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The Journal of Immunology, 2002, 169: 4594-4603.
Copyright © 2002 by The American Association of Immunologists

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-21

Akiko Uehara*, Shunji Sugawara2,*, Koji Muramoto{dagger} and Haruhiko Takada*

* Department of Microbiology and Immunology, School of Dentistry, and {dagger} Laboratory of Biomolecular Function, Graduate School of Life Sciences, Tohoku University, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 Ca2+ 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The four members of the protease-activated receptor (PAR)3 family are G protein-coupled receptors characterized by a proteolytic cleavage of the N terminus that exposes tethered ligands and autoactivates the receptor function (1, 2, 3). Three of them (PAR-1, PAR-3, and PAR-4) are activated mainly by thrombin; the fourth (PAR-2) is activated by other serine proteinases such as trypsin, tryptase, and factor Xa (1, 2, 3). It is reported that more than one family member can be present in the same cell: human platelets express PAR-1 and PAR-4 (4, 5); mouse platelets express PAR-3 and PAR-4 (6); human endothelial cells express PAR-1, PAR-2, and possibly PAR-3 (7, 8); and human oral epithelial cells express PAR-1, PAR-2, and PAR-3 (9). PAR-1 is the most widely expressed receptor among PAR family members in humans and mice, and it is reported that PAR-1 on human platelets and endothelial cells is inactivated by neutrophil serine proteinases, human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR3), by cleavage downstream of the tethered ligand (10). Because PARs are expressed in a variety of cells, it is considered that they are involved in several pathophysiological processes, including growth, development, and inflammation.

PR3 is a 29-kDa serine proteinase with homology to HLE and Cat G, all of which are stored in azurophil granules of neutrophils (11, 12, 13). PR3 also presents on the cell surface and within secretory and specific granules of neutrophils, and exposure of neutrophils to cytokines or chemoattractants induces an increase in cell surface-bound PR3 (14). PR3 is also expressed by monocytes, basophils, and mast cells (15). PR3 is a major target Ag of anti-neutrophil cytoplasmic Abs in Wegener’s granulomatosis, a debilitating autoimmune disease characterized by necrotizing vasculitis (15, 16). It has recently been shown that PR3 exhibits many biological functions, including the degradation of extracellular matrix proteins (13), regulation of myeloid differentiation (12, 17), enhancement of TNF-{alpha} and IL-1{beta} release from human monocytic cell lines (18), production of IL-8 and monocyte chemoattractant protein-1 (MCP-1) by human endothelial cells (19, 20), and antibacterial action (21), all of which indicate that cell-bound and secreted soluble PR3 actively contribute to inflammatory processes, although the underlying mechanism is unclear to date.

Oral epithelial cells are the first cells encountered by bacteria in the periodontal tissues. In addition to acting as a physical barrier against the invasion of pathogenic organisms, oral (gingival) epithelial cells in inflamed regions appear to express several proinflammatory cytokines such as IL-1{beta}, IL-6, IL-8, TNF-{alpha}, and TGF-{beta}1 (22), implying that the cells actively participate in the initiation and development of chronic oral inflammation such as periodontitis. However, only a few studies have demonstrated cytokine production by human gingival epithelial cells and related cell lines derived from the oral cavity in response to oral bacteria (23, 24, 25). In contrast to human colonic epithelial cells, human oral epithelial cells are shown to be basically unresponsive to many bacterial cell surface components, even in the presence of the soluble form of CD14, a bacterial pattern recognition receptor (26). We have recently shown that oral epithelial cells constitutively express a 24-kDa precursor form of IL-18, and that human neutrophil PR3 induced the secretion of a bioactive IL-18 from the epithelial cells in combination with LPS after priming with IFN-{gamma} (27). We extended the investigation of the underlying mechanism by which PR3 activates the epithelial cells, and in the present study, we obtained the evidence for the first time that PR3 by itself could activate the epithelial cells in culture to induce production of IL-8 and MCP-1 and expression of ICAM-1 via the PAR-2 pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Purified human neutrophil PR3 was obtained from HyTest (Turku, Finland) and from Elastin Products (Owensville, MO). Purified HLE, Cat G, phospholipase C (PLC) inhibitor U73122, and control compound U73343 were obtained from Calbiochem-Novabiochem (La Jolla, CA). The purity of three enzymes (PR3, HLE, and Cat G) was >95% by SDS-PAGE, according to the manufacturer, and further confirmed by Western blotting using anti-human PR3 (Elastin Products), HLE, and Cat G (Santa Cruz Biotechnology, Santa Cruz, CA) polyclonal Abs. The result showed that no cross-reactivity was observed in each preparation (data not shown). Human rIL-1{alpha} and rTNF-{alpha} were supplied by Dainippon Pharmaceutical (Osaka, Japan). Boc-Ala-p-nitrophenyl ester (Boc-Ala-ONp) was obtained from Bachem (Bubendorf, Germany). A low toxic serine proteinase inhibitor, Pefabloc SC, was obtained from Roche Diagnostics (Mannheim, Germany). Cycloheximide was obtained from Biomol (Plymouth Meeting, PA). Anti-CD54 (ICAM-1) (mouse IgG1) was purchased from Immunotech (Marseille, France). Neutralizing anti-CD54 84H10 (mouse IgG1) was obtained from Beckman Coulter (Miami, FL) and dialyzed against PBS. Anti-human PAR-1 mAb ATAP2 (mouse IgG) raised against aa 42–55 of human PAR-1, anti-human PAR-2 mAb SAM11 (mouse IgG2a) raised against aa 37–50 of human PAR-2, and rabbit anti-human PAR-3 polyclonal Ab raised against aa 1–103 of human PAR-3 were obtained from Santa Cruz Biotechnology. Nonenzymatic cell dissociation solution (CDS) was obtained from Sigma-Aldrich (St. Louis, MO). Cell-permeant fura 2-AM was obtained from Molecular Probes (Eugene, OR). PAR-1 and PAR-2 agonist peptides (SFLLRN and SLIGKV, respectively) were synthesized by Takara (Otsu, Japan). All other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.

Cells and cell cultures

The human oral epithelial cell lines HSC-2 (28) and KB (29), established from squamous cell carcinoma, were obtained from the Cancer Cell Repository, Institute of Development, Aging and Cancer, Tohoku University (HSC-2; Sendai, Japan) and from the Health Science Research Resources Bank (KB; Tokyo, Japan). HSC-2 was grown in RPMI 1640 with 10% heat-inactivated FCS (Life Technologies, Grand Island, NY), and KB was grown in {alpha}-MEM with 10% FCS with a medium change every 3 days. Human gingival epithelial cells were prepared from explants of normal human gingival tissues with informed consent, as described previously (27). The experimental procedure was approved by the Ethical Review Board, Tohoku University School of Dentistry.

Neutrophils from heparinized (10 U/ml) peripheral venous blood were isolated by density-gradient centrifugation on Mono-Poly resolving medium (ICN Biomedical, Costa Mesa, CA) at 300 x g for 30 min at room temperature (30). The fraction containing neutrophils was harvested and washed three times with PBS at 4°C and suspended in RPMI 1640 medium. The viability of these cells was greater than 98%, as judged by trypan blue dye exclusion. The purity of neutrophils was above 95% morphologically.

PBMCs from heparinized (10 U/ml) peripheral venous blood were isolated by Lympholyte-H (Cedarlane Laboratories, Hornby, Ontario, Canada) gradient centrifugation at 800 x g for 20 min at room temperature (31). The isolated PBMCs were washed three times with PBS at 4°C. The viability of these cells was greater than 98%, as judged by trypan blue dye exclusion.

Measurement of cytokines

Confluent oral epithelial cells were collected by CDS and washed three times in PBS. The cells (104 cells/200 µl) were seeded in culture medium in 96-well plates (Falcon; BD Labware, Lincoln Park, NJ). After incubation for 1 day at 37°C in a 5% CO2 incubator, the cells were stimulated with test materials in 200 µl medium without serum for a given period. To inhibit the enzymatic activity of PR3, it was preincubated with serine proteinase inhibitors, Pefabloc SC and {alpha}1-antitrypsin ({alpha}1-AT), and FCS for 30 min at 37°C before use. Cultivation was conducted in triplicate, and levels of IL-8 and MCP-1 in the supernatants were measured using OptEIA ELISA kits (BD PharMingen, San Diego, CA). The concentrations of the cytokines in the supernatants were determined using the Softmax data analysis program (Molecular Devices, Menlo Park, CA).

Flow cytometry

Flow cytometric analyses were performed using a FACScan cytometer (BD Biosciences, Mountain View, CA), as described (32). Oral epithelial cells were pretreated with or without cytochalasin B (30 nM) for 30 min or 1 µg/ml cycloheximide for 6 h at 37°C. Then cells were stimulated with or without PR3 (10 µg/ml) for up to 24 h in the presence or absence of cytochalasin B (30 nM) or cycloheximide (1 µg/ml) at 37°C. After the incubation, cells were collected by nonenzymatic CDS and washed in PBS. Cells were stained with anti-CD54, anti-PAR-1, anti-PAR-2, or control IgG at 4°C for 30 min, followed by FITC-conjugated goat anti-mouse IgG (BioSource International, Camarillo, CA) at 4°C for a further 30 min. For PAR-3 staining, cells were incubated with rabbit anti-PAR-3 polyclonal Ab or control IgG for 30 min, followed by FITC-conjugated swine anti-rabbit IgG (DAKO, Kyoto, Japan) at 4°C for a further 30 min. To calculate the percentage of positive cells, the baseline cursor was set at a channel that yielded less than 2% of events positive with the isotype Ab control. Fluorescence to the right was counted as specific binding. The arithmetic mean was used in the computation of the mean fluorescence intensity.

Adhesion assay

HSC-2 cells (104 cells/well) were cultured in 96-well plates for 24 h until confluent. Thereafter, cells were washed twice with PBS and cultured in RPMI 1640 medium alone or medium containing either PR3 (10 µg/ml) or TNF-{alpha} (5 ng/ml). Cells were cultured for 24 h and washed with warmed PBS. Neutrophils (3.2 x 105 cells/ml each) were then added to HSC-2 cells in RPMI 1640 medium and allowed to adhere during 30 min at 37°C under static conditions. The wells were then washed three times with warmed PBS to remove nonadherent cells. Adhesion of neutrophils was quantified using a modified myeloperoxidase assay, as described (33). Briefly, HSC-2 cells plus adherent neutrophils were washed twice with PBS without Ca2+ and Mg2+ (pH 6.0), and subsequently HSC-2 cells plus adhering cells were permeabilized in 50 µl PBS containing 0.5% hexadecyltrimethyl ammonium bromide for 30 min at room temperature. Next, 250 µl warmed O-dianisidine dihydrochloride (0.2 mg/ml in PBS, pH 6.0) containing H2O2 (0.4 mM) was added. After 15 min of incubation at 37°C, OD was read at 450 nm. Serial dilutions of neutrophils were used as a standard to calculate the number of adherent neutrophils. For the inhibition assay, HSC-2 cells were incubated with anti-CD54 mAb 84H10 or isotype control mAb (10 µg/ml each) for 120 min at 37°C before the addition of neutrophils. Next, the adhesion assay was performed as described. The mAb remained present during the adhesion assay.

Measurement of enzymatic activity and inhibition assay

The amidolytic activity of PR3 was assayed with 0.625 mM Boc-Ala-ONp for 10–30 min at 25°C in 0.1 M HEPES buffer containing 0.1 M NaCl, 10 mM CaCl2, 0.005% Triton X-100, and 5% DMSO, pH 7.5, as described previously (18, 34). The formation of the p-nitrophenol product was monitored at 405 nm using the Softmax data analysis program (Molecular Devices). One unit of enzymatic activity was defined as the liberation of 1 µmol p-nitrophenol from the substrate/min at 25°C. To inhibit the enzymatic activity of PR3, the enzyme was preincubated with different concentrations of inhibitors for 30 min before use.

RT-PCR assay

Total cellular RNA was extracted from the cells using Isogen (Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. Random hexamer-primed reverse transcription was performed on 2.5 µl total RNA in a 50-µl reaction volume, and all PCR procedures were performed in a 20-µl vol, as described previously (27). The primers used for PCR were as follows: IL-8, 5'-GATTGAGAGTGGACCACACT-3', 5'-TCTCCCGTGCAATATCTAGG-3'; MCP-1, 5'-AACTGAAGCTCGCACTCTCG-3', 5'-TCAGCACAGATCTCCTTGGC-3'; ICAM-1, 5'-CAGTCACCTATGGCAACGAC-3', 5'-ATTCAGCGTCACCTTGGCTC-3'; PAR-1, 5'-TGTGAACTGATCATGTTTATG-3', 5'-TTCGTAAGATAAGAGATATGT-3' (35); PAR-2, 5'-GCAGCCTCTCTCTCCTGCAGTGG-3', 5'-CTTGCATCTGCTTTACAGTGCG-3' (36); PAR-3, 5'-ATAACGTTTAAGAGACGGGACT-3', 5'-TAGCAGTAGATGATAAGCACA-3' (7); PAR-4, 5'-GACGAGAGCGGGAGCACC-3', 5'-CCCGTAGCACAGCAGCATGG-3' (4); and GAPDH, 5'-CTACAATGAGCTGCGTGTGG-3', 5'-AAGGAAGGCTGGAAGAGTGC-3' (27). The primers for IL-8, MCP-1, ICAM-1, PAR-1, PAR-2, PAR-3, PAR-4, and GAPDH were constructed to generate fragments of 422, 257, 243, 708, 1,066, 858, 725, and 527 bp, respectively. Cycling conditions were as follows: IL-8, 25 cycles at 94°C for 1 min, 63°C for 1 min, and 72°C for 3 min; MCP-1, ICAM-1, and GAPDH, 35 cycles at 94°C for 1.5 min, 60°C for 1 min, and 72°C for 3 min; PAR-1, 34 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and PAR-2, PAR-3, and PAR-4, 36 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 3 min. Amplified samples were visualized on 2.0% agarose gels stained with ethidium bromide and photographed under UV light.

Analysis of peptide cleavage

A peptide corresponding to a region spanning the cleavage site of the PAR-2, residues 32–45 (32SSKGRSLIGKVDGT45) (37), was synthesized by Takara. The peptide (200 µM) was incubated with proteinases for 30 min at 37°C in PBS. Each digest was separated by reversed-phase HPLC on a Wakosil 5C4-200 column (5 mm, 4.6 x 250 mm) (WAKO, Osaka, Japan) using a linear gradient from 0 to 30% acetonitrile in 0.1% trifluoroacetic acid, and the amino acid sequences of peptide fragments were analyzed by a gas phase protein sequencer (PSQ-1; Shimadzu, Kyoto, Japan) and a matrix-assisted laser desorption ionization time of flight mass spectrometry (Kompact MALDI I; Shimadzu), according to a previously described procedure (38).

Calcium mobilization

Confluent oral epithelial cells were collected by nonenzymatic CDS, washed twice with PBS, and suspended at 2 x 106 cells/ml in an extracellular medium (EM; 121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 6 mM NaHCO3, 5.5 mM glucose, 25 mM HEPES, 0.1% w/v BSA, pH 7.3). Cells were loaded with 1 µM fura 2-AM with shaking for 30 min at room temperature. After being washed with EM, cells were resuspended in EM and incubated for 30 min at room temperature. After another wash with EM, loaded cells suspended at 2 x 106 cells/ml in EM without BSA were transferred to stirred quartz cuvettes in a CAF-100 spectrofluorometer (Jasco, Tokyo, Japan) at 37°C. Fura 2 fluorescence was measured at 340 and 380 nm excitation and 510 nm emission, and the ratio of the fluorescence at the two excitation wavelengths, which is proportional to [Ca2+]i, was calculated.

Coculture of oral epithelial cells with stimulated neutrophils

Confluent HSC-2 cells in 96-well plates were cocultured with the indicated number of purified neutrophils in 200 µl serum-free medium in the presence or absence of various concentrations of FMLP for 24 h. The cocultures were also performed in the presence or absence of Pefabloc SC, {alpha}1-AT, and FCS for 24 h. After the incubation, levels of IL-8 and MCP-1 in the supernatants were measured by ELISA.

Data analysis

All experiments in this study were performed at least three times to confirm the reproducibility of the results. In most experiments, values are represented as means ± SD of triplicate assays. The statistical significance of differences between the two means was evaluated by one-way ANOVA, using the Bonferroni or Dunn method, and p values less than 0.05 were considered significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PR3 on the production of IL-8 and MCP-1 by oral epithelial cells

We first examined the effect of PR3 on the production of IL-8 and MCP-1 by oral epithelial cell lines, HSC-2 and KB. Incubation of both cell lines in the presence of various concentrations of PR3 for 24 h resulted in a dose-dependent increase in IL-8 and MCP-1 (Fig. 1GoA). PR3 at 5–10 µg/ml was most effective in stimulating the production. Incubation of HSC-2 cells with 10 µg/ml (345 nM) PR3 resulted in a time-dependent increase in the production of IL-8 and MCP-1 (Fig. 1GoB). A significant increase in production was observed from 8-h incubation, and a marked increase at 24 and 48 h. In contrast to PR3, two other neutrophil serine proteinases, HLE and Cat G, showed only marginal activity in oral epithelial cells as assessed by MCP-1 production (Fig. 1GoC), which was consistent with our recent observation (27). The PR3 from the two different sources showed the same results (Fig. 1GoD). IL-8 mRNA was already expressed in untreated cells, but MCP-1 mRNA was not expressed in untreated cells, and the expression of IL-8 and MCP-1 mRNA was increased and induced by PR3, respectively (Fig. 1GoE). IFN-{gamma} (1000 IU/ml) and IL-1{alpha} (10 ng/ml) were used as a positive control for IL-8 and MCP-1 mRNA expression, respectively. Oral epithelial cells in primary culture also produce IL-8 and MCP-1 in response to PR3, although the cytokine levels were lower than those in the epithelial cell lines (Fig. 1GoF).



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  		FIGURE 1. Effect of PR3 on the production of IL-8 and MCP-1 by human oral epithelial cells. Confluent HSC-2 and KB were incubated for 24 h in the presence of various concentrations of PR3 (A), or incubated in the presence (+) or absence (-) of PR3 (10 µg/ml) for the time indicated (B). C, Confluent HSC-2 cells were incubated in the presence of various concentrations of PR3, HLE, and Cat G for 24 h. D, Confluent HSC-2 cells were incubated in the presence of various concentrations of PR3 from HyTest (PR3 (H)) or PR3 from Elastin Products (PR3 (E)) for 24 h. Concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. E, Confluent HSC-2 and KB were incubated for 8 h with medium alone (lane 2), 10 µg/ml PR3 (lane 3), or 1000 IU/ml IFN-{gamma} for IL-8 or 10 ng/ml IL-1{alpha} for MCP-1 (lane 4), and the expression of IL-8, MCP-1, and GAPDH mRNA was analyzed by RT-PCR. Water control was loaded in lane 1. F, Confluent primary oral epithelial cells were incubated for 24 h in the presence of various concentrations of PR3. Concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. *, p < 0.05, and **, p< 0.01 compared with the respective control (medium alone). The results presented were representative of three different experiments demonstrating similar results.

 
PR3-induced ICAM-1 mediates adhesion of oral epithelial cells to neutrophils

We next examined the effect of PR3 on the expression of an adhesion molecule, ICAM-1, on HSC-2 cells by flow cytometry. Assessment of the surface expression of ICAM-1 on the untreated cells revealed no reactivity (less than 2%), but the cells treated with increasing concentrations of PR3 for 24 h showed a dose-dependent increase of ICAM-1 expression, with a plateau in the level of ICAM-1 expression reached at 1 µg/ml PR3 (Fig. 2GoA). TNF-{alpha} was used as a positive control and also enhanced ICAM-1 expression. Incubation of HSC-2 cells with 10 µg/ml PR3 resulted in a time-dependent increase in the expression of ICAM-1 (Fig. 2GoB). The expression was up-regulated from 8-h incubation, then increased linearly until 48 h, at which point almost all the cells expressed ICAM-1. A representative FACS profile of ICAM-1 expression on HSC-2 cells after given doses of PR3 treatment for 24 h is shown in Fig. 2GoC. ICAM-1 mRNA was not expressed in untreated cells and induced by PR3 (Fig. 2GoD). TNF-{alpha} (5 ng/ml) was used as a positive control. We then examined whether the ICAM-1 induced by PR3 in HSC-2 cells is involved in the adhesion to neutrophils. After the incubation of HSC-2 cells with 10 µg/ml PR3 for 24 h, a 9.1-fold increase in the adhesion of neutrophils was observed as compared with the cells incubated in the medium alone (Fig. 2GoE). TNF-{alpha} (5 ng/ml), as a positive control, also significantly promoted the adhesion to neutrophils and to HSC-2 cells. Furthermore, pretreatment of HSC-2 cells with anti-ICAM-1 mAb resulted in a complete reduction of adhesion of neutrophils, indicating that the ICAM-1 expressed on oral epithelial cells by PR3 was a major factor in the adhesion to neutrophils. KB showed the same results (data not shown). The findings shown in Figs. 1Go and 2Go indicate that PR3 activates oral epithelial cells, consequently inducing production of IL-8 and MCP-1, and expression of ICAM-1.



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  		FIGURE 2. PR3-induced ICAM-1 expression on oral epithelial cells mediates adhesion of neutrophils to oral epithelial cells. Confluent HSC-2 cells were incubated for 24 h in the presence of various concentrations of PR3 or TNF-{alpha} (A), or incubated in the presence or absence of PR3 (10 µg/ml) for the time indicated (B). The surface expression of ICAM-1 was assessed by flow cytometry. Data were expressed as percentage of ICAM-1-positive cells. C, A representative FACS profile of ICAM-1 expression on HSC-2 cells after treatment with the given doses of PR3 for 24 h is shown. D, Confluent HSC-2 cells were incubated for 8 h with medium alone (lane 2), 10 µg/ml PR3 (lane 3), or 5 ng/ml TNF-{alpha} (lane 4), and the expression of ICAM-1 and GAPDH mRNA was analyzed by RT-PCR. Water control was loaded in lane 1. E, Confluent HSC-2 cells were stimulated with medium alone or medium containing PR3 (10 µg/ml) or TNF-{alpha} (5 ng/ml) as a positive control for 24 h. HSC-2 cells washed with PBS were incubated with or without anti-CD54 mAb 84H10 or isotype control IgG (10 µg/ml each) for 30 min, and neutrophils (3.2 x 105 cells/ml each) were added. After 30 min, nonadherent cells were removed, and the number of adherent cells was quantified using a myeloperoxidase assay. **, p <0.01 compared with the respective control (medium alone). The results presented were representative of three different experiments demonstrating similar results.

 
Requirement of enzymatic activity for PR3 to induce IL-8 and MCP-1 production and ICAM-1 expression in oral epithelial cells

We then examined whether the PR3-induced activation of oral epithelial cells was due to the enzymatic activity of PR3. It has been reported that the enzymatic activity of PR3 was inhibited by sulfonyl fluoride-type serine proteinase inhibitors such as PMSF and diisopropyl fluorophosphate, and by a naturally occurring serine proteinase inhibitor, {alpha}1-AT (19, 21). Therefore, we first examined the inhibitory effects of a low toxic sulfonyl fluoride-type serine proteinase inhibitor, Pefabloc SC, and {alpha}1-AT on the enzymatic activity of PR3 using Boc-Ala-ONp as a substrate. PR3 showed substantial enzymatic activity (9.7 U/mg protein), and these inhibitors almost completely inhibited it (Fig. 3GoA). Concurrent with this, Pefabloc SC and {alpha}1-AT significantly inhibited PR3-induced IL-8 and MCP-1 production, and ICAM-1 expression by HSC-2 cells (Fig. 3Go, B–D). In addition, FCS almost completely inhibited the enzymatic activity of PR3 (Fig. 3GoA), PR3-induced IL-8, and MCP-1 production (Fig. 3Go, B and C), and ICAM-1 expression (Fig. 3GoD), probably due to naturally occurring proteinase inhibitors in the serum. By contrast, the serine proteinase inhibitors and serum did not inhibit IL-1{alpha}-induced production of MCP-1 (Fig. 3GoE). These results indicate that the enzymatic activity of PR3 is critical to the activation of oral epithelial cells.



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  		FIGURE 3. Effect of serine proteinase inhibitors and FCS on the activation of oral epithelial cells by PR3. A, PR3 was pretreated with Pefabloc SC, {alpha}1-AT, or FCS at the dose indicated for 30 min before use. Enzymatic activity was measured using Boc-Ala-ONp as a substrate, and the results are expressed as percentage of activity. The activity of PR3 was 9.7 U/mg protein. B–E, HSC-2 cells were stimulated with or without PR3 (10 µg/ml) (B–D) or rIL-1{alpha} (10 ng/ml) (E) for 24 h at 37°C. PR3 (10 µg/ml) and IL-1{alpha} (10 ng/ml) were pretreated with or without Pefabloc SC (40 µM), {alpha}1-AT (1 µg/ml), or FCS (1 and 10%) for 30 min at 37°C before use. The supernatants were collected, and the concentrations of IL-8 (B) and MCP-1 (C and E) were measured by ELISA. The cells were collected by CDS, and the surface expression of ICAM-1 was evaluated by flow cytometry (D). The percentage of cytokine production and ICAM-1 expression was calculated on the basis of the value obtained without inhibitors (B, 2.9 ± 0.2 ng/ml; C, 14.0 ± 1.1 ng/ml; D, mean fluorescence intensity = 465; E, 14.2 ± 0.9 ng/ml). Error bars indicate SD. **, p < 0.01 compared with PR3 alone. The results presented were representative of three different experiments demonstrating similar results.

 
Analysis of the PAR family in oral epithelial cells and induction of PAR-2 expression on the cell surface by PR3

Because it has been shown that PARs on human platelets and endothelial cells are activated or inactivated by various proteinases (1, 2, 3), the expression of the PAR family in oral epithelial cells was analyzed. PBMCs were used as a positive control (5). Oral epithelial cells in culture strongly expressed PAR-1 and PAR-2 mRNA, weakly expressed PAR-3 mRNA, and did not express PAR-4 mRNA, as assessed by RT-PCR analysis (Fig. 4GoA), which was consistent with the findings of immunohistochemistry using sections of human gingival tissues (9). By flow cytometric analyses, weak expression of PAR-2 was detected on the untreated cell surface, but the expression of PAR-1 and -3 on the cell surface was below the detectable limit (Fig. 4GoB). The expression of PAR-2 was markedly augmented by PR3 treatment for 1 h, an effect that was inhibited by cytochalasin B, an inhibitor of actin polymerization (39), but not by cycloheximide, a protein synthesis inhibitor. The up-regulation by PR3 was detected at 30-min incubation, and reached a plateau at 1 h (Fig. 4GoC). Exposure of the cells to PR3 for up to 24 h did not further up-regulate the expression of PAR-2, and did not induce expression of PAR-1 and -3 (data not shown). Furthermore, trypsin, an agonist for PAR-2 (1, 2, 3), and a PAR-2 agonist peptide SLIGKV also up-regulated PAR-2 expression (Fig. 4GoD), but did not up-regulate PAR-1 and -3 (data not shown) on the epithelial cell surface. These results indicate that PAR-2 is an inducible receptor from internal storage by PR3 and known PAR-2 agonists.



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  		FIGURE 4. Expression of the PAR family in oral epithelial cells. A, Total RNA was extracted from KB (lane 2), HSC-2 (lane 3), and PBMCs (lane 4), and cDNA was prepared and analyzed for the expression of PAR-1, PAR-2, PAR-3, PAR-4, and GAPDH by RT-PCR. Water control was loaded in lane 1. B, Confluent HSC-2 cells were pretreated with or without 30 nM cytochalasin B (cyto B) for 30 min or 1 µg/ml cycloheximide (CHX) for 6 h at 37°C. Then cells were stimulated with or without PR3 (10 µg/ml) for 1 h in the presence or absence of 30 nM cytochalasin B or 1 µg/ml cycloheximide. After the incubation, cells were collected by CDS, and the expression of PAR-1, PAR-2, and PAR-3 of the cells was analyzed by flow cytometry. C and D, Confluent HSC-2 cells were stimulated with or without 10 µg/ml PR3 for the time indicated (C), or 1 µM trypsin or 100 µM PAR-2 agonist peptide (SLIGKV, PAR-2AP) for 1 h (D). After the incubation, cells were collected by CDS, and the expression of PAR-2 of the cells was analyzed by flow cytometry. The number in B–D indicates the degree of increases in PAR expression after the treatments evaluated on the basis of the value obtained with untreated cells. The results presented were representative of four different experiments demonstrating similar results.

 
Cleavage of human PAR-2 peptide with exposure of its tethered ligand by PR3

The above observation suggests that PR3 cleaves PAR-2 at the specific site and exposes its tethered ligand. To examine this possibility, a peptide corresponding to region surrounding the cleavage site of the human PAR-2 (Fig. 5GoA) was incubated with PR3, and proteolytic fragments were analyzed. Trypsin, an agonist for PAR-2 (1, 2, 3), was used as a positive control. The PAR-2 peptide was rapidly cleaved at the site, R36-S37, by 5 nM PR3 or trypsin (Fig. 5GoB). The major peptide fragment was identified to be SLIGKVDGT, the PAR-2 tethered ligand, by sequencing. The measured molecular mass (890.8) was in good agreement with the calculated value (890.2). The fragment, SSKGR, was not detected probably because it was further cleaved to SSK and GR. Upon digestion with 500 nM PR3 or trypsin, the large fragment was cleaved at the site, K41-V42, to give another major peak, which corresponded to SLIGK (measured value, 517.9; calculated value, 517.8). PR3 from two different sources showed the identical result. In contrast, thrombin, which does not activate PAR-2 (1, 2, 3), did not cleave the PAR-2 peptide.



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  		FIGURE 5. Analysis of cleavage products of PAR-2 peptide. A, The peptide sequence of human PAR-2 used in this study. The number refers to the sequence region of the intact receptor. The slash indicates a putative trypsin cleavage site that leads to exposure of the tethered ligand domain (underlined residues) in the intact receptor (37 ). B, The peptide (200 µM) was incubated with the indicated concentrations of PR3, trypsin, and thrombin for 30 min at 37°C, and separated by reversed-phase HPLC. The flow rate was 1 ml/min. AUFS, absorbance units at full scale. Each peak (P1 and P2) was then analyzed by a protein sequencer and a mass spectrometry. P1, no cleaved peptide; P2, SLIGKVDGT. The results presented were representative of three different experiments demonstrating similar results.

 
Involvement of PAR-2 in the PR3-induced activation of oral epithelial cells

To confirm that PR3-induced activation of oral epithelial cells is mediated by PAR-2, Ca2+ mobilization in the cells was measured on exposure of KB cells to PR3, trypsin, and synthetic PAR agonist peptides. Trypsin induced a Ca2+ response, and abolished the response to a second application of trypsin, although the cells responded to the PAR-1 agonist peptide, SFLLRN (Fig. 6GoA). PR3 as well as the PAR-2 agonist peptide SLIGKV induced the mobilization of Ca2+ in the cells the same as trypsin (Fig. 6Go, B and F). SLIGKV stimulation rendered cells refractory to subsequent stimulation with PR3, but not SFLLRN (Fig. 6GoC), while stimulation with SFLLRN had no such effect (Fig. 6GoD). Furthermore, trypsin inhibited a subsequent response to PR3 and to SLIGKV (Fig. 6GoE), and PR3 also desensitized responses to PAR-2 agonists, trypsin (Fig. 6GoG), and SLIGKV (Fig. 6GoH). HSC-2 and PR3 from two different sources showed the same results, although the Ca2+ mobilization in HSC-2 was lower than that in KB (data not shown). These results clearly indicated that the PR3-induced activation of the cells is mediated by PAR-2.



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  		FIGURE 6. Effect of trypsin, PR3, and PAR agonist peptides on calcium mobilization in oral epithelial cells. A–H, Fura 2-loaded KB cells were exposed to 1 µM trypsin, 10 µg/ml PR3, 100 µM PAR-1 agonist peptide (SFLLRN, PAR-1AP), and 100 µM PAR-2 agonist peptide (SLIGKV, PAR-2AP) as indicated sequence, and the change in intracellular calcium was monitored. The results presented were representative of three different experiments demonstrating similar results.

 
PR3 induced only marginal response in KB cells at 0.01 µM, and induced a good response at 1 µM, which pattern was the same as that with trypsin, as assessed by Ca2+ mobilization (Fig. 7Go), indicating that the potency and efficacy are basically the same as those of trypsin. PAR-2 agonist peptide SLIGKV required 100 times greater concentration to induce the same response.



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  		FIGURE 7. Potency and efficacy of PR3 compared with those of other PAR-2 agonists. Fura 2-loaded KB cells were exposed to PR3, trypsin, and PAR-2 agonist peptide (SLIGKV, PAR-2AP) at the concentrations indicated, and the change in intracellular calcium was monitored. PR3, 1 µM = 29 µg/ml; trypsin, 1 µM = 24 µg/ml; PAR-2AP, 100 µM = 61.6 µg/ml. The results presented were representative of three different experiments demonstrating similar results.

 
In common with many G protein-coupled receptors, the principal mechanism of PAR-mediated activation is through G{alpha}q proteins, resulting in activation of PLC (1, 2, 3). To examine that PLC is also involved in PR3-induced activation of oral epithelial cells, HSC-2 cells were stimulated with PR3 in the presence of the PLC inhibitor U73122 or the control compound U73343 for 24 h. The PAR-1 and -2 agonist peptides were used as control, and both peptides induced production of IL-8 and MCP-1 (Fig. 8Go), indicating that the epithelial cells are activated through not only PAR-2, but also PAR-1. The inhibition of PLC completely abolished the production of cytokine induce by both agonist peptides and PR3.



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  		FIGURE 8. Effect of PLC inhibition on the PR3-induced production of IL-8 and MCP-1 by oral epithelial cells. Confluent HSC-2 cells were pretreated with or without U73122 or U73343 at the dose indicated for 30 min. Then cells were stimulated with the PAR-1 agonist peptide (PAR-1AP; 100 µM), the PAR-2 agonist peptide (PAR-2AP; 100 µM), or PR3 (10 µg/ml) for 24 h. Concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. Error bars indicate SD. **, p < 0.01 compared with the respective control (PAR-1AP, PAR-2AP, or PR3 alone). The results presented were representative of three different experiments demonstrating similar results.

 
Activation of oral epithelial cells by stimulated neutrophils

To examine whether stimulated neutrophils activate oral epithelial cells, confluent monolayers of HSC-2 were cocultured with indicated number of neutrophils in the presence or absence of FMLP for 24 h. Neutrophils at 0.5 x 105 cells with 0.01 µM FMLP started to induce production of IL-8 from the cells, and increasing concentrations of FMLP resulted in a dose-dependent increase of the IL-8 production (Fig. 9GoA). A significant increase in the production of MCP-1 was observed at 5 x 105 cells of neutrophils with 0.01 µM FMLP, and the MCP-1 production reached a plateau at 0.1 µM FMLP (Fig. 9GoB). Unstimulated neutrophils lacked activity regardless of number of neutrophils. In contrast to the soluble PR3, as shown in Fig. 3Go, the production of IL-8 and MCP-1 induced by stimulated neutrophils was only partially inhibited by serine proteinase inhibitors and serum (Fig. 9Go, C and D).



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  		FIGURE 9. Activation of oral epithelial cells with stimulated neutrophils. Confluent HSC-2 cells were cocultured with the indicated number of neutrophils in the presence or absence of FMLP at the dose indicated for 24 h, and concentrations of IL-8 (A) and MCP-1 (B) in the culture supernatants were determined by ELISA. Confluent HSC-2 cells were cultured with 106 of neutrophils and 10 µM FMLP in the presence or absence of Pefabloc SC (40 µM), {alpha}1-AT (1 µg/ml), or FCS (1 and 10%) for 24 h, and concentrations of IL-8 (C) and MCP-1 (D) in the culture supernatants were determined by ELISA. The percentage of cytokine production was calculated based on the value obtained without inhibitors (C, 4.9 ± 0.03 ng/ml; D, 7.3 ± 0.09 ng/ml). Error bars indicate SD. **, p < 0.01 compared with no inhibitor. The results presented were representative of three different experiments demonstrating similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that human neutrophil PR3 activates oral epithelial cells to induce production of IL-8 and MCP-1 and expression of ICAM-1 through PAR-2. Although it is reported that PR3 inactivates PAR-1 by cleaving downstream of the tethered ligand (10), there was no evidence that it does the same to PAR-2. The present study proves for the first time that the PR3-induced activation of oral epithelial cells is mediated by PAR-2, based on the following evidence: 1) oral epithelial cells strongly express PAR-1 and PAR-2 mRNA and weakly express PAR-3 mRNA, and PAR-2 expression on the cell surface was induced by PR3; 2) activation of the cells by PR3 was inhibited by serine proteinase inhibitors and serum; 3) PR3 and trypsin, an agonist for PAR-2, cleaved the PAR-2 peptide with exposure of its tethered ligand; 4) trypsin and a synthetic PAR-2 agonist peptide induced the mobilization of intracellular Ca2+, and rendered cells refractory to subsequent stimulation with PR3; and 5) a PAR-2 agonist peptide as well as PR3 induced cytokine production by the cells, which was inhibited by the PLC inhibitor.

Lactate dehydrogenase activity was not detected in the supernatant of the epithelial cells after PR3 treatment, as previously reported (27), which indicates that PR3 has no adverse effect on the epithelial cell viability. Evidence for the activation of oral epithelial cells by PR3 includes: 1) the expression of IL-8, and MCP-1 and ICAM-1 mRNA was increased and induced by PR3, respectively (Figs. 1Go and 2Go), and 2) PR3 induced the mobilization of intracellular Ca2+ in the epithelial cells (Fig. 6Go). In contrast to PR3, two other neutrophil serine proteinases, HLE and Cat G, showed only marginal activity on oral epithelial cells, as assessed from the production of MCP-1 (Fig. 1GoC). HLE and Cat G also induced the mobilization of intracellular Ca2+ in oral epithelial cells to a small extent as compared with PR3 and trypsin, and PR3, trypsin, and a PAR-2 agonist peptide rendered cells refractory to subsequent exposure to HLE and Cat G (data not shown), although it is reported that HLE and Cat G also inactivate PAR-1 by cleaving downstream of the tethered ligand (10). The findings indicate that PR3 is most involved in activating oral epithelial cells through PAR-2 among the three neutrophil serine proteinases.

It is reported that PR3 has an elastase-like specificity for Ala, Ser, and Val at the P1 site (13), but there was no evidence that it cleaves Arg-X and Lys-X bonds. The present study showed that PR3 rapidly cleaved between Arg and Ser and relatively inefficiently between Lys and Val, which was the same specificity as trypsin and tryptase (Fig. 5GoB) (37), indicating that the R36-S37 site and surrounding the site of the PAR-2 are structurally accessible by trypsin, tryptase, and PR3, but not by thrombin.

The present study showed that PR3 promoted the surface expression of PAR-2 on oral epithelial cells, which was inhibited by cytochalasin B, but not by cycloheximide (Fig. 4GoB). The longer incubation with PR3 for up to 24 h neither up-regulated PAR-2 expression nor induced PAR-1 and -3 on the cell surface (data not shown). This indicates that PAR-2 was inducible receptor from intracellular storage, and not required de novo synthesis among the PAR family. In support of this, it was reported that the expression of PAR-2 on human endothelial cells was also up-regulated by LPS as well as IL-1{alpha} and TNF-{alpha} without an effect on PAR-1 expression (40), and that PAR-2 expression in asthmatic bronchial epithelium was significantly increased in comparison with normal epithelium (41). Recently, the PAR-2-induced activation of keratinocytes was shown to be mediated by the mitogen-activated protein kinase and NF-{kappa}B pathways (42), which are important for the gene expression of cytokines. Thus, it is conceivable that the activation of oral epithelial cells by PR3 is mediated by these pathways involved in producing inflammatory cytokines and expressing adhesion molecules on the cell surface.

PR3 is secreted as a soluble form by activated neutrophils, and also exists as a membrane-bound form on neutrophils. Previous study demonstrated that each azurophil granule of neutrophils contains PR3 at 13.4 mM, and the activation of neutrophils resulted in about a 10-fold increase in membrane-bound PR3 (14), indicating that the local concentration of PR3 around activated neutrophils is high enough to activate PAR2. In addition, although serum contains abundant naturally occurring proteinase inhibitors, membrane-bound PR3 is substantially resistant to inhibition by naturally occurring inhibitors such as {alpha}1-AT and elafin, even when these inhibitors are used at a 100- to 300-fold molar excess over the enzyme (14). In support of this possibility, the results in Fig. 9Go showed that activation of oral epithelial cells by FMLP-stimulated neutrophils was only partially inhibited by serine proteinase inhibitors and serum, suggesting that the activation is likely to occur in vivo.

The present study showed that oral epithelial cells are activated to produce IL-8 and MCP-1 and highly express ICAM-1 on the cell surface in response to PR3 through the PAR-2 pathway. IL-8 is a major chemokine responsible for the activation of neutrophils and migration of neutrophils and T cells to inflammatory sites (43). MCP-1 plays a critical role in the activation and migration of monocytes, T cells, and NK cells, and is an important factor in the development of Th1 and Th2 responses (44, 45). ICAM-1 is one of the major adhesion molecules interacting with the {beta}2 integrin family, including LFA-1 and Mac-1 present on neutrophils, monocytes, and T cells (46). Fig. 2Go shows that ICAM-1 expressed on oral epithelial cells in response to PR3 contributes extensively to the interaction with neutrophils (via Mac-1 and LFA-1), and the ICAM-1-mediated interaction may further augment the activation of oral epithelial cells through PAR-2. IL-18 is another cytokine closely involved in controlling Th1 and Th2 responses (47), and we recently showed that oral epithelial cells produce an active IL-18 when stimulated by PR3 in combination with LPS after IFN-{gamma} priming (27). The inflamed site is characterized by an infiltration of neutrophils. Infiltration of neutrophils into gingival tissues is an early event in gingival inflammation, and neutrophils are the predominant leukocytes in the gingival crevicular fluid (GCF) (~90%) (48). It is also evident that GCF has neutrophil serine proteinase activities against MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide for HLE and Suc-Ala-Ala-Pro-Phe-p-nitroanilide for Cat G (49). Because MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide is a PR3 substrate as well (13), PR3 is most likely present in GCF. Therefore, the present study as well as our previous study (27) suggest that the PAR-2-mediated activation of oral epithelial cells by PR3, which is released by active neutrophils, further augments accumulation of neutrophils and immune cells by controlling Th1 or Th2 responses at periodontitis sites, and consequently plays an important role in host defense against periodontal pathogens.

PAR-2 is expressed in the gastrointestinal tract, pancreas, kidney, liver, airway, prostate, ovary, eye, and skin, and is found in epithelial and endothelial cells, smooth muscle cells, keratinocytes, T cell lines, and certain tumor cells (1, 2, 3). It was recently reported that stimulation of PAR-2 with an agonist peptide activates human keratinocytes (42), eosinophils (50), and respiratory epithelial cells (51) to induce inflammatory mediators, and up-regulates keratinocyte phagocytosis (52). PR3 is a major target Ag of anti-neutrophil cytoplasmic Abs (15, 16) and degrades extracellular matrix proteins (13). PR3 is also shown to activate many types of cells (12, 17, 18, 19, 20), although the underlying mechanism was unclear. Therefore, the present study may provide one of the mechanisms of the cell activation by PR3 through PAR-2, and suggests that PR3 could activate the PAR-2-expressing cells, and regulate a number of inflammatory processes, and that the control of PAR-2-activating proteinases including PR3 at inflammatory sites might be beneficial in the regulation of inflammation.


   Acknowledgments
 
We thank N. Takahashi and Y. Iwami (Tohoku University School of Dentistry) for helpful advice concerning calcium mobilization assay and for generously allowing us the use of the spectrofluorometer, and E. Nemoto (Tohoku University School of Dentistry) for helpful discussion.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (12470380 and 13671894). Back

2 Address correspondence and reprint requests to Dr. Shunji Sugawara, Department of Microbiology and Immunology, Tohoku University School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail address: sugawars{at}mail.cc.tohoku.ac.jp Back

3 Abbreviations used in this paper: PAR, protease-activated receptor; {alpha}1-AT, {alpha}1-antitrypsin; Boc-Ala-ONp, Boc-Ala-p-nitrophenyl ester; Cat G, cathepsin G; CDS, cell dissociation solution; EM, extracellular medium; GCF, gingival crevicular fluid; HLE, human leukocyte elastase; MCP-1, monocyte chemoattractant protein-1; PLC, phospholipase C; PR3, proteinase 3. Back

Received for publication April 29, 2002. Accepted for publication August 15, 2002.


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