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The Journal of Immunology, 2004, 173: 4179-4189.
Copyright © 2004 by The American Association of Immunologists

Proinflammatory Cytokines Induce Proteinase 3 as Membrane-Bound and Secretory Forms in Human Oral Epithelial Cells and Antibodies to Proteinase 3 Activate the Cells through Protease-Activated Receptor-21

Akiko Uehara*,{dagger}, Yumiko Sugawara{ddagger}, Takashi Sasano{ddagger}, Haruhiko Takada{dagger} and Shunji Sugawara2,*

Divisions of * Oral Immunology and {dagger} Oral Microbiology, Department of Oral Biology, and {ddagger} Division of Oral Diagnosis, Department of Oral Medicine and Surgery, Tohoku University Graduate School of Dentistry, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Anti-neutrophil cytoplasmic Abs targeting proteinase 3 (PR3) have been detected in relation to a wide range of inflammatory conditions such as periodontitis, and interaction of anti-PR3 Abs with endothelial and epithelial cells provokes cell activation, although the underlying mechanism has been unclear. The present study showed that human oral epithelial cells expressed PR3 mRNA after treatment with proinflammatory cytokines such as IL-1{alpha}, TNF-{alpha}, IFN-{alpha}, IFN-{beta}, and IFN-{gamma}. A 29-kDa PR3 was expressed on the cell surface and released into culture supernatants by the cells upon stimulation with these cytokines. The membrane and supernatant fractions of oral epithelial cells exhibited enzymatic activity, which was inhibited by serine proteinase inhibitors, but not by a cysteine proteinase inhibitor or secretory leukocyte protease inhibitor. Addition of anti-PR3 Abs to cytokine-primed oral epithelial cells in culture induced remarkable secretion of IL-8 and monocyte chemoattractant protein 1 and aggregation of PR3 on the cells. RNA interference targeted to protease-activated receptor-2 mRNA and intracellular Ca2+ mobilization assays revealed that anti-PR3 Abs activated the epithelial cells through protease-activated receptor-2, a family of G protein-coupled receptors. The anti-PR3 Ab-mediated cell activation was completely abolished by RNA interference targeted to PR3 mRNA and by inhibition of phospholipase C and NF-{kappa}B. Immunohistochemistry showed that inflamed oral epithelium actually expresses PR3 protein. These results suggest that oral epithelial cells express functional PR3 in the inflamed sites and respond to anti-PR3 Abs detected in diseased sera, and that these mechanisms may actively participate in the inflammatory process, including periodontitis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Proteinase 3 (PR3;3 EC 3.4.21.76) is a 29-kDa serine proteinase with a high degree of homology to human leukocyte elastase (HLE; EC 3.4.21.37) and cathepsin G (Cat G; EC 3.4.21.20) (1). PR3 exhibits many biological activities, including the degradation of extracellular matrix proteins (2), regulation of myeloid differentiation (3, 4), enhancement of TNF-{alpha} and IL-1{beta} release by human monocytic cell lines (5) and IL-8 and MCP-1 production by human endothelial cells (6, 7), and antibacterial activity (8). These findings suggest that cell-bound and soluble PR3 actively contribute to inflammatory processes. In experimental models, PR3 caused severe tissue damage (1), and the cytotoxic effects of PR3 on endothelial cells are partially mediated by apoptosis (9). PR3 is mainly stored in azurophil granules of neutrophils and is also generated by monocytes, basophils, and mast cells (10). Recently, human renal tubular epithelial cells were reported to express PR3 (11, 12). In contrast, Brouwer et al. (13) reported that PR3 detected in renal tissue was originally derived from neutrophils, and renal cells merely took up the released PR3. Therefore, it is still controversial whether or not PR3 is produced by nonhemopoietic cells (14, 15).

Anti-neutrophil cytoplasmic Abs (ANCA) were first described in 1982 by Davies et al. (16) in patients with necrotizing glomerulonephritis. ANCA is an autoantibody directed against the enzymes located in the primary granules of neutrophils and lysosomes of monocytes. PR3 is a major target Ag of ANCA in Wegener’s granulomatosis (WG), a debilitating autoimmune disease characterized by necrotizing vasculitis (10, 17). Since then, ANCA have been detected in relation to a wide range of inflammatory, infectious, and neoplastic conditions (18, 19, 20, 21). Novo et al. (22, 23) described a high rate of occurrence of ANCA in serum of patients with periodontal disease. In contrast, Hattar et al. (12) reported that PR3 was detected in human renal tubular epithelial cells treated with TNF-{alpha}, and the primed cells responded to anti-PR3 Abs with activation of the phosphoinositide-related signal transduction pathway.

Protease-activated receptor (PAR) family members are G protein-coupled receptors characterized by a proteolytic cleavage of the N terminus that exposes tethered ligands and autoactivates the receptor function, and therefore, certain proteinases activate cells through the PAR family (24, 25, 26). Recently, we demonstrated that PR3 activates human epithelial and nonepithelial cells via the PAR-2 pathway (27, 28). Taken the above findings all together into consideration, we hypothesized that anti-PR3 Abs might activate inflamed oral epithelial cells bearing endogenous PR3 through the PAR-2 pathway. To test this hypothesis, we first examined whether oral epithelial cells express and produce PR3 upon stimulation with various proinflammatory cytokines. We demonstrated that proinflammatory cytokines induced expression of PR3 mRNA in oral epithelial cells in culture and PR3 protein with enzymatic activity on the cell surface and in the culture supernatants of the cells. Then we investigated the possibility that anti-PR3 Abs induce the production of chemokines IL-8 and MCP-1 by the epithelial cells through the PAR-2 pathway.


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

Purified human PR3 and rabbit anti-human PR3 polyclonal Abs were obtained from Elastin Products (Owensville, MO). Mouse anti-human PR3 mAb (CLB-12.8) was obtained from CLB (Amsterdam, The Netherlands). Phospholipase C (PLC) inhibitor U73122, its control compound U73343, and an NF-{kappa}B inhibitor, pyrrolidine dithiocarbamate (PDTC), were obtained from Calbiochem-Novabiochem (La Jolla, CA). Human natural IFN-{alpha} and IFN-{gamma} were kindly provided by Hayashibara Biochemical Laboratories (Okayama, Japan). Natural IFN-{beta} was provided by Toray (Tokyo, Japan). Human rIL-1{alpha} and rTNF-{alpha} were supplied by Dainippon Pharmaceutical (Osaka, Japan). An ultrapurified LPS from Salmonella enterica serovar Abortus-equi (29) was a gift from C. Galanos (Max Plank Institut für Immunbiologie, Freiburg, Germany). A low toxic serine proteinase inhibitor, Pefabloc SC, and a cysteine proteinase inhibitor, E-64, were obtained from Roche Diagnostics (Indianapolis, IN). Recombinant human secretory leukocyte protease inhibitor (SLPI) was purchased from R&D Systems (Minneapolis, MN). Nonenzymatic cell dissociation solution (CDS) was obtained from Sigma-Aldrich (St. Louis, MO). PAR-1 agonist peptide (PAR-1AP; SFLLRN) and PAR-2 agonist peptide (PAR-2AP; SLIGKV) were synthesized by Takara Shuzo (Otsu, Japan). All other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.

Cells and cell culture

Human oral epithelial cell lines HSC-2 (30), HO-1-u-1 (31), and KB (32), established from squamous cell carcinoma, were obtained from the Cancer Cell Repository, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). A human colon adenocarcinoma cell line SW620 (CCL-227) and a human cervical epitheloid carcinoma HeLa (JCRB9004) were obtained from the American Type Culture Collection (Rockville, MD) and Health Science Research Resources Bank (Osaka, Japan), respectively. HSC-2, HO-1-u-1, SW620, and HeLa were grown in RPMI 1640 with 10% FCS (Invitrogen Life Technologies, Grand Island, NY), and KB cells were grown in {alpha}-MEM with 10% FCS.

Human PBMCs from heparinized (10 U/ml) peripheral venous blood of a healthy volunteer were isolated by Lympholyte-H (Cedarlane Laboratories, Hornby, Canada) gradient centrifugation, as described previously (33).

RNA extraction and RT-PCR assay

Total cellular RNA was obtained using Isogen (Nippon Gene, Tokyo, Japan) and reverse transcripted using random hexamer primers and avian myeloblastosis virus reverse-transcriptase XL (Life Sciences, St. Petersburg, FL), as described previously (27). The primers used for PCR were as follows: PR3, forward, 5'-ATCGTGGGCGGGCACGAGGCG-3', and reverse, 5'-GCGGCCAGGGACGAAAGTGCA-3' (7); HLE, forward, 5'-AGTGCCTGGCCATGGGCTGG-3', and reverse, 5'-CACCGGGGCAAAGGCATCGG-3' (34); Cat G, forward, 5'-TGAGAGTGCAGAGGGATAGG-3', and reverse, 5'-CAGGAAACTTGAGACCCTGG-3' (34); plasminogen activator, forward, 5'-ATGACAGAGGATTCAGGTAC-3', and reverse, 5'-GAAGGTGAAGTCATTGAAGA-3'; chymotrypsin-like enzyme, forward, 5'-CTTCTGCGGTGGTTCTCTCAT-3', and reverse, 5'-GAGCACGGTCCCACTGAG-3'; trypsin, forward, 5'-CTCATCAGCGAACAGTGG-3', and reverse, 5'-TTGGTGTAGACTCCAGGC-3'; PAR-2, forward, 5'-GCAGCCTCTCTCTCCTGCAGTGG-3', and reverse, 5'-CTTGCATCTGCTTTACAGTGCG-3' (35); and GAPDH, forward, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', and reverse, 5'-CATGTGGGCCATGAGGTCCACCAC-3' (36). The primers for PR3, HLE, Cat G, plasminogen activator, chymotrypsin-like enzyme, trypsin, PAR-2, and GAPDH were designed to generate fragments of 662, 257, 216, 150, 468, 538, 1066, and 983 bp, respectively. PCR was performed for 35 cycles for 1 min at 94°C, 1 min at 55°C for HLE, Cat G, trypsin, and PAR-2; at 60°C for PR3 and GAPDH; and at 64°C for plasminogen activator and chymotrypsin-like enzyme; and 1 min at 72°C. Amplified samples were visualized on 2.0% agarose gels stained with ethidium bromide and photographed under UV light. The bands on the photographs were scanned and then analyzed using an Image Master 1D (Pharmacia Biotech, Uppsala, Sweden). The results are expressed as relative mRNA accumulation corrected with reference to GAPDH mRNA as an internal standard.

Flow cytometry

Flow cytometric analyses were performed using a FACScan cytometer (BD Biosciences, Mountain View, CA). Oral epithelial cells were stimulated with or without cytokines or LPS for up to 2 h at 37°C. After the incubation, cells were collected by nonenzymatic CDS and washed in PBS. Cells were stained with anti-PR3 polyclonal Ab at 4°C for 30 min, followed by FITC-conjugated swine anti-rabbit IgG (DakoCytomation, Kyoto, Japan) at 4°C for a further 30 min.

Immunoblot analysis

Cell-free supernatants were precipitated with 10% TCA for 30 min at 4°C. After centrifugation at 13,000 x g for 30 min, pellets were washed in acetone. Oral epithelial cells were collected by CDS and washed three times with PBS. The precipitated culture supernatants (equivalent to 3 x 105 cells each) and cell pellets (3 x 105 cells each) were solubilized with Laemmli sample buffer (37), and subjected to Western blotting using rabbit anti-human PR3 polyclonal Ab, as described previously (33).

Measurement of LDH activity

For the quantification of plasma membrane damage, lactate dehydrogenase (LDH) activity in the epithelial cell culture supernatants treated with cytokines was measured with a cytotoxicity detection kit (Roche Diagnostics), according to the manufacturer’s instructions, as described previously (33).

Measurement of enzymatic activity

Cell membrane fractions were prepared by Dounce homogenization, as described previously (33). Amidolytic activities of proteinases in culture supernatants and membrane fractions of oral epithelial cells were assayed at 25°C for 10–60 min with 0.625 mM Boc-Ala-p-nitrophenyl ester (Boc-Ala-ONp; Bachem, Bubendorf, Germany) 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. The liberation of p-nitrophenol was monitored at 405 nm using the Softmax data analysis program (Molecular Devices, Menlo Park, CA). One unit of enzymatic activity was defined as the amount that liberated 1 µmol of p-nitrophenol from the substrate per minute at 25°C. To inhibit the enzymatic activities, test specimens were preincubated with different concentrations of inhibitors for 30 min before use.

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 Discovery Labware, Lincoln Park, NJ). After incubation for 1 day at 37°C in a 5% CO2 incubator, the cells were stimulated with anti-PR3 Abs or LPS in 200 µl of the medium without serum for a given period. 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).

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 (Molecular Probes, Eugene, OR) with shaking for 30 min at room temperature. After being washed 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.

RNA interference

Transfection for targeting endogenous PAR-2, PR3, and Lamin A/C was conducted using Lipofectamine 2000 (Invitrogen Life Technologies) and small interfering RNA (siRNA) (final concentration, 200 nM), according to the manufacturer’s instructions. The sequence of the target for PAR-2 and PR3 mRNA used in this study was 5'-AAAGAGCTTCCCCATAAATCC-3' and 5'-AACCTCAGTGCGTCCGTCACC-3', respectively, and siRNA for PAR-2 and PR3 were synthesized and purified by Silencer siRNA Construction Kit (Ambion, Austin, TX). siRNA of Lamin A/C-targeted 5'-CTGGATTTCCAGAAGAACA-3' was purchased from B-Bridge International (San Jose, CA).

Assay for NF-{kappa}B activity

Activated NF-{kappa}B was measured with an NF-{kappa}B assay kit specific for the p65 subunit, according to the manufacturer’s instructions (Active Motif, Carlsbad, CA). Briefly, samples of whole cell extracts (1–10 µg of protein/well) were added to 96-well plates coated with an oligonucleotide containing the NF-{kappa}B consensus site (5'-GGGACTTTCC-3'), and incubated for 1 h at room temperature with mild agitation. After washing three times, NF-{kappa}B p65 Ab was added and incubated for 1 h without agitation, followed by addition of HRP-conjugated anti-mouse IgG1. Colorimetric reactions were developed, stopped, and measured at 450 nm. The specificity of binding was also examined using an oligonucleotide containing a wild-type or mutated NF-{kappa}B consensus binding site.

Immunohistochemistry

Human gingival tissues were obtained with informed consent from five adult periodontitis patients undergoing periodontal surgery. Immunohistochemistry was conducted, as follows. Tissues were fixed in periodate-lysine-4% paraformaldehyde for 4 h at 4°C. After washing in PBS containing sucrose, fixed tissues were embedded in OCT compound (Sakura, Tokyo, Japan), and immediately frozen. Six-micrometer frozen tissue sections were incubated with rabbit anti-human PR3 polyclonal Ab or anti-human integrin {beta}2 (CD18) mAb P4H9 (mouse IgG3) (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. After that, sections were treated with secondary Abs such as the goat anti-rabbit Envision +/HRP kit or goat anti-mouse Envision +/HRP kit (DakoCytomation). The chromogen used was 3',3-diaminobenzidine tetrahydrochloride (DakoCytomation). The sections were counterstained with hematoxylin. As a negative control, rabbit Ig or mouse Ig (DakoCytomation) was used. Double enzyme-linked immunohistochemistry was performed, as follows. First, anti-PR3 polyclonal Ab was applied overnight with the goat anti-rabbit Envision +/HRP kit. The chromogen used was 3',3-diaminobenzidine tetrahydrochloride. Then anti-integrin {beta}2 mAb was applied overnight, followed by application of goat anti-mouse Envision +/HRP kit. The chromogen in this second system was TrueBlue (Kirkegaard & Perry Laboratories, Gaithersburg, MD). After drying, the specimens were mounted. As negative controls, rabbit and mouse control Abs were used in each step of double immunohistochemistry. The Ethical Review Board of Tohoku University Graduate School of Dentistry (Sendai, Japan) approved the experimental procedures.

Immunostaining

Oral epithelial HSC-2 cells were cultured on eight-chamber glass slides (Falcon) until confluent and treated with TNF-{alpha} (10 ng/ml) for 2 h, washed three times with PBS, and incubated with rabbit anti-human PR3 polyclonal Ab for the time indicated at 37°C in a 5% CO2 incubator. After fixation with 4% paraformaldehyde for 15 min at room temperature, cells were treated with anti-human PAR-2 mAb (mouse IgG2a) (1/500; Santa Cruz Biotechnology) for 2 h at room temperature without permeabilizing. As a control, HSC-2 cells were treated with or without TNF-{alpha} for 2 h, and fixed with 4% paraformaldehyde for 15 min at room temperature, and anti-PR3 polyclonal Ab and anti-PAR-2 mAb for 2 h at room temperature. Samples were then washed and incubated with Alexa Fluor 546 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG2a (1/400; Molecular Probes). Nuclei were visualized by staining with 4',6-diamidino-2-phenylindole (Molecular Probes). Samples were photographed with a Leica DC 200 cooled charge-coupled device camera mounted on a Leica DMR microscope using the application Leica Qfluoro system (Leica Microsystems, Solms, Germany).

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 Dunnett method, and values of p < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of PR3 mRNA in oral epithelial cells in response to proinflammatory cytokines

We first examined by RT-PCR whether human oral epithelial cells express PR3 mRNA. Untreated human oral epithelial HSC-2, KB, and HO-1-u-1 cells did not express PR3 mRNA, and expression of PR3 mRNA was clearly induced after treatment with proinflammatory cytokines such as IL-1{alpha}, TNF-{alpha}, IFN-{alpha}, IFN-{beta}, and IFN-{gamma} for 2 h (Fig. 1A). However, treatment with LPS did not induce PR3 mRNA expression in oral epithelial cells. Significant induction of PR3 mRNA expression was observed in a concentration-dependent manner when the cells were treated with these cytokines for 2 h, and the optimum concentration was 10 ng/ml IL-1{alpha} and TNF-{alpha}, and 1000 IU/ml IFN-{alpha}, IFN-{beta}, and IFN-{gamma} (Fig. 1B). Treatment with the cytokines for 1 h was not sufficient for PR3 expression in HSC-2 cells, whereas following the cytokine treatments for 2 h, the cells strongly expressed PR3 mRNA (Fig. 1C). As maximal PR3 expression was noted after 2-h treatment with the above concentrations of reagents, the subsequent experiments were performed under these conditions. In contrast to PR3 mRNA, the mRNAs of two other neutrophil serine proteinases, HLE and Cat G, were not induced by the cytokines in three oral epithelial cell lines, whereas PBMCs used as a positive control showed clear bands of the expected size (Fig. 1D, and data not shown).



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  		FIGURE 1. Expression of PR3 mRNA in oral epithelial cell lines treated with proinflammatory cytokines. A, Confluent HSC-2, KB, and HO-1-u-1 cells were incubated with or without IL-1{alpha} (10 ng/ml, lane 3); TNF-{alpha} (10 ng/ml, lane 4); IFN-{alpha}, IFN-{beta}, and IFN-{gamma} (1000 IU/ml, lanes 5–7, respectively); and LPS (100 ng/ml, lane 8) for 2 h. Cells were then collected, and the expression of PR3 and GAPDH mRNAs was analyzed by RT-PCR. A water control and a control with medium alone in the cultures were loaded in lanes 1 and 2, respectively. B and C, HSC-2 cells were incubated with IL-1{alpha}, TNF-{alpha}, IFN-{alpha}, IFN-{beta}, and IFN-{gamma} at the indicated concentrations for 2 h (B) or incubated with IL-1{alpha} (10 ng/ml), TNF-{alpha} (10 ng/ml), IFN-{alpha} (1000 IU/ml), IFN-{beta} (1000 IU/ml), and IFN-{gamma} (1000 IU/ml) for the time indicated (C), and the expression of PR3 and GAPDH mRNAs was analyzed by RT-PCR. Bands were further quantified using an imaging analyzer. The results are expressed relative to GAPDH mRNA as an internal standard. D, Total RNA was extracted from HSC-2 (lane 2), KB (lane 3), HO-1-u-1 (lane 4), and PBMCs (lane 5), all of which were treated with IL-1{alpha} (10 ng/ml) for 2 h, and the expression of HLE, Cat G, and GAPDH mRNAs was analyzed by RT-PCR. A water control was loaded in lane 1. Three additional experiments gave results similar to those shown here. **, p < 0.01 compared with respective control (medium alone).

 
Surface expression and secretion of bioactive PR3 by oral epithelial cells treated with proinflammatory cytokines

We next examined the cell surface expression of PR3 by flow cytometry. Consistent with the results of RT-PCR, unstimulated and LPS-treated HSC-2 cells did not express PR3 on the cell surface, whereas treatment with IL-1{alpha}, TNF-{alpha}, IFN-{alpha}, IFN-{beta}, or IFN-{gamma} caused a marked increase in the expression of PR3 on the cell surface (Fig. 2A). KB and HO-1-u-1 cells showed similar results (data not shown). The induction of PR3 expression by these cytokines was detected at 30 min of incubation, and reached a plateau at 2 h (Fig. 2B, and data not shown). Furthermore, immunoblot analysis showed that 29-kDa PR3 protein was detected in the cell lysates and culture supernatants of the cytokine-treated HSC-2 cells; this is the same molecular mass as that of purified PR3 (Fig. 2C). KB and HO-1-u-1 cells showed the same results (data not shown). LDH assays showed that no LDH activity was detected in the supernatants upon stimulation with cytokines, indicating that the epithelial cell membrane was not damaged by the cytokine treatment (Fig. 2D). These results suggest that PR3 is induced on the cell surface of oral epithelial cells and secreted by the cells in response to proinflammatory cytokines.



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  		FIGURE 2. PR3 expression on the cell surface and detection of PR3 in cell lysate and in the culture supernatant of the oral epithelial cells treated with proinflammatory cytokines. A, Confluent HSC-2 cells were incubated for 2 h in the absence or presence of IL-1{alpha} (10 ng/ml); TNF-{alpha} (10 ng/ml); and IFN-{alpha}, IFN-{beta}, and IFN-{gamma} (1000 IU/ml each). The surface expression of PR3 was assessed by flow cytometry. Data were expressed as percentage of positive cells. B, Confluent HSC-2 cells were stimulated with or without IL-1{alpha} (10 ng/ml) for the time indicated, and the expression of PR3 was assessed by flow cytometry. C, Confluent HSC-2 cells were stimulated with IL-1{alpha} (10 ng/ml, lane 2); TNF-{alpha} (10 ng/ml, lane 3); IFN-{alpha}, IFN-{beta}, and IFN-{gamma} (200 IU/ml, lanes 4–6, respectively); or LPS (100 ng/ml, lane 7) for 2 h. The whole cell lysates (3 x 105 cells each) and TCA-precipitated supernatants (equivalent to 3 x 105 cells each) were then subjected to Western blotting using anti-PR3 mAb. Purified PR3 (2 ng/lane, lane 8) and medium alone (lane 1) were loaded as a control. D, LDH activity in the supernatants, as in B, was measured. Percentage of LDH activity was expressed based on maximum releasable LDH activity in the cells induced by 1% Triton X-100 for 24 h. ND, Not detected. The results are representative of four different experiments with similar results.

 
We next examined whether the PR3 of oral epithelial cells induced by proinflammatory cytokines had enzymatic activity using Boc-Ala-ONp as a substrate (2). As shown in Fig. 3A, the culture supernatants of TNF-{alpha}-treated cells showed substantial enzymatic activity. The enzymatic activities of the culture supernatants and cell membrane fractions were 0.3 U and 2.9 U/105 cells, respectively, and the enzymatic activity was significantly inhibited by a low toxic sulfonyl fluoride-type serine proteinase inhibitor, Pefabloc SC, at 4 µM, and a naturally occurring serine proteinase inhibitor, {alpha}1-antitrypsin ({alpha}1-AT), at 1 µg/ml (0.02 µM) (Fig. 3, B and C), as reported previously (6, 27, 28, 38). By contrast, treatment with a high dose of a cysteine proteinase inhibitor, E-64 (up to 100 µM), did not inhibit the enzymatic activity of the culture supernatants or membrane fractions. Furthermore, SLPI, which inhibits HLE and Cat G, but not PR3 (2) (up to 20 µM), did not influence the enzymatic activity. In addition, oral epithelial cells used in this study did not express mRNAs of other serine proteases such as plasminogen activator, chymotrypsin-like enzyme, and trypsin (Fig. 3D), which are reported to be expressed in epithelial cells (39, 40, 41). Intestinal SW620 cells and crevicular HeLa cells were used as positive controls. These results suggest that the enzymatic activity derived from oral epithelial cells is due to PR3. The enzymatic activity of the culture supernatant and membrane fractions induced by other cytokines (IL-1{alpha}, IFN-{alpha}, IFN-{beta}, and IFN-{gamma}) showed the same results (data not shown).



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  		FIGURE 3. Detection of bioactive PR3 in the culture supernatant and cell membrane fraction from oral epithelial cells treated with cytokines. A, Confluent oral epithelial HSC-2 cells were treated with TNF-{alpha} (10 ng/ml). After incubation for the time indicated ({blacksquare}, 0; {blacktriangleup}, 2; {diamondsuit}, 4; {square}, 6; {circ}, 8; {triangleup}, 12; {diamond}, 24 h), the culture supernatant was collected. Enzymatic activities of the cell culture supernatant were measured using Boc-Ala-ONp as a substrate. Substrate alone ({blacksquare}) was assessed as a control. The culture supernatants (B) and membrane fraction (C) of the cells treated with TNF-{alpha} (10 ng/ml) were pretreated with Pefabloc SC, {alpha}1-AT, E-64, or SLPI at the dose indicated for 30 min before use. Then enzymatic activity was measured, and the results are expressed as percentage of activity. **, p < 0.01 compared without inhibitors. D, Confluent HSC-2, KB, HO-1-u-1, SW620, and HeLa cells were collected, and the expression of plasminogen activator (PA), chymotrypsin-like enzyme (Chymo), trypsin, and GAPDH mRNAs was analyzed by RT-PCR. No reverse transcriptase as a negative control was loaded in lane 1. The results are representative of three different experiments with similar results.

 
Activation of oral epithelial cells by anti-PR3 Abs

We next examined whether the addition of anti-PR3 Abs caused cellular activation. Although nonprimed oral epithelial cells were not activated by anti-PR3 Abs (data not shown), anti-PR3 Abs provoked marked production of IL-8 and MCP-1 by TNF-{alpha}-pretreated oral epithelial cells, whereas comparable amounts of control IgG and LPS were completely ineffective (Fig. 4A). Anti-PR3 Abs from two different sources showed the same results. Consistent with the results in Fig. 3, B and C, the activation induced by anti-PR3 Abs was significantly inhibited by {alpha}1-AT and Pefabloc, but not SLPI or E-64 (Fig. 4B), indicating that the enzymatic activity of PR3 is required for the anti-PR3-mediated cell activation.



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  		FIGURE 4. Anti-PR3 Abs activated oral epithelial cells primed with cytokines to produce IL-8 and MCP-1. A, Confluent HSC-2 cells were pretreated with TNF-{alpha} (10 ng/ml) for 2 h. After being washed three times with PBS, cells were incubated for 24 h in the presence or absence of various concentration of anti-PR3 Abs from Elastin Products ({alpha}-PR3 Ab (E)) and CLB ({alpha}-PR3 Ab (C)), control Ab (10 µg/ml), and LPS (100 ng/ml) at 37°C. B, TNF-{alpha} (10 ng/ml; 2 h)-primed HSC-2 cells were pretreated with or without Pefabloc (40 µM), {alpha}1-AT (1 µg/ml), SLPI (20 µM), or E-64 (10 µM) for 1 h, then incubated for 24 h in the presence or absence of anti-PR3 Abs (10 µg/ml), control Ab (10 µg/ml), or LPS (100 ng/ml) at 37°C, and the inhibitors remained present during incubation for 24 h. Concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. **, p < 0.01 compared with the respective control (medium alone). The results are representative of three different experiments with similar results.

 
Involvement of PAR-2 and NF-{kappa}B in the anti-PR3 Ab-induced oral epithelial cell activation

As we recently showed that PR3 activates oral epithelial cells through PAR-2 (27), we next examined whether the cell activation triggered by anti-PR3 Ab occurs through PAR-2. Ca2+ mobilization in the cytokine-primed KB cells was measured after exposure to anti-PR3 Abs (from different sources), exogenous PR3, trypsin, or synthetic PAR agonist peptides. Anti-PR3 Abs rapidly induced a Ca2+ response and abolished the response to a second application of PAR-2AP and exogenous PR3 (Fig. 5, A–C). Exogenous PR3, trypsin as a reference PAR-2-activating serine proteinase, and PAR-2AP rendered cells refractory to subsequent stimulation with anti-PR3 Abs (Fig. 5, D–F), while stimulation with PAR-1AP did not cause such an effect (Fig. 5G). HSC-2 and HO-1-u-1 cells showed the same results, although the Ca2+ mobilization was less pronounced than that in KB cells (data not shown). These results clearly indicated that the anti-PR3 Ab-induced cell activation is mediated by PAR-2.



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  		FIGURE 5. Effects of anti-PR3 Abs, PR3, trypsin, and PAR agonist peptides on calcium mobilization in cytokine-primed oral epithelial cells. A–G, Fura 2-loaded KB cells were exposed to 10 µg/ml anti-PR3 Abs ({alpha}-PR3) from Elastin Products (E) or CLB (C), 100 µM PAR-1AP and PAR-2AP, 1 µM PR3, and 1 µM trypsin, as indicated in sequence, and the change in intracellular calcium was monitored. The results are representative of three different experiments with similar results.

 
To further confirm the involvement of PAR-2, we used siRNA to block the expression of PAR-2. Transfection of HSC-2 cells with PAR-2-specific siRNA resulted in inhibition of PAR-2 mRNA, but not GAPDH mRNA from 8 h up to 72 h of culture (Fig. 6A). In the transfected cells, anti-PR3 Ab-induced activation of primed oral epithelial cells was almost completely inhibited by PAR-2-specific siRNA (Fig. 6B). 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 (24, 25, 26). To examine whether PLC is also involved in the Ab-induced activation of oral epithelial cells, TNF-{alpha}-pretreated HSC-2 cells were treated with Abs in the presence of the PLC inhibitor U73122 or the control compound U73343 for 24 h. As shown in Fig. 6C, the inhibition of PLC completely abolished the production of IL-8 and MCP-1 induced by anti-PR3 Abs. PAR-2AP was used as a positive control and showed the same results as those with anti-PR3 Abs (Fig. 6, B and C). Furthermore, PR3-specific siRNA blocked the expression of PR3 in the TNF-{alpha}-treated HSC-2 cells (Fig. 7A) and subsequently inhibited anti-PR3 Ab-induced production of IL-8 and MCP-1 (Fig. 7B) and Ca2+ mobilization (Fig. 7C), while PAR-2AP was still effective on the cells. Lamin A/C-specific siRNA was used as a negative control (Fig. 7, A and B). These results clearly indicated that endogenous PR3 was responsible for the anti-PR3 Ab-induced activation of PAR-2.



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  		FIGURE 6. Involvement of PAR-2 in anti-PR3 Ab-induced oral epithelial cell activation. A, Confluent HSC-2 cells were pretreated with TNF-{alpha} (10 ng/ml) for 2 h. After being washed three times with PBS, the cells were transfected with PAR-2-specific siRNA. After the transfection for the time indicated, the expression of PAR-2 and GAPDH mRNA was analyzed by RT-PCR. B, TNF-{alpha}-pretreated HSC-2 cells were transfected with PAR-2-specific siRNA for 24 and 48 h, and were incubated for an additional 24 h in the presence or absence of anti-PR3 Abs, as described in Fig. 5, or with control Ab (10 µg/ml each) at 37°C. PAR-2AP (100 µM) was used as a positive control. C, TNF-{alpha}-pretreated HSC-2 cells were incubated with or without 10 µM U73122 or control U73343 for 30 min. Then the cells were stimulated with anti-PR3 Abs (10 µg/ml) for 24 h. PAR-2AP (100 µM) was used as a positive control. Concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. **, p < 0.01 compared with the respective control (medium alone). The results are representative of three different experiments with similar results.

 


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  		FIGURE 7. Involvement of endogenous PR3 in anti-PR3 Ab-induced oral epithelial cell activation. A, Confluent HSC-2 cells were pretreated with TNF-{alpha} (10 ng/ml) for 2 h. After being washed three times with PBS, the cells were transfected with PR3- or Lamin A/C-specific siRNA. After transfection for the time indicated, the expression of PR3 and GAPDH mRNA was analyzed by RT-PCR. B, TNF-{alpha}-pretreated HSC-2 cells were transfected with PR3-specific siRNA for 24 and 48 h or Lamin A/C-specific siRNA for 48 h, and were incubated for an additional 24 h in the presence or absence of anti-PR3 Abs ({alpha}-PR3) from Elastin Products (E) or CLB (C) or with control Ab (10 µg/ml each) at 37°C. PAR-2AP (100 µM) was used as a positive control. Concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. **, p < 0.01 compared with the respective control. C, TNF-{alpha}-pretreated KB cells were transfected with PR3-specific siRNA for 24 and 48 h, and the cells were loaded with fura 2. Fura 2-loaded KB cells were exposed to 10 µg/ml anti-PR3 Abs ({alpha}-PR3) from Elastin Products (E) or CLB (C), or 100 µM PAR-2AP, and the change in intracellular calcium was monitored. The results are representative of three different experiments with similar results.

 
It has been reported that agonists of PAR-2 induced activation of NF-{kappa}B in human dermal microvascular endothelial cells (42). As shown in Fig. 8A, addition of anti-PR3 Abs clearly activated NF-{kappa}B in oral epithelial cells. TNF-{alpha}-induced surface expression of PR3 was not affected by treatment with PDTC, an inhibitor of NF-{kappa}B (Fig. 8B), but PDTC markedly reduced the production of IL-8 and MCP-1 induced by anti-PR3 Abs as well as PAR-2AP on oral epithelial cells (Fig. 8C). These results exclude the possibility that inhibition of NF-{kappa}B interfered with PR3 expression on oral epithelial cells and indicate that anti-PR3 Ab-induced activation of the cells involves NF-{kappa}B downstream of PAR-2 signaling.



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  		FIGURE 8. Involvement of NF-{kappa}B in production of IL-8 and MCP-1 induced by anti-PR3 Abs in human oral epithelial cells. A, Confluent HSC-2 cells were pretreated with TNF-{alpha} (10 ng/ml) for 2 h. After being washed three times with PBS, cells were incubated for 1 h in the presence or absence of anti-PR3 Abs ({alpha}-PR3), as described in Fig. 5, or control Ab (10 µg/ml each) at 37°C, and active NF-{kappa}B was measured. PAR-2AP (100 µM) was used as a positive control. B, Confluent HSC-2 cells were pretreated with TNF-{alpha} (10 ng/ml) for 2 h. After being washed three times with PBS, cells were incubated for 2 h in the absence or presence of PDTC. The surface expression of PR3 was assessed by flow cytometry. C, Confluent HSC-2 cells were pretreated with TNF-{alpha} (10 ng/ml) for 2 h. After being washed three times with PBS, cells were treated with or without PDTC for 30 min at 37°C. After incubation, cells were stimulated for 24 h in the absence or presence of anti-PR3 Abs ({alpha}-PR3) from Elastin Products (E) or CLB (C), or control Ab (10 µg/ml each) at 37°C. The inhibitor remained present during incubation for 24 h. PAR-2AP (100 µM) was used as a positive control. Concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. **, p < 0.01 compared with the respective control. The results are representative of three different experiments with similar results.

 
Aggregation of PR3 on oral epithelial cells by anti-PR3 Ab

To elucidate the mechanism of anti-PR3-induced activation of PAR-2, we examined mobilization of PR3 and PAR-2 by immunostaining. Untreated HSC-2 cells express PAR-2, but not PR3 (Fig. 9A). Treatment with TNF-{alpha} induced expression of PR3 (Fig. 9B), which is consistent with the results of RT-PCR (Fig. 1) and flow cytometry (Fig. 2). Addition of anti-PR3 polyclonal Ab induced aggregation of PR3, which peaked at 15–30 min and decreased thereafter (Fig. 9, C–F). The Ab treatment did not induce aggregation of PAR-2. These results indicated the aggregation of membrane-bound PR3 by anti-PR3 Ab is important for PAR-2 activation.



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  		FIGURE 9. Aggregation of PR3 on oral epithelial cells by anti-PR3 Ab. Oral epithelial HSC-2 cells were left untreated (A) or treated with TNF-{alpha} (10 ng/ml) for 2 h (B). After being washed three times with PBS, the treated cells were incubated with rabbit anti-PR3 polyclonal Ab for 15 min (C), 30 min (D), 1 h (E), and 2 h (F). After fixation, cells were treated with anti-PAR-2 mAb. PR3 and PAR-2 were then visualized with anti-Alexa Fluor 546 (red) and anti-Alexa Fluor 488 (green), respectively. Nuclei were visualized by staining with 4',6-diamidino-2-phenylindole in blue. Yellow staining shows colocalization of PR3 and PAR-2. The results are representative of three different experiments with similar results.

 
Expression of PR3 in human gingival tissues of adult periodontitis

Finally, we examined by immunohistochemistry whether human oral (gingival) tissues actually express PR3 protein. PR3 was scarcely expressed in the cryosections of slightly inflamed gingival tissue from adult periodontitis patients (Fig. 10A). In contrast, PR3 was diffusely expressed in prickle cell layer of the epithelium in the severely inflamed epithelial tissues (Fig. 10B). In high magnification of the section, PR3 was expressed in epithelial cells surrounded by intercellular bridge, and strongly in infiltrating segmented cells (Fig. 10C). Integrin {beta}2 was expressed by infiltrating segmented cells, but not by epithelial cells (Fig. 10, D and E). Double staining demonstrated that the segmented cells were double positive, but the epithelial cells were positive for only PR3 (Fig. 10, F and G). In addition, infiltrating cells in subepithelial tissues were also double positive (Fig. 10, B, D, and F). The results clearly showed that inflamed gingival epithelial cells as well as inflammatory segmented cells express RP3 in vivo.



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  		FIGURE 10. Expression of PR3 protein in human gingival tissues. A–G, Cryosections of gingival tissues from patients with adult periodontitis with slightly inflamed (A) and severely inflamed gingival tissues (B–G) were stained with anti-PR3 Ab (brown) (A–C) and anti-integrin {beta}2 mAb (brown) (D and E), or double stained with anti-PR3 Ab (brown) and anti-integrin {beta}2 mAb (blue) (F and G). The sections were counterstained with hematoxylin in blue (A–E). C, E, and G, Prickled cell layer in B, D, F, respectively. Scale bars: 200 µm (A, B, D, and F); 20 µm (C, E, and G).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we clearly demonstrated that human oral epithelial cells express PR3 on the cell surface, secrete it into the supernatant under inflammatory conditions, i.e., in the presence of IL-1{alpha}, TNF-{alpha}, IFN-{alpha}, IFN-{beta}, or IFN-{gamma}, and could be activated by Ab directed to the surface PR3, resulting in marked production of chemokines IL-8 and MCP-1 through PAR-2.

The induction of membrane PR3 expression by cytokines such as TNF-{alpha} is a well-established phenomenon in neutrophils and monocytes, but the ability of nonhemopoietic cells to express PR3 has remained controversial. PR3 was found histologically in tubular epithelial cells as well as endothelial cells in inflamed human kidneys in patients with WG (13) and glomerulonephritis (43), although it was assumed that PR3 in these cells might be due to the property of the cells to take up cationic proteins. TNF-{alpha} levels were elevated during active phases and not detected in remission in WG (14), and transcription of the TNF-{alpha} gene in PBMCs of patients with systemic vasculitis and WG was enhanced compared with that in healthy blood donors (44). PR3 mRNA and protein were detected in kidney biopsies of distal tubules and glomerular epithelia by in situ hybridization techniques (11). Furthermore, some studies have demonstrated PR3 mRNA and surface protein expression in endothelial (14, 45, 46, 47, 48) and epithelial cells in culture (11, 12, 49) treated with TNF-{alpha} or IL-1{alpha}. Consistent with these observations, we first demonstrated that cytokine exposure results in surface expression and secretion of enzymatically active PR3 by oral epithelial cells (Figs. 2 and 3). In support of this, PR3 expression was clearly found in inflamed gingival epithelium of patients with adult periodontitis (Fig. 10).

In this study, we used Boc-Ala-ONp as a substrate of serine protease including PR3 (2), and enzymatic activities of culture supernatants and cell membrane fractions were inhibited by Pefabloc SC at 4 µM and {alpha}1-AT at 1 µg/ml (0.02 µM), but not by SLPI at 20 µM or E64 at 100 µM (Fig. 3). We previously showed that 1 µg/ml {alpha}1-AT inhibited 10 µg/ml (345 nM) HLE, Cat G, and PR3, and 2 µM SLPI inhibited 10 µg/ml HLE and Cat G, but not PR3 (27). It is reported that epithelial cells possess other serine proteases, such as plasminogen activator (39), chymotrypsin-like enzyme (40), and trypsin (41), and Boc-Ala-ONp cannot distinguish between different serine proteases. However, oral epithelial HSC-2, KB, and HO-1-u-1 used in this study did not express these epithelial serine proteases (Fig. 3D). These observations suggest that the enzymatic activity was due to PR3. To clarify this point, we blocked PR3 by RNA interference, and the results showed that PR3-specific siRNA inhibited anti-PR3 Ab-induced production of IL-8 and MCP-1 and Ca2+ mobilization (Fig. 7). In contrast, transfection of Lamin A/C-specific siRNA did not influence the expression of PR3 mRNA and anti-PR3 Ab-induced production of IL-8 and MCP-1 (Fig. 7, A and B). These results clearly indicated that endogenous PR3 was responsible for anti-PR3 Ab-induced activation of oral epithelial cells.

Abbott et al. (50) first described in 1989 that a mAb to neutrophil cytoplasmic Ag (PR3) and WG sera bound to human cultured glomerular epithelial and endothelial cells as well as to HUVEC. These investigators concluded that the Ag recognized by the ANCA-positive sera is also expressed on vascular surface structures, suggesting a direct pathogenic role of ANCA. This finding, together with the evidence that neutrophils are not necessarily the exclusive source of PR3, acted as an impetus for investigations of the interaction between endothelial Ags and ANCA. Thereafter, Mayet et al. showed that binding of anti-PR3 Abs to TNF-{alpha}-treated endothelial cells led to a marked increase of neutrophil adhesion via induction of E-selectin (45), anti-PR3 Abs displayed lytic activity against endothelial cells treated with TNF-{alpha} (46), incubation of TNF-{alpha}-treated endothelial cells with anti-PR3 Abs led to a marked increase of endothelial VCAM-1 expression (47), and binding of anti-PR3 Abs to TNF-{alpha}-treated tubular epithelial cells led to a marked increase of adhesion molecules, ICAM-1 and VCAM-1 (48), although the underlying mechanisms have remained unclear.

As ANCA have been detected in a wide range of autoimmune diseases, tumors, inflammatory conditions, and infection, ANCA might be involved in many steps of inflammatory processes together with other mediators. Hattar et al. (12) reported that the interaction of ANCA with human renal tubular epithelium contributes to the development of renal lesions associated with WG. In fact, Novo et al. (22, 23) found that 75% of the sera of patients with adult periodontitis were positive for ANCA, while none of the healthy controls was positive. The present study showed that binding of anti-PR3 Abs to oral epithelial cells led to a marked increase of the production of chemokines IL-8 and MCP-1 (Fig. 4), suggesting that the expression of PR3 is induced on the membranes of oral epithelial cells by proinflammatory cytokines, and that these cells may in turn become targets of circulating ANCA. Nonspecific complement-dependent activation of oral epithelial cells could be ruled out, because all the experiments in this study were performed under serum-free conditions. As untreated oral epithelial cells, lacking PR3 surface expression, were not activated by anti-PR3 treatment, specific targeting of PR3 is a prerequisite for ANCA-mediated activation of these epithelial cells. Isotype-matched control IgG was not capable of eliciting oral epithelial signaling, suggesting that ligation of solely Fc IgGRs is not sufficient for inducing oral epithelial activation. Therefore, the present study provides a possible mechanism for active interplay of ANCA with oral epithelial cells.

Activation of cellular signal transduction pathways by anti-PR3 Abs has been described in neutrophils with activation of protein kinase C and tyrosine phosphorylation of numerous proteins in relation to the activation of the neutrophil respiratory burst by these autoantibodies (51). Although some studies showed that anti-PR3 Abs interact with HUVEC (52) and renal epithelial cells (12), thereby provoking pronounced activation of the phosphoinositide-related signal transduction pathway, the signaling pathway elicited by the anti-PR3 Abs has remained unclear. We have recently shown that human epithelial and nonepithelial cells are activated by PR3 through PAR-2, resulting in activation of PLC, which is a common pathway among G protein-coupled receptors, including PARs (27, 28). RNA interference targeted to PAR-2 mRNA and the intracellular Ca2+ mobilization assays shown in this study provided the first evidence that cross-linking of PR3 on the epithelial cell surface with anti-PR3 Abs activated PAR-2, and consequently provoked the PAR-2 signaling pathway, initiating the activation of PLC (Figs. 5 and 6). Immunostaining demonstrated that PR3 and PAR-2 coexpressed on TNF-{alpha}-treated oral epithelial cells, and that the addition of anti-PR3 polyclonal Ab induced aggregation of PR3, but not PAR-2, on the epithelial cells (Fig. 9). The results indicate that anti-PR3 Abs concentrated enzymatic activity of PR3 via aggregation, consequently leading to activation of PAR-2. PAR-2 is present in many types of cells, including smooth muscle cells, stromal cells, and endothelial and epithelial cells (53). PAR-2 expression is significantly increased in asthmatic bronchial epithelium in comparison with normal epithelium (54). PAR-2 deficiency in mice attenuates allergic dermatitis (55), delays the onset of inflammation (56), and decreases eosinophil infiltration and hyperreactivity in allergic inflammation of the airway (57). These findings suggest that PAR-2 plays a crucial role in the regulation of inflammation.

Chemokines represent a group of chemotactic cytokines that induce cellular locomotion. The total amount of IL-8 in gingival crevicular fluid (GCF) in adult periodontitis was significantly higher than that in GCF in clinically healthy gingiva (58), and MCP-1 expression was induced in GCF in adult periodontitis (59), and the chemokines in GCF were derived from gingival tissues and other sources, such as leukocytes. Although there are no data concerning basal tissue amounts of IL-8 and MCP-1 in oral epithelium, it was reported that the IL-8 concentration in GCF in adult periodontitis is about twice that in GCF from healthy subjects (317 and 189 ng/ml, respectively) (58). The present study showed that there was a ~3-fold increase in production of IL-8 and MCP-1 from cytokine-primed oral epithelial cells by adding anti-PR3 Abs in vitro. Findings in in vitro confluent monolayer culture do not necessarily apply to in vivo stratified oral epithelium, but we could consider the findings using cultured cells to be a reflection of conditions in vivo especially in periodontitis patients, whose serum levels of ANCA are higher than those of healthy donors. In addition, activation of NF-{kappa}B is required for the production of both IL-8 and MCP-1 (60). Agonists of PAR-2 induce IL-6 and IL-8 release and activation of NF-{kappa}B in human dermal microvascular endothelial cells (39). The present study also showed that production of IL-8 and MCP-1 from oral epithelial cells caused by anti-PR3 Abs was mediated through activation of NF-{kappa}B. As NF-{kappa}B is a crucial transcriptional factor among several signaling pathways, including production of proinflammatory cytokines, interaction of ANCA and host cells may also cause activation of molecules other than IL-8 and MCP-1.

ANCA have been detected in a wide range of inflammatory, infectious, and neoplastic conditions, and PR3 is present in many types of cells other than epithelial cells, including neutrophils, monocytes, basophils, and mast cells, independently of tissue type (10, 11). In addition, PAR-2 is also present in many types of cells (52). Therefore, endogenous PR3-mediated cell activation triggered by ANCA via PAR-2 may occur in various cells and play a principal role in tissue-specific inflammatory processes and physiological responses.


   Acknowledgments
 
We thank Dr. C. Galanos for providing the purified Salmonella LPS. We also thank N. Takahashi and Y. Iwami (Tohoku University Graduate School of Dentistry) for helpful advice concerning calcium mobilization assay and for generously allowing us to use the spectrofluorometer.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (14370576 and 15390551) and the 21st Century Center of Excellence Program Special Research Grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. A.U. is supported by research fellowships of the Japan Society for the Promotion of Science for Young Scientists (6961). Back

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

3 Abbreviations used in this paper: PR3, proteinase 3; {alpha}1-AT, {alpha}1-antitrypsin; ANCA, anti-neutrophil cytoplasmic Ab; Cat G, cathepsin G; CDS, cell dissociation solution; EM, extracellular medium; GCF, gingival crevicular fluid; HLE, human leukocyte elastase; LDH, lactate dehydrogenase; PAR, protease-activated receptor; PAR-1AP/PAR-2AP, PAR-1/2 agonist peptide; PDTC, pyrrolidine dithiocarbamate; PLC, phospholipase C; siRNA, small interfering RNA; SLPI, secretory leukocyte protease inhibitor; WG, Wegener’s granulomatosis. Back

Received for publication December 1, 2003. Accepted for publication July 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kao, R. C., N. G. Wehner, K. M. Skubitz, B. H. Gray, J. R. Hoidal. 1988. Proteinase 3: a distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J. Clin. Invest. 82:1963.
  2. Rao, N. V., N. G. Wehner, B. C. Marshall, W. R. Gray, B. H. Gray, J. R. Hoidal. 1991. Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase: structural and functional properties. J. Biol. Chem. 266:9540.[Abstract/Free Full Text]
  3. Bories, D., M.-C. Raynal, D. H. Solomon, Z. Darzynkiewicz, Y. E. Cayre. 1989. Down-regulation of a serine protease, myeloblastin, causes growth arrest and differentiation of promyelocytic leukemia cells. Cell 59:959.[Medline]
  4. Sköld, S., B. Rosberg, U. Gullberg, T. Olofsson. 1999. A secreted proform of neutrophil proteinase 3 regulates the proliferation of granulopoietic progenitor cells. Blood 93:849.[Abstract/Free Full Text]
  5. Coeshott, C., C. Ohnemus, A. Pilyavskaya, S. Ross, M. Wieczorek, H. Kroona, A. H. Leimer, J. Cheronis. 1999. Converting enzyme-independent release of tumor necrosis factor {alpha} and IL-1{beta} from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proc. Natl. Acad. Sci. USA 96:6261.[Abstract/Free Full Text]
  6. Berger, S. P., M. A. J. Seelen, P. S. Hiemstra, J. S. J. Gerritsma, E. Heemskerk, F. J. van der Woude, M. R. Daha. 1996. Proteinase 3, the major autoantigen of Wegener’s granulomatosis, enhances IL-8 production by endothelial cells in vitro. J. Am. Soc. Nephrol. 7:694.[Abstract]
  7. Taekema-Roelvink, M. E. J., C. van Kooten, M. C. Janssens, E. Heemskerk, M. R. Daha. 1998. Effect of anti-neutrophil cytoplasmic antibodies on proteinase 3-induced apoptosis of human endothelial cells. Scand. J. Immunol. 48:37.[Medline]
  8. Lundqvist, C., V. Baranov, S. Teglund, S. Hammarström, M.-L. Hammarström. 1994. Cytokine profile and ultrastructure of intraepithelial {gamma}{delta} T cells in chronically inflamed human gingiva suggest a cytotoxic effector function. J. Immunol. 153:2302.[Abstract]
  9. Yang, J. J., R. Kettritz, R. J. Falk, J. C. Jennette, M. L. Gaido. 1996. Apoptosis of endothelial cells induced by the neutrophil serine proteases proteinase 3 and elastase. Am. J. Pathol. 149:1617.[Abstract]
  10. Braun, M. G., E. Csernok, W. L. Gross, H.-K. Müller-Hermelink. 1991. Proteinase 3, the target antigen of anticytoplasmic antibodies circulating in Wegener’s granulomatosis: immunolocalization in normal and pathologic tissues. Am. J. Pathol. 139:831.[Abstract]
  11. Schwarting, A., D. Hagen, M. Odenthal, H. Brockmann, H.-P. Dienes, E. Wandel, H.-J. Rumpelt, K.-H. Meyer zum Büschenfelde, P. R. Galle, W. Mayet. 2000. Proteinase-3 mRNA expressed by glomerular epithelial cells correlates with crescent formation in Wegener’s granulomatosis. Kidney Int. 57:2412.[Medline]
  12. Hattar, K., U. Grandel, A. Bickenbach, A. Schwarting, W.-J. Mayet, J. Bux, S. Jessen, C. Fischer, W. Seeger, F. Grimminger, U. Sibelius. 2002. Interaction of antibodies to proteinase 3 (classic anti-neutrophil cytoplasmic antibody) with human renal tubular epithelial cells: impact on signaling events and inflammatory mediator generation. J. Immunol. 168:3057.[Abstract/Free Full Text]
  13. Brouwer, E., M. G. Huitema, A. H. L. Mulder, P. Heeringa, H. van Goor, J. W. C. Tervaert, J. J. Weening, C. G. M. Kallenberg. 1994. Neutrophil activation in vitro and in vivo in Wegener’s granulomatosis. Kidney Int. 45:1120.[Medline]
  14. Mayet, W.-J., E. Csernok, C. Szymkowiak, W. L. Gross, K.-H. Meyer zum Büschenfelde. 1993. Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener’s granulomatosis. Blood 82:1221.[Abstract/Free Full Text]
  15. King, W. J., D. Adu, M. R. Daha, C. J. Brooks, D. J. Radford, A. A. Pall, C. O. S. Savage. 1995. Endothelial cells and renal epithelial cells do not express the Wegener’s autoantigen, proteinase 3. Clin. Exp. Immunol. 102:98.[Medline]
  16. Davies, D. J., J. E. Moran, J. F. Niall, G. B. Ryan. 1982. Segmental necrotizing glomerulonephritis with antineutrophil antibody: possible arbovirus aetiology?. Br. Med. J. 285:606.
  17. Lüdemann, J., B. Utecht, W. L. Gross. 1990. Anti-neutrophil cytoplasm antibodies in Wegener’s granulomatosis recognize an elastinolytic enzyme. J. Exp. Med. 171:357.[Abstract/Free Full Text]
  18. Hagen, E. C., B. E. P. B. Ballieux, L. A. van Es, M. R. Daha, F. J. van der Woude. 1993. Antineutrophil cytoplasmic autoantibodies: a review of the antigens involved, the assays, and the clinical and possible pathogenetic consequences. Blood 81:1996.[Free Full Text]
  19. Savige, J., D. Davies, R. J. Falk, J. C. Jennette, A. Wiik. 2000. Antineutrophil cytoplasmic antibodies and associated diseases: a review of the clinical and laboratory features. Kidney Int. 57:846.[Medline]
  20. Ardiles, L. G., G. Valderrama, P. Moya, S. A. Mezzano. 1997. Incidence and studies on antigenic specificities of antineutrophil-cytoplasmic autoantibodies (ANCA) in poststreptococcal glomerulonephritis. Clin. Nephrol. 47:1.[Medline]
  21. Bartunková, J., V. Tesar, A. Sedivá. 2003. Diagnostic and pathogenetic role of antineutrophil cytoplasmic autoantibodies. Clin. Immunol. 106:73.[Medline]
  22. Novo, E., E. G.-M. Gregor, S. Nava, L. Perini. 1997. A possible defective estimation of antineutrophil cytoplasmic antibodies in systemic lupus erythematosus due to the coexistence of periodontitis: preliminary observations. P. R. Health Sci. J. 16:369.[Medline]
  23. Novo, E., N. Viera. 1996. Antineutrophil cytoplasmic antibodies: a missing link in the pathogenesis of periodontal disease?. J. Periodont. Res. 31:365.[Medline]
  24. Déry, O., C. U. Corvera, M. Steinhoff, N. W. Bunnett. 1998. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am. J. Physiol. 274:C1429.
  25. Coughlin, S. R.. 2000. Thrombin signalling and protease-activated receptors. Nature 407:258.[Medline]
  26. O’Brien, P. J., M. Molino, M. Kahn, L. F. Brass. 2001. Protease activated receptors: theme and variations. Oncogene 20:1570.[Medline]
  27. Uehara, A., S. Sugawara, K. Muramoto, H. Takada. 2002. Activation of human oral epithelial cells by neutrophil proteinase 3 through protease-activated receptor-2. J. Immunol. 169:4594.[Abstract/Free Full Text]
  28. Uehara, A., K. Muramoto, H. Takada, S. Sugawara. 2003. Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor-2. J. Immunol. 170:5690.[Abstract/Free Full Text]
  29. Galanos, C., O. Lüderitz, O. Westphal. 1979. Preparation and properties of a standardized lipopolysaccharide from Salmonella abortus equi (Novo-Pyrexal). Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. Reihe A 243:226.
  30. Momose, F., T. Araida, A. Negishi, H. Ichijo, S. Shioda, S. Sasaki. 1989. Variant sublines with different metastatic potentials selected in nude mice from human oral squamous cell carcinomas. J. Oral Pathol. Med. 18:391.[Medline]
  31. Miyauchi, S., T. Moroyama, T. Sakamoto, T. Okamoto, K. Takada. 1985. Establishment of human tumor cell line (Ueda-1) derived from squamous cell carcinoma of the floor of the mouth. Jpn. J. Oral Maxillofac. Surg. 31:1347.
  32. Eagle, H.. 1955. Propagation in a fluid medium of a human epidermoid carcinoma, strain KB. Proc. Soc. Exp. Biol. Med. 89:362.
  33. Sugawara, S., A. Uehara, T. Nochi, T. Yamaguchi, H. Ueda, A. Sugiyama, K. Hanzawa, K. Kumagai, H. Okamura, H. Takada. 2001. Neutrophil proteinase 3-mediated induction of bioactive IL-18 secretion by human oral epithelial cells. J. Immunol. 167:6568.[Abstract/Free Full Text]
  34. Hirata, R. K., S.-T. Chen, S. C. Weil. 1993. Expression of granule protein mRNAs in acute promyelocytic leukemia. Hematol. Pathol. 7:225.[Medline]
  35. Storck, J., B. Küsters, M. Vahland, C. Morys-Wortmann, E. R. Zimmermann. 1996. Trypsin induced von Willebrand factor release from human endothelial cells is mediated by PAR-2 activation. Thromb. Res. 84:463.[Medline]
  36. Uehara, A., S. Sugawara, H. Takada. 2002. Priming of human oral epithelial cells by interferon-{gamma} to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans. J. Med. Microbiol. 51:626.[Abstract/Free Full Text]
  37. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
  38. Campbell, E. J., M. A. Campbell, C. A. Owen. 2000. Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition. J. Immunol. 165:3366.[Abstract/Free Full Text]
  39. Ghosh, S., R. Brown, J. C. R. Jones, S. M. Ellerbroek, M. S. Stack. 2000. Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by {alpha}3{beta}1 integrin in oral keratinocytes. J. Biol. Chem. 275:23869.[Abstract/Free Full Text]
  40. Firth, J. D., E. S. Sue, E. E. Putnins, D. Oda, V.-J. Uitto. 1996. Chymotrypsin-like enzyme secretion is stimulated in cultured epithelial cells during proliferation and in response to Actinobacillus actinomycetem comitans. J. Periodont. Res. 31:345.[Medline]
  41. Koshikawa, N., S. Hasegawa, Y. Nagashima, K. Mitsuhashi, Y. Tsubota, S. Miyata, Y. Miyagi, H. Yasumitsu, K. Miyazaki. 1998. Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse. Am. J. Pathol. 153:937.[Abstract/Free Full Text]
  42. Shpacovitch, V. M., T. Brzoska, J. Buddenkotte, C. Stroh, C. P. Sommerhoff, J. C. Ansel, K. Schulze-Osthoff, N. W. Bunnett, T. A. Luger, M. Steinhoff. 2002. Agonists of proteinase-activated receptor 2 induce cytokine releas