Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2 This information is current as Akiko Uehara, Shunji Sugawara, Koji Muramoto and of October 2, 2021. Haruhiko Takada J Immunol 2002; 169:4594-4603; ; doi: 10.4049/jimmunol.169.8.4594 http://www.jimmunol.org/content/169/8/4594 Downloaded from References This article cites 52 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/169/8/4594.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-21 Akiko Uehara,* Shunji Sugawara,2* Koji Muramoto,† and Haruhiko Takada* 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 Downloaded from -a synthetic PAR-2 agonist peptide induced intracellular Ca2؉ mobilization, and rendered cells refractory to subsequent stimu lation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein-coupled PAR-2 and actively participates in the process of inflammation such as periodontitis. The Journal of Immunol- ogy, 2002, 169: 4594–4603. http://www.jimmunol.org/ he four members of the protease-activated receptor is considered that they are involved in several pathophysiological pro- (PAR)3 family are G protein-coupled receptors character- cesses, including growth, development, and inflammation. T ized by a proteolytic cleavage of the N terminus that ex- PR3 is a 29-kDa serine proteinase with homology to HLE and poses tethered ligands and autoactivates the receptor function (1– Cat G, all of which are stored in azurophil granules of neutrophils 3). Three of them (PAR-1, PAR-3, and PAR-4) are activated (11–13). PR3 also presents on the cell surface and within secretory mainly by thrombin; the fourth (PAR-2) is activated by other and specific granules of neutrophils, and exposure of neutrophils to serine proteinases such as trypsin, tryptase, and factor Xa (1–3). It cytokines or chemoattractants induces an increase in cell surface- is reported that more than one family member can be present in the bound PR3 (14). PR3 is also expressed by monocytes, basophils, by guest on October 2, 2021 same cell: human platelets express PAR-1 and PAR-4 (4, 5); and mast cells (15). PR3 is a major target Ag of anti-neutrophil mouse platelets express PAR-3 and PAR-4 (6); human endothelial cytoplasmic Abs in Wegener’s granulomatosis, a debilitating au- cells express PAR-1, PAR-2, and possibly PAR-3 (7, 8); and hu- toimmune disease characterized by necrotizing vasculitis (15, 16). man oral epithelial cells express PAR-1, PAR-2, and PAR-3 (9). It has recently been shown that PR3 exhibits many biological func- PAR-1 is the most widely expressed receptor among PAR family tions, including the degradation of extracellular matrix proteins members in humans and mice, and it is reported that PAR-1 on (13), regulation of myeloid differentiation (12, 17), enhancement human platelets and endothelial cells is inactivated by neutrophil of TNF-␣ and IL-1␤ release from human monocytic cell lines (18), serine proteinases, human leukocyte elastase (HLE), cathepsin G production of IL-8 and monocyte chemoattractant protein-1 (Cat G), and proteinase 3 (PR3), by cleavage downstream of the teth- (MCP-1) by human endothelial cells (19, 20), and antibacterial ered ligand (10). Because PARs are expressed in a variety of cells, it action (21), all of which indicate that cell-bound and secreted sol- uble PR3 actively contribute to inflammatory processes, although the underlying mechanism is unclear to date. *Department of Microbiology and Immunology, School of Dentistry, and †Laboratory Oral epithelial cells are the first cells encountered by bacteria in of Biomolecular Function, Graduate School of Life Sciences, Tohoku University, the periodontal tissues. In addition to acting as a physical barrier Sendai, Japan against the invasion of pathogenic organisms, oral (gingival) epi- Received for publication April 29, 2002. Accepted for publication August 15, 2002. thelial cells in inflamed regions appear to express several proin- The costs of publication of this article were defrayed in part by the payment of page flammatory cytokines such as IL-1␤, IL-6, IL-8, TNF-␣, and charges. This article must therefore be hereby marked advertisement in accordance ␤ with 18 U.S.C. Section 1734 solely to indicate this fact. TGF- 1 (22), implying that the cells actively participate in the 1 This work was supported in part by Grants-in-Aid for Scientific Research from the initiation and development of chronic oral inflammation such as Japan Society for the Promotion of Science (12470380 and 13671894). periodontitis. However, only a few studies have demonstrated cy- 2 Address correspondence and reprint requests to Dr. Shunji Sugawara, Department of tokine production by human gingival epithelial cells and related Microbiology and Immunology, Tohoku University School of Dentistry, 4-1 Seiryo- cell lines derived from the oral cavity in response to oral bacteria machi, Aoba-ku, Sendai 980-8575, Japan. E-mail address: [email protected]. tohoku.ac.jp (23–25). In contrast to human colonic epithelial cells, human oral 3 ␣ ␣ epithelial cells are shown to be basically unresponsive to many Abbreviations used in this paper: PAR, protease-activated receptor; 1-AT, 1- antitrypsin; Boc-Ala-ONp, Boc-Ala-p-nitrophenyl ester; Cat G, cathepsin G; CDS, bacterial cell surface components, even in the presence of the sol- cell dissociation solution; EM, extracellular medium; GCF, gingival crevicular fluid; HLE, human leukocyte elastase; MCP-1, monocyte chemoattractant protein-1; PLC, uble form of CD14, a bacterial pattern recognition receptor (26). phospholipase C; PR3, proteinase 3. We have recently shown that oral epithelial cells constitutively Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 4595 ␣ ␣ express a 24-kDa precursor form of IL-18, and that human neu- teinase inhibitors, Pefabloc SC and 1-antitrypsin ( 1-AT), and FCS for 30 trophil PR3 induced the secretion of a bioactive IL-18 from the min at 37°C before use. Cultivation was conducted in triplicate, and levels epithelial cells in combination with LPS after priming with IFN-␥ of IL-8 and MCP-1 in the supernatants were measured using OptEIA ELISA kits (BD PharMingen, San Diego, CA). The concentrations of the (27). We extended the investigation of the underlying mechanism cytokines in the supernatants were determined using the Softmax data anal- by which PR3 activates the epithelial cells, and in the present ysis program (Molecular Devices, Menlo Park, CA). study, we obtained the evidence for the first time that PR3 by itself could activate the epithelial cells in culture to induce production of Flow cytometry IL-8 and MCP-1 and expression of ICAM-1 via the PAR-2 Flow cytometric analyses were performed using a FACScan cytometer pathway. (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 ␮g/ml cycloheximide for6hat37°C. Then cells were stimulated with or Materials and Methods without PR3 (10 ␮g/ml) for up to 24 h in the presence or absence of Reagents cytochalasin B (30 nM) or cycloheximide (1 ␮g/ml) at 37°C. After the incubation, cells were collected by nonenzymatic CDS and washed in PBS. Purified human neutrophil PR3 was obtained from HyTest (Turku, Finland) Cells were stained with anti-CD54, anti-PAR-1, anti-PAR-2, or control IgG and from Elastin Products (Owensville, MO). Purified HLE, Cat G, phos- at 4°C for 30 min, followed by FITC-conjugated goat anti-mouse IgG pholipase C (PLC) inhibitor U73122, and control compound U73343 were (BioSource International, Camarillo, CA) at 4°C for a further 30 min.