been demonstrated in different cell types (mast cells, monocytes in response to lipopolysaccharide) (Parente and Perretti, 2003); therefore, we cannot rule out the possibility that COX-1 is overexpressed by a specific cell type in response to PAR2 agonist.
Porphyromonas gingivalis (Pg) has been recognized as the major periodontopathogen responsible for human chronic periodontitis. Although the use of the Pg-infection model in rats is very close to human disease and involves numerous proteases released by pathogens, we chose to use a mechanically based model of periodontitis. The major reason we chose this model was that the numerous proteases released by Pg could have caused degradation of the PAR2-activating peptide, thereby masking the potential enhancing effects of PAR2 activation on periodontitis. The model we used (ligature) is a very well-established model of periodontitis. The ligature acts not only by causing mechanical trauma on the dentogingival area, but also by promoting plaque accumulation, thus increasing the number of bacteria. This model takes into account an important part of the representation of the human disease: the intense host-plaque interactions.
The gingival sulcus can be an environment rich in proteases derived from periodontopathogens. These highly proteolytic enzymes have been implicated as important factors in eliciting host responses that result in the destruction of periodontal tissues (Ekuni et al., 2003). The exact mechanism by which bacterial proteases mediate periodontitis is far from being understood in detail. Based on the results obtained from the present study, one possible explanation could be that bacterial proteases can mediate periodontal breakdown through the activation of the PAR2 receptor in host cells. This hypothesis is further supported by the fact that gingipain, a protease released by P. gingivalis, has been shown to activate PAR2 in vitro (Lourbakos et al., 2001). However, we cannot rule out the possibility that PAR2 in host gingival tissues might be activated by host proteases such as trypsin, which can be released by damaged endothelial cells, or tryptase, which is released upon mast cell degranulation.
In conclusion, we have shown that PAR2 activation in periodontal tissues constitutes a destructive signal, causing all the signs of periodontitis. Since PAR2 can be activated by periodontal pathogen proteases (Lourbakos et al., 2001), these results strongly suggest a role for PAR2 activation in the development of periodontal diseases. Inhibition of PAR2 activation or even proteolytic activity may thus represent a novel therapeutic alternative, particularly for the treatment of the most aggressive forms of periodontitis. PAR2 inhibition could constitute an alternative approach to modulate host response to periodontal pathogens.
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ACKNOWLEDGMENTS |
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Received July 5, 2004; Last revision October 20, 2004; Accepted November 27, 2004
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REFERENCES |
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