Expression of protease-activated receptors 1 and 2 in individuals with healthy gingiva and chronic periodontitis.

BACKGROUND: Protease-activated receptors (PAR-1 and PAR-2) are found to be immensely exhibited in the periodontal tissues. These receptors are involved in the inflammatory and repair processes. Activation of PARs is mainly by the bacterial and endogenous enzymes. The aim of the study was to determine the role of PAR-1 and PAR-2 in initiating periodontal inflammation and to immunolocalize these receptors in the gingival tissues of healthy and chronic periodontitis individuals.
MATERIALS AND METHODS: A total of 50 patients were selected for this study, of which 25 were healthy controls and 25 were chronic periodontitis patients. Gingival tissues were excised from the marginal gingiva and interdental papilla under local anesthesia (xylocaine with 2% adrenaline) during crown lengthening procedure or during periodontal therapy depending on the respective groups. Immunohistochemical analyses of PARs were done by staining the samples with hematoxylin and eosin and with primary and secondary antibody for PAR-1 and PAR-2.
RESULTS: The Hematoxylin and Eosin staining showed more inflammatory changes in the periodontitis group compared to healthy gingiva. In chronic periodontitis, PAR-1-positive cells were seen in the basal layer with a weak expression and were showing negative expression in the superficial layer. In consideration of PAR-2, there was a very strong expression up to the superficial layer of the epithelium, compared to PAR-1. On comparing the intensity of staining in the connective tissue of chronic periodontitis sample, there was an increased expression of PAR-2 compared with PAR-1. A low positive expression of PAR-1 and PAR-2 was observed in the epithelium and connective tissue of the healthy tissues.
CONCLUSION: The results clearly demonstrated the role of PAR-1 and 2 in periodontal inflammation.

INTRODUCTION

Periodontal diseases are multifactorial; complex diseases cause an upregulated or maladapted immunoinflammatory response to bacterial plaque which predisposes the patients to periodontal tissue breakdown; mediated by endogenous host enzymes and bacterial proteinases present in the periodontal pocket, for example, neutrophils serine proteinase 3, gingipains, and mast cell tryptase.[12]

More recently, it was shown that the pharmacological activities of these proteinases are mediated through specificprotease-activated receptors (PARs) activation. PARs can act as a first “alarm system” since they are capable of recognizing bacteria in the environment. However, in addition, some of the periodontal pathogens, such as Porphyromonas gingivalis, secrete proteases that are recognized by cells through the family of PARs. PARs are known to be expressed in the epithelium of gingiva and have been implicated in the pathogenesis of periodontal diseases.[345]

PARs are seven-transmembrane domain, G protein–coupled receptors that mediate cellular responses to extracellular proteinases.[6] They are activated by proteolytic cleavage of N-terminal domain, generating a new N-terminal receptor that serves as a “tethered ligand” that binds to one of the extracellular loops of the receptor itself resulting in its autoactivation.[78]

Four PARs have been identified, as PAR-1, PAR-2, PAR-3, and PAR-4. PAR-1 is expressed on osteoblasts, astrocytes, fibroblasts, epithelial cells, platelets, endothelial cells and also has an important role in the tissues injury, bone repair, homeostasis and also leads to granulocytes attraction by inducing the chemokines.[9101112] PAR-2 is activated by trypsin, mast cell tryptase, neutrophil protease 3, tissue factor/factor VIIa/factor Xa, membrane-tethered serine protease-1, and gingipains. Previous studies by Holzhausen et al.[13] exhibited an increased PAR-2 expression in chronic periodontitis patients. Recent studies have shown that PAR-2 activation plays an important role in the inflammatory process and tissue breakdown in periodontal tissues.[141516]

Since PAR-3 and PAR-4 are less abundant than PAR-1 and PAR-2 in the periodontal disease, we investigated the expression of PAR-1 and PAR-2 in individuals with healthy gingiva and chronic periodontitis. Most of studies on PARs expression were detected mainly in the epithelium; our study is one among few studies to investigate the expression in the connective tissue since the microbial enzymes and the elevated proinflammatory mediators in the soft tissue may lead to the disarray in the epithelial tissue, enables the bacteria and their toxic substances to enter the subepithelial connective tissue. The vulnerability of the inhabitat periodontal connective tissue cells to the bacterial agents can modify them into vital contributors in the pathophysiological process of demolition of the periodontal tissues.

Therefore, the aim of the present study was to determine the role of PARs 1 and 2 in initiating periodontal inflammation and to immunolocalize PARs 1 and 2 in gingival tissues of healthy and chronic periodontitis individuals.

MATERIALS AND METHODS

This case–control study was conducted from November 2009 to June 2010. A total of 50 patients were selected, comprises of two groups: Group-I 25 healthy controls and Group-II 25 chronic periodontitis patients. Informed consent was obtained from all the individuals who have participated in the study.

The individuals for the study were selected from patients attending the Outpatient Department of Periodontics. The study was approved by Institutional Ethics Committee. The individuals were chosen based on specific inclusion and exclusion criteria. The presence of at least 10 natural teeth, sites with probing depth of more than or equal to 5 mm, and attachment loss of more than 2 mm were included for chronic periodontitis group, and for the healthy group, absence of bleeding on probing, probing depth of < 3 mm and no loss of attachment, systemically healthy and no previous history of periodontal disease were included in the study. Oral Hygiene Index (OHI) simplified was done to check for debris and calculus with the explorer to assess the thickness of the plaque at the cervical margin of tooth; all four surfaces were examined and recorded. History of tobacco usage, individuals who have taken antibiotics in the past 6 months, individuals who have taken analgesics in the past 1 week, pregnant and lactating women, presence of any other systemic disease were excluded from the study. Gingival tissues were excised from the marginal gingiva and interdental papilla using Bard parker no. 15 under local anesthesia (xylocaine with 2% adrenaline) during crown lengthening procedure or during periodontal therapy depending on the respective groups. The excised tissue samples were immediately fixed in 10% buffered formalin solution. The tissues were then transformed to the oral pathology department for paraffin embedding. From each block, 3 serial sections were prepared on coated slides, 1 section for Hematoxylin and Eosin staining and the other two were used for immunohistochemistry (IHC) (PAR-1 and PAR-2) analysis. 4-μm human gingival tissue sections were made onto APES-coated slides. The tissue sections were deparaffinized by immersing the slides in 3 changes of fresh xylene each for 5 min and processed for routine IHC analysis.[17]

The PAR antibody (PAR-1 and PAR-2) stained the epithelium and the underlying connective tissue brown against blue background in positive cells of formalin-fixed, paraffin-embedded tissue sections. Membranous staining pattern in breast carcinoma was used as the standard to interpret the study sections. Endothelial capillaries, collagen, fibroblasts, nerve tissue, inflammatory cells, and mast cells were evaluated under IHC staining. The PAR staining was graded as mild (+), moderate (++), severe (+++), and intense (++++) according to the intensity and pattern of staining by a pathologist.[18] The sections which had no stain were graded as negative. Immunohistochemically, positivity was recorded as a percentage of cells staining with mild-intense immunoreactivity per ×10 and ×40 field, with enumeration of at least 200 cells/sample.

Statistical analysis

Statistical analysis was performed using SPSS software (version 15). The intensity of staining for PAR-1 and PAR-2 was compared using Pearson Chi-square test. Pearson's values were considered to be significant when P < 0.05.

RESULTS

The demographic data for the study group were depicted in Table 1; on comparison, clinical parameters such as OHI and plaque index were significantly higher in the chronic periodontitis group compared with the control group.

Table 1

Demographic data for the study group

The intensity of staining in the suprabasal layer of epithelium of PAR-1 was compared between healthy and chronic periodontitis samples; it was found to be statistically significant [P = 0.009, Table 2].

Table 2

Intensity of staining in the suprabasal layer of epithelium of protease-activated receptor-1 in healthy and chronic periodontitis sample

Intensity of staining in the suprabasal layer of epithelium of PAR-2 was compared between healthy and chronic periodontitis sample; it was found to be statistically significant [P =0.000, Table 3].

Table 3

Intensity of staining in the suprabasal layer of epithelium of protease-activated receptor-2 in healthy and chronic periodontitis sample

When comparing the intensity of staining of PAR-1 in the superficial layer (stratum corneum) of epithelium and connective tissue in control and chronic periodontitis group, it shows a low positive expression of PAR-1 in the epithelium and connective tissue of the healthy tissues, while positive expression of PAR-1 was seen around areas of collagen destruction of chronic periodontitis [Figures 1 and 2].

Figure 1

Section showing low positive expression of protease-activated receptor 1 in the epithelium and connective tissue of the control tissue (×10)

Figure 2

Section showing positive expression of protease-activated receptor 1 around areas of collagen destruction of chronic periodontitis (×10)

On comparing the intensity of staining in the superficial layer (stratum corneum) of epithelium and connective tissue of PAR-2 between control and chronic periodontitis group, a low positive expression of PAR-2 in the epithelium and connective tissue of the healthy tissues was observed. Considering PAR-2 in chronic periodontitis, increased expression of PAR-2 around nerve tissue and areas of collagen destruction of chronic periodontitis was perceived [Figures 3 and 4].

Figure 3

Section showing low positive expression of protease-activated receptor 2 in the epithelium and connective tissue of the control tissue (×10)

Figure 4

Section showing increased expression of protease-activated receptor 2 around nerve tissue and areas of collagen destruction of chronic periodontitis (×10)

Differentiation of staining intensity was noted in the superficial layer of the epithelium in PAR-1 and PAR-2 in the chronic periodontitis sample. Negative expression of PAR-1 in the superficial layer was detected. However, very strong expression of PAR-2 in the epithelium and connective tissue around areas of destruction was noted [Figures 5 and 6].

Figure 5

Section showing negative expression of protease-activated receptor 1 in the superficial layer of chronic periodontitis (×40)

Figure 6

Section showing very strong expression of protease-activated receptor 2 in the epithelium and connective tissue around areas of destruction (×10)

Intensity of staining in the connective tissue in PAR-1 and PAR-2 was compared in the periodontitis sample; there was an increased expression of PAR-2, but it was not statistically significant [P = 0.345, Table 4].

Table 4

Comparison in the intensity of staining in the connective tissue in protease-activated receptor-1 and protease-activated receptor-2

DISCUSSION

The PARs are found to be expressed on the gingival epithelial cells and in the connective tissues as a part of the immunosurveillance system. PARs are part of the innate immune receptors, which can recognize specific bacterial or endogenous serine proteases which lead to the initiation of defensive immune responses. The main PAR-activating proteinases found in the periodontal environment are neutrophil proteinase 3, thrombin, plasmin, tryptase, matrix metalloproteinase (MMP)-1, MMP-13, and gingipains. The neutrophil proteinase 3 shows a dissimilar way of activation of PAR-1 and PAR-2 by a biased signaling mechanism leading to “noncanonical” receptor activating tethered ligand sequences, causing contradictory signaling pathways from thrombin and trypsin.[1920] Receptors of the PAR family accommodate as sensors of serine proteinase of the blood clotting system in the target cells involved in inflammation. Thrombin is an endogenous serine proteinase, activates PARs and guides in healing, control inflammation, and fibrosis.

Activation of PAR-1 by thrombin and of PAR-2 by factor X a leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. It has been found that the presence of PAR receptors on mast cells contributes to the association of inflammation and blood clotting processes. Since limited literature available related to PAR-1 and PAR-2 in periodontal disease, our study was aimed to determine the role of PAR-1 and PAR-2 in initiating periodontal inflammation and to immunolocalize them in the gingival tissues of healthy and chronic periodontitis individuals.

In our study, there was a weak expression of PAR-1 around areas of mast cell, collagen, and blood capillaries; in contrast PAR-2 detected a strong expression around the mast cell of chronic periodontitis samples. Holzhausen et al.[21] by selectively inhibiting tryptase with nafamostat mesilate shows a downregulation of reduction in alveolar bone loss, granulocyte infiltration, and also a reduced PAR-2 expression in the gingival tissue of rats, proposed that tryptase may play a role in the pathogenesis of chronic periodontal disease through PAR-2 activation.

The present study was done to show the different layers of expression of PAR-1 and PAR-2 in staining of the layers of epithelium and the connective tissue in individuals with healthy gingiva and chronic periodontitis. IHC analysis was done to detect the expression of PARs in the gingival tissues, and it was indicated that both receptors are vigorously expressed in gingival epithelial cells and in subepithelial connective tissue, but there was an increased expression of PAR-2 observed in the superficial layer of the squamous epithelium; PAR-1 expression seems to be reduced from the basal to superficial layer. The reason for this intense expression of PAR-2 could be due to the fact that PAR-2 receptor most likely makes first contact with P. gingivalis binding to oral epithelium. No significant staining was observed in the basal layer. Our study was in agreement with Lourbakos et al.[2] who reported that overexpression of PAR-2 was noted on the superficial layer of epithelium.

Uehara et al.[22] reported that hepatocyte growth factor increases the production of matrix metalloproteinases by the activation of PAR-1 by stimulating it with P. gingialis. Thrombin has the ability to inhibiting osteoblast differentiation and apoptosis; synthesis and secretion of growth factors and cytokines are through the activation of PAR-1. On the other hand, it induces the synthesis of osteoprotegerin, helps to regulate bone homeostasis, and prevents osteoclastogenesis.[10] Conversely, Uehara et al.[23] showed gingipains synergistically increase the secretion of proinflammatory cytokines through PAR-1 and PAR-2 in combination with Toll-like receptors or NOD agonists.

In the underlying connective tissue, low positive expression of PAR-1 was found to be seen in the areas of collagen destructions and around inflammatory cells; contradictorily, intense staining was observed for PAR-2 in areas of collagen destruction, increased angiogenesis, around young capillaries, and inflammatory cells. This could be due to the increased presence of tissue factor/factor VIIa, which activates PAR-2 and it generates thrombin through the action of extrinsic coagulation pathway on fibroblasts, inturn activates PAR-1.[22223]

The most striking observation from the present study was the increased presence of PAR-2 compared with PAR-1, which were positively concentrated around areas of inflammatory cells, collagen eradications, and blood vessels. Yun et al.[24] showed that RgpA activated PARs and induced T-cell activation, suggesting that P. gingivalis, through its gingipains, makes use of the host cell PAR-2 to exacerbate inflammation during chronic periodontal disease. Furthermore, gingipains has the ability to activate PAR-2 on oral epithelial cells leading to the production of proinflammatory mediators, such as interleukin (IL)-6 and IL-8 that could result in periodontal tissue breakdown. Current literature by Tada et al.[25] found that the expression of IL-33 increases Th2 cytokine-mediated inflammatory responses, during P. gingivalis infection in human gingival epithelial cells through PAR-2 through gingipain-dependent activation.

In the current study, the superficial layer of the epithelium showed a negative expression for PAR-1 in both the groups. On considering PAR-2, an increase in the staining intensity was observed in the superficial layer in chronic periodontitis group. Increased PAR-2 receptor expression was higher in the deeper periodontal pockets compared with the shallower pockets; this would suggest that PAR-2 overexpression is possibly correlated with periodontal inflammation severity. These results were in congruent with Holzhausen et al.[13] PAR-2 expression was seen to be more intracellular compared to PAR-1 which was expressed on the cell surface. PAR-2 has been expressed vigorously on mast cells; it has been shown that activation of PAR-2 on mast cells leads to degranulation by these cells, causing the release of pro-inflammatory compounds that kill pathogens and upregulate the immune replication. Therefore, these cells could play a primary role in periodontitis by causing the activation of the receptor on other cells in the periodontal tissues. Our study result coincided with Wong et al.[26] who stated that PAR-2 may also have a role in mast cell differentiation or infiltration into tissues. Therefore, the regulation of T-cells and mast cells by PAR-2 suggests a pivotal role in the pathogenesis of the periodontal disease.

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 perforate gingival tissue and activate PAR-2 on epithelial, endothelial, and connective tissue cells, thus causing an increased production of proinflammatory mediators.[2327] Second, mast cells attracted to the site of inflammation could additionally recognize P. gingivalis, release TNF-α, which would recruit neutrophils to the site of infection, activate PAR-2 on surrounding cells.

Furthermore, the neutrophil serine protease-3 activates PAR-2 urges the release of pro-inflammatory cytokines not only affects the periodontal destruction but can also increase the expression of MMPs by acting indirectly. Therefore, there is convincing evidence in the literature showing that gingipains and neutrophil serine protease 3 make use of host cell PAR-2 to exacerbate the inflammation seen in chronic periodontal disease.[2829]

Many disputes exist over the role of PARs in periodontal tissues. It has the capacity to play both pro- and anti-inflammatory effect which is possible with the future development of antagonists of human PAR-2-activating proteases as a potential disease-modifying therapeutic agents for chronic periodontitis. In addition to periodontal therapy, doxycycline in a subantimicrobial dose inhibits the activity of MMPs and thus reduces the degradation of collagen.[30]

CONCLUSION

PAR-1 and PAR-2 have significant roles in the pathogenesis of periodontal disease. The findings in the present study provided the evidence that PAR-1 and PAR-2 are expressed on the periodontal tissue in the epithelium and connective tissue. Consequently, the detailed mechanisms underlying the effects should be understood and their correlation with cytokines requires further research. However, further in vivo studies with increased sample size are needed to demonstrate the role of PAR-1 and PAR-2 in periodontal disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.