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* Department of Microbiology and Immunology, Graduate School of Dentistry and
Laboratory of Biomolecular Function, Graduate School of Life Sciences, Tohoku University, Sendai, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophil-derived serine proteinases, human leukocyte elastase (HLE; EC 3.4.21.37), cathepsin G (Cat G; EC 3.4.21.20), and proteinase 3 (PR3; EC 3.4.21.76) are stored in the azurophilic granules of neutrophils as active enzymes. The major physiological function of the proteinases is commonly thought to be the intralysosomal degradation of engulfed cell debris or microorganisms (5, 6, 7). It has become evident that HLE, Cat G, and PR3 play crucial roles in extracellular proteolytic processes at sites of inflammation. Over the past few years, evidence, however, has accumulated that various proteins are also natural substrates of these proteases (5, 6, 7). Furthermore, we have recently shown that PR3 activates oral epithelial cells through PAR-2, although HLE and Cat G were less effective in the activation of the epithelial cells (8).
Human secretory leukocyte protease inhibitor (SLPI) is an 11.7-kDa polypeptide originally isolated from parotid saliva (9). SLPI is considered as a product of epithelial cells, including oral epithelial cells, that maintains activity against leukocyte serine proteinases in the protected space (10, 11). In addition, macrophages and neutrophils from nonepithelial tissues also express SLPI (12, 13, 14, 15, 16). SLPI functions as an inhibitor of serine proteinases including HLE and Cat G but does not inhibit PR3 (5). These observations raised the possibility that SLPI from human oral epithelial cells may account for PR3-specific activation of the epithelial cells via PAR-2 (8) and led to the hypothesis that HLE and Cat G also have the ability to activate nonepithelial cells in the same manner as PR3. We therefore examined the hypothesis using human gingival fibroblasts (HGF) as nonepithelial cells. HGF are the major constituents of periodontal tissues and produce various inflammatory cytokines such as IL-1, IL-6, and IL-8 upon stimulation with bacteria and their components (17, 18, 19). These findings imply that HGF actively participate in the initiation and development of chronic oral inflammation such as periodontitis.
The present study showed for the first time that HLE and Cat G as well as PR3 activated nonepithelial HGF to induce production of IL-8 and monocyte chemoattractant protein 1 (MCP-1) via the PAR-2 pathway.
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Materials and Methods |
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Purified human neutrophil PR3 was obtained 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 was 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, as previously described (8). Goat anti-human SLPI polyclonal Ab and rSLPI were obtained by R&D Systems (Minneapolis, MN). 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) and 
1-antitrypsin (1-AT) were obtained from Sigma-Aldrich (St. Louis, MO). Cell permeant fura 2-acetoxymethyl ester 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
HGF were prepared from the explants of normal gingival tissues of 6- to 10-year-old patients under informed consent, which also had been given by the parents because of the age of the donors (18). The explants were cut into pieces and cultured in 100-nm-diameter tissue culture dishes (Falcon; BD Labware, Lincoln Park, NJ) in -MEM supplemented with 10% heat-inactivated FCS with a medium change every 3 days for 10–15 days until confluent cell monolayers were formed. After three to four subcultures by CDS, homogeneous, slim, spindle-shaped cells grown in characteristic swirls were obtained. The cells were used as confluent monolayers at subculture levels 2 through 7. The human oral epithelial cell lines HSC-2 (20), HO-1-u-1 (21), and KB (22), established from squamous cell carcinoma, were obtained from the Cancer Cell Repository, Institute of Development, Aging and Cancer (Tohoku University (HSC-2 and HO-1-u-1, Sendai, Japan) and from the Health Science Research Resources Bank (KB; Tokyo, Japan). HSC-2 and HO-1-u-1 were grown in RPMI 1640 with 10% FCS (Life Technologies, Grand Island, NY) and KB was grown in -MEM with 10% FCS with a medium change every 3 days. The experimental procedure was approved by the Ethical Review Board (Tohoku University Graduate School of Dentistry).
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 (23). The isolated PBMCs were washed three times with PBS at 4°C. The viability of these cells was >98%, as judged by trypan blue dye exclusion.
Measurement of cytokines
Confluent HGF 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). After incubation for 1 day at 37°C in a 5% CO2 incubator, the cells were stimulated with test materials in 200 µl of the medium without serum for a given period. To inhibit the enzymatic activity of serine proteinases, it was preincubated with 1-AT or rSLPI 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).
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 of total RNA in a 50-µl reaction vol, and all PCR procedures were performed in a 20-µl vol as described previously (8). The primers used for PCR were as follows: SLPI, 5'-ATGAAGTCCAGCGGCCTCTT-3', 5'-ATGGCAGGAATCAAGCTTTC-3' (24); PAR-1, 5'-TGTGAACTGATCATGTTTATG-3', 5'-TTCGTAAGATAAGAGATATGT-3' (25); PAR-2, 5'-GCAGCCTCTCTCTCCTGCAGTGG-3', 5'-CTTGCATCTGCTTTACAGTGCG-3' (26); PAR-3, 5'-ATAACGTTTAAGAGACGGGACT-3', 5'-TAGCAGTAGATGATAAGCACA-3' (27); PAR-4, 5'-GACGAGAGCGGGAGCACC-3', 5'-CCCGTAGCACAGCAGCATGG-3' (28); and GAPDH, 5'-CTACAATGAGCTGCGTGTGG-3', 5'-AAGGAAGGCTGGAAGAGTGC-3' (23). The primers for SLPI, PAR-1, PAR-2, PAR-3, PAR-4, and GAPDH were constructed to generate fragments of 408, 708, 1,066, 858, 725, and 527 bp, respectively. Cycling conditions were as follows: SLPI, 35 cycles at 94°C for 1 min, 54°C for 1 min, and 72°C for 3 min; 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.
Immunoblot analysis
Cell pellets (3 x 105 cells each) were used for the detection of SLPI. All samples were solubilized with Laemmli sample buffer (29). SDS-PAGE was performed in a 15% polyacrylamide slab gel containing 0.1% SDS under reducing conditions according to the method of Laemmli (29). Proteins were transferred to a polyvinylidene difluoride membrane by a semidry system (ATTO Instruments, Tokyo, Japan). The blot was blocked for 2 h with 1% w/v nonfat dry milk, 1% w/v BSA, and 0.05% Tween 20 in PBS (Blotto/Tween) and incubated with anti-SLPI polyclonal Ab at 2 µg/ml in Blotto/Tween overnight at 4°C. The blot was washed four times with Blotto/Tween and then incubated for 2 h with HRP-conjugated affinity-purified rabbit anti-goat IgG at 1/2000 (Jackson ImmunoResearch Laboratories, West Grove, PA) in Blotto/Tween. After being washed, SLPI was visualized with diaminobenzidine in the presence of 0.03% CoCl2. The Mr of the proteins was estimated by comparison with the position of a standard (Bio-Rad, Hercules, CA).
Flow cytometry
Flow cytometric analyses were performed using a FACScan cytometer (BD Biosciences, Mountain View, CA) as described elsewhere (8). HGF were stimulated with or without HLE or Cat G (10 µg/ml) for up to 24 h at 37°C. After the incubation, cells were collected by nonenzymatic CDS and washed in PBS. Cells were stained with 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 an additional 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 another 30 min. To calculate the percentage of positive cells, the baseline cursor was set at a channel that yielded <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.
Analysis of peptide cleavage
A peptide corresponding to a region spanning the cleavage site of the PAR-2, residues 32–45 (32SSKGRSLIGKVDGT45) (30), was synthesized by Takara. The peptide (200 µM) was incubated with proteinases for 30 min at 37°C in 10 mM Tris-HCl (pH 8.0). 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 (31).
Calcium mobilization
Confluent HGF 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, and 0.1% w/v BSA, pH 7.3). Cells were loaded with 1 µM fura 2-acetoxymethyl ester 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 intracellular Ca2+ concentration, was calculated according to a previously described procedure (8).
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently reported that neutrophil PR3 activates human oral epithelial cells through PAR-2 (8). Since PR3 has high homology with two other well-characterized neutrophil serine proteinases, HLE and Cat G (5), we examined whether the three proteinases have the ability to activate nonepithelial cells using HGF, a major constituent of gingival connective tissues. In contrast to the epithelial cells, which mainly responded to PR3, but not to HLE and Cat G (8), incubation of HGF with HLE and Cat G for 24 h resulted in a significant increase in the production of IL-8 and MCP-1 with the same potency as PR3 (Fig. 1A). SLPI is an inhibitor of HLE and Cat G but not PR3 and is mainly expressed in epithelial cells (10). Therefore, we examined the expression of SLPI in oral epithelial cells and HGF. Oral epithelial cells expressed SLPI mRNA (Fig. 1B, lane 3) and an 11.7-kDa SLPI protein (Fig. 1C, lanes 4–6), but HGF did not express SLPI in mRNA (Fig. 1B, lane 2) or protein form (Fig. 1C, lanes 1--3). HLE-, Cat G-, and PR3-induced MCP-1 production was significantly inhibited by pretreatment with a naturally occurring serine proteinase inhibitor, 1-AT (32) (Fig. 1D). The results indicate that enzymatic activities of the proteinases are critical to the cell activation, as described previously (8, 23). Pretreatment of the proteinases with rSLPI clearly inhibited the production of MCP-1 from HGF induced by HLE and Cat G, but not by PR3 (Fig. 1D). These results indicate that HLE and Cat G as well as PR3 have the ability to activate nonepithelial cells, and HLE and Cat G could not activate the epithelial cells, probably due to the expression of SLPI by the cells.
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We next examined the expression of the PAR family in HGF. HGF in culture expressed PAR-1 and PAR-2 mRNA but not PAR-3 and PAR-4 mRNA as assessed by RT-PCR analysis (Fig. 2A). Furthermore, PAR-1 and PAR-2 were clearly expressed on the cell surface, but the expression of PAR-3 on the cell surface could not be detected by flow cytometric analyses (Fig. 2B). It must be mentioned that the expression of PAR-1 and -2 was not affected after the cells were exposed to HLE and Cat G for 1 h (Fig. 2B) and after the longer incubation with HLE and Cat G for up to 24 h (data not shown).
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These results suggest that HLE and Cat G cleave PAR-2 at a specific site with exposure of its tethered ligand, as in the case of PR3 (8). To examine this possibility, a peptide corresponding to the region surrounding the cleavage site of the human PAR-2 (Fig. 3A) was incubated with HLE and Cat G and proteolytic fragments were analyzed. Trypsin, an agonist for PAR-2 (1, 2, 3, 4), was used as a positive control. The PAR-2 peptide was rapidly cleaved at the site, R36-S37 by 5 nM HLE, Cat G, or trypsin (Fig. 3B). The major peptide fragment was identified to be SLIGKVDGT, the PAR-2 tethered ligand, by sequencing, which was the same result as with PR3 (8). The measured molecular mass (HLE, 890.7; Cat G, 890.9; trypsin, 890.5) 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. In contrast, thrombin, which does not activate PAR-2 (1, 2, 3, 4), did not cleave the PAR-2 peptide.
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To further examine whether HLE and Cat G activate HGF through PAR-2, Ca2+ mobilization in the cells was measured on exposure of HGF to HLE, Cat G, trypsin, and synthetic PAR-2 agonist peptide SLIGKV. HLE and Cat G induced a Ca2+ response and abolished the response to a second application of the same proteinase (Fig. 4, A and E) as well as the PAR-2 agonist peptide (Fig. 4, B and F) and trypsin, known to PAR-2-activating serine proteinase (Fig. 4, C and G). Trypsin stimulation rendered cells refractory to subsequent stimulation with HLE or Cat G (Fig. 4, D and H) while stimulation with thrombin, known to PAR-1-activating serine proteinase, had no such effect (Fig. 4, I–K). Furthermore, HLE inhibited a subsequent response to Cat G but not to thrombin (Fig. 4L). These results clearly indicated that the HLE- and Cat G-induced activation of HGF are mediated by PAR-2.
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q proteins, resulting in activation of PLC (1, 2, 3, 4). To examine whether PLC is also involved in HLE- and Cat G-induced activation of HGF, HGF were stimulated with HLE and Cat G 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 controls, and the peptides induced production of IL-8 and MCP-1 (Fig. 6). The result indicates that HGF could be activated through both PAR-1 and -2 pathways, which is consistent with the result of the expression of PAR-1 and -2 by the cells (Fig. 2). The inhibition of PLC completely abolished the production of cytokine induced by the agonist peptides, HLE and Cat G, as in the case of PR3 (8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to PR3, two other neutrophil serine proteinases, HLE and Cat G, showed only marginal activity in human oral epithelial cells as assessed by production of inflammatory cytokine (8). In addition, PR3 but not HLE and Cat G induced production of bioactive IL-18 from the epithelial cells on costimulation with LPS after IFN-
priming (23). Nonepithelial HGF, however, were equally activated by the three neutrophil serine proteinases (Fig. 1A). These observations led to the question of which factor causes the different responses between the epithelial and nonepithelial cells. Host tissues are protected from unregulated proteolysis by HLE, Cat G, and other proteinases by multiple proteinase inhibitors. It is noteworthy that there is at least one proteinase inhibitor, SLPI, which is able to gain access to the protected space. SLPI is present in many epithelial secretions (10) and thought to be one of the major inhibitors of serine proteinases including HLE and Cat G but not PR3 (5), protecting the epithelium from attack by the inflammatory cells. The present study showed that human oral epithelial cells but not HGF constitutively express SLPI mRNA by RT-PCR (Fig. 1B) and the 11.7-kDa form of SLPI protein by immunoblot analysis (Fig. 1C), and that rSLPI inhibited the production of MCP-1 induced by HLE and Cat G, but not by PR3 (Fig. 1D). These results indicate that the ineffectiveness of HLE and Cat G on oral epithelial cells was due to SLPI secreted by the cells and that the three neutrophil serine proteinases have basically a similar ability to activate human nonepithelial cells. In support of this, Fig. 5 showed that potency and efficacy are basically the same among these three serine proteinases, as assessed by intracellular calcium mobilization in HGF.
Skin-derived antileukoproteinase (SKALP), also known as elafin, was first discovered in keratinocytes from hyperproliferative human epidermidis (33, 34) and is a potent epithelial-specific inhibitor of PR3 as well as HLE (35). With regard to SKALP/elafin, a previous report showed that keratinizing gingival epithelium was moderately expressed SKALP/elafin in three or four of the most differentiated layers but staining was never seen in the other layer of the epithelium by immunohistochemistry (36). In support of this, primary oral epithelial cells and oral epithelial cell lines, KB, HSC-2, and HO-1-u-1 cells, which were used for our previous (8) and this study, did not express SKALP/elafin mRNA as assessed by RT-PCR (data not shown). HGF also did not express SKALP/elafin mRNA by RT-PCR (data not shown), which is consistent with a previous study (37). The observations exclude the possibility that SKALP/elafin participates in the neutrophil serine proteinase-mediated activation of oral epithelial cells and HGF.
Because HLE, Cat G, and PR3 belong to a group of endoproteolytic enzymes with a comparatively broad substrate specificity (38, 39, 40), it is widely held that these enzymes are capable of cleaving nearly all proteins in an unspecific manner. With regard to the effect of neutrophil serine proteinases on the PAR family, it is reported that PAR-1 on human platelets and endothelial cells is inactivated by HLE, Cat G, and PR3 by cleavage downstream of the tethered ligand (41) and that HLE and Cat G inactivate human PAR-3 (42). It is also predicted that HLE, Cat G, and PR3 are able to inactivate human PAR-2 by cleavage downstream of the tethered ligand using the recombinant receptors (43). However, the report (43) drew this prediction based on observations at an enzyme/substrate ratio of 1:800, which was much higher than in our study (1:4000; Fig. 3B). Furthermore, we did not observe a reduction in PAR-1 or PAR-2 on the surface of HGF after the treatment with HLE and Cat G (Fig. 2B), in contrast to the previous report using human platelets (41). Although it was reported that PR3 has an elastase-like specificity for Ala, Ser, and Val at the P1 site (5, 40), our recent study showed that PR3 cleaves the PAR-2-tethered ligand between Arg36 and Ser37 to activate human oral epithelial cells (8), and the present study showed that HLE and Cat G clearly and rapidly cleaved between Arg36 and Ser37, which was the same specificity as trypsin and PR3 (Fig. 3B) (8). These results indicate that the Arg36-Ser37 site and surrounding area of PAR-2 are structurally accessible to neutrophil serine proteinases and that the sensitivity of the PAR family to neutrophil serine proteinases may differ among cell types.
The inflamed site is characterized by an infiltration of neutrophils. Infiltration of neutrophils into gingival tissues is an early event in gingival inflammation (44). Previous studies clearly show that HLE and Cat G are found in gingival tissues and these proteinases increase in inflamed gingival tissues during periodontal diseases (45, 46, 47). The concentration of HLE and Cat G and PR3 in neutrophils exceeds 5 mM (48) and 13 mM (42), respectively. When neutrophils are activated by stimulants such as TNF-, platelet-activating factor, FMLP, and IL-8, HLE, Cat G, and PR3 are rapidly released from cytoplasmic granules into the extracellular space, and the activation of neutrophils resulted in an increase in the membrane-bound form of the proteinases on the cell surface (32, 49, 50). The membrane-bound proteinases are catalytically active and are remarkably resistant to inhibition by naturally occurring proteinase inhibitors (32, 49, 50). We have recently shown that activation of human oral epithelial cells by FMLP-stimulated neutrophils was only partially inhibited by serine proteinase inhibitors and serum in the coculture system (8). These results suggest that activation of nonepithelial cells by neutrophil serine proteinases is likely to occur in vivo. The present study showed that HGF are activated to produce IL-8 and MCP-1 in response to HLE and Cat G 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 (51). 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 (52, 53). Therefore, PAR-2-mediated activation of HGF by neutrophil serine proteinases, which are released by activated neutrophils, may play an important role in innate immunity against periodontal pathogens in controlling Th1 and Th2 responses at the periodontitis site.
PAR-2 is present in many types of cells including smooth muscle of vascular and nonvascular origin, stromal cells from a variety of tissues, endothelial and epithelial cells independent of tissue type, and throughout the gastrointestinal tract (54). The expression of PAR-2 on human endothelial and epithelial cells was up-regulated by the activation of cells without an effect on PAR-1 expression (23, 55). Another study demonstrated that PAR-2 expression was significantly increased in asthmatic bronchial epithelium in comparison to normal epithelium (56). Furthermore, it has been recently shown that PAR-2 deficiency in mice attenuated allergic dermatitis (57), delayed the onset of inflammation (58), and decreased eosinophil infiltration and hyperreactivity in allergic inflammation of the airway (59). These findings suggest that PAR-2 plays a crucial role in the regulation of inflammation. Considering these observations, the present study implies that neutrophil serine proteinases secreted from activated neutrophils at the site of inflammation could regulate the PAR-2 activity, resulting in the control of a number of inflammatory processes. Manipulation of the proteinases might be more beneficial in the regulation of inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shunji Sugawara, Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan. E-mail address: sugawars{at}mail.cc.tohoku.ac.jp
3 Abbreviations used in this paper: PAR, protease-activated receptor; HLE, human leukocyte elastase; Cat G, cathepsin G; PR3, proteinase 3; SLPI, secretory leukocyte protease inhibitor; HGF, human gingival fibroblasts; MCP-1, monocyte chemoattractant protein 1; PLC, phospholipase C; CDS, cell dissociation solution; 1-AT, 1-antitrypsin; EM, extracellular medium; SKALP, skin-derived antileukoproteinase.
Received for publication December 30, 2002. Accepted for publication March 25, 2003.
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 E. Csernok, M. Ai, W. L. Gross, D. Wicklein, A. Petersen, B. Lindner, P. Lamprecht, J. U. Holle, and B. Hellmich Wegener autoantigen induces maturation of dendritic cells and licenses them for Th1 priming via the protease-activated receptor-2 pathway Blood, June 1, 2006; 107(11): 4440 - 4448. [Abstract] [Full Text] [PDF]
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E. Camerer, I. Cornelissen, H. Kataoka, D. N. Duong, Y.-W. Zheng, and S. R. Coughlin Roles of protease-activated receptors in a mouse model of endotoxemia Blood, May 15, 2006; 107(10): 3912 - 3921. [Abstract] [Full Text] [PDF]
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M. Chignard and D. Pidard Neutrophil and Pathogen Proteinases versus Proteinase-Activated Receptor-2 Lung Epithelial Cells: More Terminators than Activators. Am. J. Respir. Cell Mol. Biol., April 1, 2006; 34(4): 394 - 398. [Abstract] [Full Text] [PDF]
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 M. Holzhausen, L. C. Spolidorio, R. P. Ellen, M.-C. Jobin, M. Steinhoff, P. Andrade-Gordon, and N. Vergnolle Protease-Activated Receptor-2 Activation: A Major Role in the Pathogenesis of Porphyromonas gingivalis Infection Am. J. Pathol., April 1, 2006; 168(4): 1189 - 1199. [Abstract] [Full Text] [PDF]
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 E. B. Kelso, J. C. Lockhart, T. Hembrough, L. Dunning, R. Plevin, M. D. Hollenberg, C. P. Sommerhoff, J. S. McLean, and W. R. Ferrell Therapeutic Promise of Proteinase-Activated Receptor-2 Antagonism in Joint Inflammation J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1017 - 1024. [Abstract] [Full Text] [PDF]
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 G. R. Martin and J. L. Wallace Gastrointestinal Inflammation: A Central Component of Mucosal Defense and Repair Experimental Biology and Medicine, February 1, 2006; 231(2): 130 - 137. [Abstract] [Full Text] [PDF]
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 Y. Hirota, Y. Osuga, T. Hirata, M. Harada, C. Morimoto, O. Yoshino, K. Koga, T. Yano, O. Tsutsumi, and Y. Taketani Activation of protease-activated receptor 2 stimulates proliferation and interleukin (IL)-6 and IL-8 secretion of endometriotic stromal cells Hum. Reprod., December 1, 2005; 20(12): 3547 - 3553. [Abstract] [Full Text] [PDF]
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 K. Ikawa, T. Nishioka, Z. Yu, Y. Sugawara, J. Kawagoe, T. Takizawa, V. Primo, B. Nikolic, T. Kuroishi, T. Sasano, et al. Involvement of neutrophil recruitment and protease-activated receptor 2 activation in the induction of IL-18 in mice J. Leukoc. Biol., November 1, 2005; 78(5): 1118 - 1126. [Abstract] [Full Text] [PDF]
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A. Uehara, K. Muramoto, T. Imamura, K. Nakayama, J. Potempa, J. Travis, S. Sugawara, and H. Takada Arginine-Specific Gingipains from Porphyromonas gingivalis Stimulate Production of Hepatocyte Growth Factor (Scatter Factor) through Protease-Activated Receptors in Human Gingival Fibroblasts in Culture J. Immunol., November 1, 2005; 175(9): 6076 - 6084. [Abstract] [Full Text] [PDF]
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G. J. Laurent No Bit PARt for PAR-1 Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 213 - 215. [Full Text] [PDF]
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T. Suzuki, T. J. Moraes, E. Vachon, H. H. Ginzberg, T.-T. Huang, M. A. Matthay, M. D. Hollenberg, J. Marshall, C. A. G. McCulloch, M. T. H. Abreu, et al. Proteinase-Activated Receptor-1 Mediates Elastase-Induced Apoptosis of Human Lung Epithelial Cells Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 231 - 247. [Abstract] [Full Text] [PDF]
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I. Kuwahara, E. P. Lillehoj, A. Hisatsune, W. Lu, Y. Isohama, T. Miyata, and K. C. Kim Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L355 - L362. [Abstract] [Full Text] [PDF]
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S. Tancharoen, K. P. Sarker, T. Imamura, K. K. Biswas, K. Matsushita, S. Tatsuyama, J. Travis, J. Potempa, M. Torii, and I. Maruyama Neuropeptide Release from Dental Pulp Cells by RgpB via Proteinase-Activated Receptor-2 Signaling J. Immunol., May 1, 2005; 174(9): 5796 - 5804. [Abstract] [Full Text] [PDF]
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E. Sokolova, Z. Grishina, F. Buhling, T. Welte, and G. Reiser Protease-activated receptor-1 in human lung fibroblasts mediates a negative feedback downregulation via prostaglandin E2 Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L793 - L802. [Abstract] [Full Text] [PDF]
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 Y. Hirota, Y. Osuga, T. Hirata, K. Koga, O. Yoshino, M. Harada, C. Morimoto, E. Nose, T. Yano, O. Tsutsumi, et al. Evidence for the Presence of Protease-Activated Receptor 2 and Its Possible Implication in Remodeling of Human Endometrium J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1662 - 1669. [Abstract] [Full Text] [PDF]
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 M. Holzhausen, L.C. Spolidorio, and N. Vergnolle Proteinase-activated Receptor-2 (PAR2) Agonist Causes Periodontitis in Rats Journal of Dental Research, February 1, 2005; 84(2): 154 - 159. [Abstract] [Full Text] [PDF]
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A. Uehara, Y. Sugawara, T. Sasano, H. Takada, and S. Sugawara 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-2 J. Immunol., September 15, 2004; 173(6): 4179 - 4189. [Abstract] [Full Text] [PDF]
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 V. S. OSSOVSKAYA and N. W. BUNNETT Protease-Activated Receptors: Contribution to Physiology and Disease Physiol Rev, April 1, 2004; 84(2): 579 - 621. [Abstract] [Full Text] [PDF]
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