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The Journal of Immunology, 2001, 167: 6568-6575.
Copyright © 2001 by The American Association of Immunologists

Neutrophil Proteinase 3-Mediated Induction of Bioactive IL-18 Secretion by Human Oral Epithelial Cells1

Shunji Sugawara2,*, Akiko Uehara*, Tomonori Nochi{dagger}, Takahiro Yamaguchi{dagger}, Haruyasu Ueda{ddagger}, Akiko Sugiyama*, Kazuo Hanzawa§, Katsuo Kumagai, Haruki Okamura{ddagger} and Haruhiko Takada*

* Department of Microbiology and Immunology, School of Dentistry, and {dagger} Department of Animal Production Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; {ddagger} Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; § Seiryo Dental Clinic, Sendai, Japan; and T-Cell Research Institute, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-18, a potent IFN-{gamma}-inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-{gamma}-inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-{gamma}-priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-{gamma}-priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-18 was originally identified as an IFN-{gamma}-inducing factor (1) and is now shown to be a multifunctional cytokine that augments both innate and acquired immunity (2). Its major role is reported to be strong induction of IFN-{gamma} production from Th1 and NK cells in concert with IL-12 (1, 2, 3), but pleiotropy and redundancy, common characteristics of many cytokines, also apply to IL-18 (2). It also induces GM-CSF, IL-4, IL-6, IL-13, and histamine, augments NK activity, and stimulates Fas ligand expression (3, 4, 5, 6). IL-18 is intracellularly produced as a 24-kDa biologically inactive precursor (proIL-18)3 (3) and secreted as an 18-kDa active form after cleavage by caspase-1 (7, 8), originally designated as IL-1{beta}-converting enzyme (9). Recently, it was reported that IL-18 possesses proinflammatory properties, directly inducing the production of TNF-{alpha} by T and NK cells with subsequent release of IL-1{beta} and IL-8 from monocytes (10). IL-18 is produced not only by activated macrophages (1) but also by dermal keratinocytes (11), osteoblasts (12), adrenal cortex cells (13), and intestinal epithelial cells (14, 15), implying that it plays important physiological roles as well as a part in immune regulation.

Proteinase 3 (PR3; 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), all of which are stored in azurophil granules of polymorphonuclear neutrophils (PMNs) (16). PR3 also presents on the cell surface and within secretory and specific granules of PMNs, and exposure of PMNs to cytokines or chemoattractants induces an increase in cell surface-bound PR3 (17). In addition, PR3 is expressed by monocytes, basophils, and mast cells (18). PR3 is a major target Ag of anti-neutrophil cytoplasmic Abs in Wegener’s granulomatosis, a debilitating autoimmune disease characterized by necrotizing vasculitis (18, 19). It has recently been shown that PR3 has many functions, including degradation of extracellular matrix proteins (20), regulation of myeloid differentiation (21, 22), platelet activation (23), induction of apoptosis (24, 25), and enhancement of TNF-{alpha} and IL-1{beta} release (26). It is also reported that PR3 interacts with a 111-kDa membrane molecule of HUVEC (27), enhances IL-8 production by the cells (28), and exhibits antibacterial action (29) that is independent of its enzymatic action. Thus, these findings suggest that cell-bound and secreted soluble PR3 actively contribute to inflammatory processes.

Oral epithelial cells are thought to act as a physical barrier against the entry of pathogenic organisms. In addition, inflamed oral (gingival) epithelial cells appear to express several proinflammatory cytokines, such as IL-1{beta}, IL-6, IL-8, TNF-{alpha}, and TGF-{beta}1 (30), implying that the cells potentially participate in the initiation and development of oral chronic inflammations, e.g., periodontitis. Considering the biological role and tissue distribution of IL-18, we hypothesized that oral epithelial cells express IL-18 and secrete the active form at the site of inflammation, and that neutrophil serine proteinases are involved in this process because the inflamed site is characterized by the infiltration of PMNs. In this study, we obtained evidence for the first time that human oral epithelial cells constitutively express IL-18 as a 24-kDa proIL-18, and revealed that the secretion of bioactive IL-18 from the epithelial cells was induced by combined treatment with PR3 and LPS after IFN-{gamma}-priming via a caspase-1-independent pathway.


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

Human neutrophil PR3 was obtained from HyTest (Turku, Finland). HLE and Cat G were obtained from Calbiochem-Novabiochem (La Jolla, CA). Purity of the three enzymes was >95% by SDS-PAGE, according to the manufacturer. Cell-permeable Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO), a caspase-1 inhibitor, and cycloheximide (CHX) were purchased from Biomol (Plymouth Meeting, PA). Z-Val-Ala-Asp-fluoromethyl ketone (ZVAD-FMK), a caspase family protease inhibitor, and a mAb against the active form of human IL-18 125-2H (mouse IgG1) were obtained from Medical & Biological Laboratories (Nagoya, Japan). Human natural IFN-{alpha}, natural IFN-{gamma}, rIL-18, and anti-human IL-18 mAb 2-10C (mouse IgG1) were kindly provided by Hayashibara Biochemical Laboratories (Okayama, Japan). Human rIFN-{beta} and rIL-12 were kindly provided by Toray Silicon (Tokyo, Japan) and by M. Kobayashi (Genetics Institute, Cambridge, MA), respectively. An ultrapurified LPS prepared from Salmonella enterica serovar abortus-equi (Novo-Pyrexal) (31) was a gift from C. Galanos (Max Planck Institut für Immunbiologie, Freiburg, Germany). Boc-Ala-p-nitrophenyl ester (Boc-Ala-ONp) was obtained from Bachem (Bubendorf, Switzerland). A low toxic serine proteinase inhibitor, Pefabloc SC, was obtained from Roche Diagnostics (Mannheim, Germany). Rabbit anti-human PR3 polyclonal Ab was obtained from Elastin Products (Owensville, MO). Isotype control mouse mAb was purchased from Beckman Coulter (Miami, FL) and dialyzed against PBS. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.

Immunohistochemistry

Human gingival tissues were obtained from adult periodontitis patients undergoing periodontal surgery with informed consent. Immunohistochemistry was performed as described previously (32). Briefly, the tissues were quickly frozen in acetone dry ice at -70°C, and cryostat sections, 5 µm thick, were prepared from the frozen tissues. The sections were incubated with 5 µg/ml of anti-IL-18 mAb 2-10C for 14 h at 4°C. After three washes with PBS, FITC-conjugated goat anti-mouse IgG diluted at 1/400 was added and incubated at room temperature for 45 min. To investigate the specificity of immunostaining, the primary Ab was replaced by an irrelevant mouse IgG at the same protein concentration. Nuclei were stained with propidium iodide. The immunoreactivity was observed using a confocal laser microscope (Bio-Rad, Hercules, CA). Normal and inflamed regions of the specimens were pathologically confirmed.

Cells and cell lines

Human gingival epithelial cells were prepared from explants of normal human gingival tissues with informed consent as described previously (33). In brief, the explants were cut into pieces and cultured in keratinocyte serum-free medium (Life Technologies, Grand Island, NY) containing bovine pituitary extract (0.05%) and recombinant human epidermal growth factor (820 µM) supplemented with kanamycin (200 µg/ml) with a medium change every 3–5 days until confluent cell monolayers were formed. Human oral epithelial cell lines HSC-2, HO-1-u-1, and KB, 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% heat-inactivated FCS (Life Technologies). KB was grown in {alpha}-MEM with 10% FCS.

Human PBMCs were isolated from heparinized peripheral blood of healthy adult donors by Lympholyte-H (Cedarlane Laboratories, Hornby, Ontario, Canada) gradient centrifugation at 800 x g for 20 min at room temperature (34). The isolated PBMCs were washed three times with PBS and suspended in RPMI 1640 medium.

RNA extraction and RT-PCR

Total cellular RNA was prepared from oral epithelial cells with Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Random hexamer-primed reverse transciption was performed on 2.5 µl of total RNA in a 50-µl reaction volume, and all PCR procedures were performed in a 20-µl volume as described previously (34). The primers used for PCR had the following sequences: IL-18, 5'-GCTTGAATCTAAATTATCAGTC-3', 5'-GAAGATTCAAATTGCATCTTAT-3'; and human GAPDH, 5'-CATCACCATCTTCCAGGAGC-3', 5'-CATGAGTCCTTCCACCATACC-3'. Cycling conditions were as follows: with IL-18, 35 cycles at 94°C for 2 min, 55°C for 1 min, and 72°C for 1 min for amplifying a 342-bp product; and with GAPDH, 35 cycles at 94°C for 30 s, 56°C for 40 s, and 72°C for 2 min for amplifying a 286-bp product. Amplified samples were visualized on 2.0% agarose gels stained with ethidium bromide and photographed under UV light.

Cell treatment for IL-18 production

Primary oral epithelial cells and oral epithelial cell lines (5 x104 cells/0.5 ml) were seeded in the culture medium in wells of 24-well plates (Falcon; BD Labware, Franklin Lakes, NJ). After incubation for 1 day at 37°C in a 5% CO2 incubator, cells were incubated with 1000 IU/ml IFNs for 3 days. After the incubation, three washes of the cells with PBS were followed by the addition of LPS (100 ng/ml, unless otherwise indicated) and PR3, HLE, or Cat G (10 µg/ml each, unless otherwise indicated) in 400 µl of the medium without serum for 24 h at 37°C. For the inhibition of caspases and protein synthesis, IFN-{gamma}-primed cells in 24-well plates were preincubated with given concentrations of caspase inhibitors for 1 h and 1 µg/ml CHX for 6 h, respectively, at 37°C. They were then stimulated with PR3 (10 µg/ml) and LPS (100 ng/ml) for 24 h at 37°C. To inhibit the enzyme activity of PR3, PR3 was preincubated with serine proteinase inhibitors, Pefabloc SC and {alpha}1-antitrypsin ({alpha}1-AT), for 30 min at 37°C before use.

After the incubation, the supernatants were collected and the level of IL-18 in the supernatants was determined with a human IL-18 ELISA kit for determination of active IL-18 (Medical & Biological Laboratories) (35). Then cells were collected with 0.25% trypsin/1 mM EDTA (Life Technologies), washed with PBS, and used for immunoblot analysis.

Preparation of cell membrane and cytoplasm fractions

Oral epithelial cells after the treatment were collected by trypsinization. Cells were suspended in hypotonic buffer (10 mM Tris-HCl (pH 7.4), 1 mM PMSF, 10 µM aprotinin, and 1 mM MgCl2) and incubated on ice for 30 min. Cells were then homogenized in a Dounce homogenizer in 15 strokes, and sucrose was added to a final concentration of 0.25 M. The homogenate was centrifuged at 500 x g for 5 min at 4°C twice to remove nuclei. Supernatants were centrifuged at 15,000 x g at 4°C for 30 min, and pellets were used as cell membrane fractions. Supernatants were concentrated by Vivaspin 2 concentrators (Vivascience, Lincoln, U.K.) at 6,000 x g at 4°C and used as cytoplasm fractions.

Immunoblot analysis

Cell pellets (3 x 105 cells each) and vacuum-dried supernatants (equivalent to 3 x 105 cells each) were used for the detection of IL-18 and caspase-1, and cell membrane and cytoplasm fractions (equivalent to 5 x 105 cells each) were used for the detection of PR3. All samples were solubilized with Laemmli sample buffer (36). SDS-PAGE was performed in a 15% polyacrylamide slab gel containing 0.1% SDS under reducing conditions, according to the method of Laemmli (36). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by a semidry transblot system (Atto Instruments, Tokyo, Japan). The blot was blocked for 2 h with 5% w/v nonfat dry milk and 0.05% Tween 20 in PBS (Blotto/Tween) and incubated with anti-IL-18 mAb 2-10C at 2 µg/ml, rabbit anti-human caspase-1 p10 polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA) at 1/100, or rabbit anti-human PR3 polyclonal Ab at 1/200 in Blotto/Tween overnight with 5% w/v nonfat dry milk at 4°C. The blot was washed four times with Blotto/Tween and then incubated for 2 h with HRP-conjugated affinity-purified goat anti-mouse IgG at 1/2000 (Jackson ImmunoResearch Laboratories, West Grove, PA) or goat anti-rabbit IgG at 1/500 (Santa Cruz Biotechnology) in Blotto/Tween with 5% w/v nonfat dry milk. After being washed, IL-18, caspase-1, and PR3 were 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).

Assay for IFN-{gamma}-inducing activity of IL-18

Assay for IFN-{gamma}-inducing activity of IL-18 was performed as described previously (35). Briefly, human PBMCs (3 x 105 cells/well) were incubated with culture supernatants of oral epithelial cells at 1/4, LPS (25 ng/ml) or PR3 (2.5 µg/ml) with 10 ng/ml of rIL-12 in the presence or absence of 1 µg/ml of anti-IL-18 mAb 125-2H or isotype control mouse IgG1 in 96-well plates (total volume, 200 µl) for 48 h, and the level of IFN-{gamma} in the supernatant was determined with a human IFN-{gamma} ELISA kit (BioSource International, Camarillo, CA).

Measurement of LDH activity

For the quantification of plasma membrane damage, lactate dehydrogenase (LDH) activity in the epithelial cell culture supernatants prepared for IL-18 ELISA was measured with a cytotoxicity detection kit (Roche Diagnostics) according to the manufacturer’s instructions. Percentage of LDH activity in the supernatants was calculated as [(experimental value - LDH activity released from untreated cells) / (maximum releasable LDH activity in the cells by 1% Triton X-100 - LDH activity released from untreated cells)] x100.

Measurement of enzyme activity

Amidolytic activities of proteinases were assayed at 25°C for 10–30 min with 0.625 mM Boc-Ala-ONp in 0.1 M HEPES buffer containing 0.1 M NaCl, 10 mM CaCl2, 0.005% Triton X-100, and 5% DMSO, pH 7.5, as described previously (26, 37). The liberation of p-nitrophenol was monitored at 405 nm using the Softmax data analysis program (Molecular Devices, Menlo Park, CA). One unit of enzyme activity was defined as the liberation of 1 µmol of p-nitrophenol from the substrate per min at 25°C. To inhibit the enzymatic activities of PR3 and HLE, the enzymes were preincubated with Pefabloc SC and {alpha}1-AT for 30 min before use.

Statistical analysis

All of the experiments in this study were conducted at least three times. The data shown are representative results. Experimental values are given as means ± SD. The statistical significance of differences between two means was evaluated by Student’s unpaired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-18 in human oral epithelial cells as a precursor

We first examined whether human oral epithelial cells express IL-18 protein by immunohistochemistry. In the cryosections of gingival tissue from the adult periodontitis patients, IL-18 was detected in the cytoplasm of the cells in the normal gingival stratified squamous epithelium (Fig. 1GoA). In the inflamed region of the section, although many inflammatory cells had infiltrated the lamina propria, the signal of IL-18 staining was comparable to that in the normal region (Fig. 1GoB). Because IL-18 is synthesized as a 24-kDa biologically inactive proIL-18 and converted to an 18-kDa bioactive form by caspase-1 similar to the case of IL-1{beta}, and because the immunohistochemistry could not distinguish between proIL-18 and active IL-18, we next examined which type of IL-18 is expressed using primary oral epithelial cells in culture and the oral epithelial cell lines, KB, HSC-2, and HO-1. All of the cells were found to constitutively express IL-18 mRNA by RT-PCR (Fig. 2GoA) and to constitutively express the 24-kDa proIL-18 but not 18-kDa active form by immunoblot analysis (Fig. 2GoB).



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  		FIGURE 1. Expression of IL-18 protein in human oral epithelial cells. Cryosections of gingival tissue from adult periodontitis patients were stained with the anti-IL-18 mAb 2-10C (green). Cell nuclei were counterstained with propidium iodide (red). Normal (A and B) and inflamed (C and D) regions of the section stained with control mAb (A and C) and expression of IL-18 (B and D). GE, gingival epithelium; LP, lamina propria. Bar = 25 µm.

 


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  		FIGURE 2. Expression of IL-18 mRNA and proIL-18 in primary oral epithelial cells and oral epithelial cell lines. Primary oral epithelial cells in culture (lane 1) and oral epithelial cell lines (lane 2, KB; lane 3, HSC-2; lane 4, HO-1) were collected by trypsinization. A, Total RNA was extracted from the cells, and cDNA was prepared and analyzed for the expression of IL-18 mRNA by RT-PCR. Water control was loaded in lane 5. B, The cell pellets (3 x 105 cells each) were solubilized with Laemmli sample buffer, subjected to 15% SDS-PAGE, and transferred to PVDF membrane. rIL-18 (2 ng) was loaded as a control (lane 5). The blot was probed with anti-IL-18 mAb 2-10C. The results are representative of four different experiments with similar results.

 
Detection of bioactive IL-18 in the culture supernatant of oral epithelial cells treated with PR3 and LPS after IFN-{gamma} priming

The above observation leads to the question of whether bioactive IL-18 is generated in the extracellular supernatant of oral epithelial cells; therefore, the cells were stimulated under various conditions. LPS treatment alone (0.1–100 µg/ml) for 24 h could not generate IL-18 in the supernatants of primary oral epithelial cells and the three oral epithelial cell lines as determined by ELISA (Fig. 3GoA). IFN-{gamma} (1000 IU/ml) treatment for 3 days was also ineffective in generating IL-18. Following the IFN-{gamma} treatment, stimulation with LPS alone (0.1–100 µg/ml) for 24 h was ineffective but, strikingly, stimulation with PR3 (10 µg/ml) in combination with LPS (0.1 µg/ml, the lowest concentration used in this study) for 24 h generated a marked increase in IL-18 in the supernatants of these cells (Fig. 3GoA). This priming by IFN-{gamma} was the most effective in inducing IL-18, and neither IFN-{alpha} nor IFN-{beta} pretreatment generated IL-18 in the supernatants in response to PR3 and LPS (data not shown). The IL-18 ELISA kit used in this study detects only active IL-18 (35). To confirm that IL-18 induced in the supernatants is bioactive, we examined whether the supernatant induced production of IFN-{gamma} by PBMCs in the presence of rIL-12, the principal activity of IL-18 (1). As shown in Fig. 3GoB, the supernatant induced production of IFN-{gamma} along with rIL-12 from PBMCs, and the IFN-{gamma}-inducing activity was completely inhibited by the anti-IL-18 neutralizing mAb 125-2H. rIL-12 at 10 ng/ml alone, and LPS and/or PR3 at the same concentration as in the supernatants of PBMC culture even in the presence of rIL-12 did not induce production of IFN-{gamma} by PBMCs, while stimulation with rIL-18 (0.5 and 1 ng/ml) along with rIL-12 did (Fig. 3GoC), as previously described (1, 2, 35), indicating that the IFN-{gamma}-inducing activity was due to IL-18 in the supernatant. In contrast to PR3, other neutrophil serine proteinases, HLE and Cat G, were ineffective in inducing active IL-18 in the supernatants in combination with LPS after the IFN-{gamma} priming (Fig. 3GoD), indicating that the IL-18-inducing activity was specific to PR3. The results obtained using HSC-2 are depicted in Fig. 3GoD and in additional experiments, except those for Fig. 4GoA, because similar results were obtained from primary oral epithelial cells and two other oral epithelial cell lines.



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  		FIGURE 3. Detection of bioactive IL-18 in the culture supernatant of primary oral epithelial cells and oral epithelial cell lines treated with PR3 and LPS after IFN-{gamma} priming. A, Subconfluent primary oral epithelial cells and oral epithelial cell lines (HSC-2, HO-1, and KB) were incubated in the presence or absence of 1000 IU/ml IFN-{gamma} for 3 days. After being washed, cells were stimulated with LPS at indicated doses and/or PR3 (10 µg/ml) for 24 h and the level of IL-18 in the supernatant was measured by ELISA. PBMCs were incubated with the supernatants in which IL-18 was detected by ELISA in A at 1/4 with or without 10 ng/ml rIL-12 in the presence or absence of 1 µg/ml anti-IL-18 mAb 125-2H or isotype control mouse IgG1 for 48 h (B), or incubated with rIL-18 (0.1 and 1 ng/ml), LPS (25 ng/ml), and/or PR3 (2.5 µg/ml) with or without rIL-12 (10 ng/ml) for 48 h (C), and the levels of IFN-{gamma} in the supernatants were analyzed by ELISA. D, Subconfluent HSC-2 cells were incubated in the presence or absence of IFN-{gamma} (1000 IU/ml) for 3 days. After being washed, cells were stimulated with 10 µg/ml of PR3, HLE, or Cat G in the presence or absence of LPS (100 ng/ml) for 24 h, and the level of IL-18 in the supernatant was determined by ELISA. The results are representative of three different experiments with similar results. ND, Not detected. **, p < 0.01.

 


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  		FIGURE 4. Induction of 18-kDa IL-18 in the cell lysate and supernatant of oral epithelial cells by PR3 in combination with LPS followed by IFN-{gamma} priming. A, LDH activity in the supernatants as in Fig. 3GoA 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 as described in Materials and Methods. ND, Not detected. B, Subconfluent HSC-2 cells were incubated in the presence or absence of IFN-{gamma} (1000 IU/ml) for 3 days. After being washed, cells were stimulated with LPS (100 ng/ml) and/or PR3 (10 µg/ml) for 24 h. The cell pellets (3 x 105 cells each) and vacuum-dried supernatants (equivalent to 3 x 105 cells each) were then solubilized with Laemmli sample buffer, subjected to 15% SDS-PAGE, and transferred to PVDF membrane. The blots were probed with anti-IL-18 mAb 2-10C. rIL-18 (2 ng) was loaded as a control. The results are representative of four different experiments with similar results.

 
Extracellular secretion of active IL-18 from oral epithelial cells on treatment with PR3 and LPS after IFN-{gamma} priming

The above observation raises the question of whether PR3 is involved in induction of the secretion of bioactive IL-18 from the epithelial cells or cleaves proIL-18 directly outside of the cells. To clarify which is the case, we first examined whether the cells become leaky after the PR3 treatment by measuring LDH activity in the culture supernatants. No LDH activity was detected in the supernatants on stimulation with PR3 and LPS for 24 h irrespective of IFN-{gamma} priming (Fig. 4GoA), indicating that the epithelial cell membrane was not damaged after the treatment. PR3 itself had no effect on LDH activity (data not shown). Next, the Mr of IL-18 protein in both the cell lysate and extracellular supernatant after the above treatment was examined by immunoblot analysis. The cells expressed 18-kDa IL-18 with almost the same Mr as rIL-18 in addition to 24-kDa proIL-18 only on costimulation with PR3 and LPS after IFN-{gamma} priming (Fig. 4GoB). Following this treatment, the supernatant contained only the 18-kDa active form, and 24-kDa proIL-18 was not detected in the supernatants after any of the treatments used. IL-18 in the supernatant was markedly increased at 18 and 24 h when 10 µg/ml PR3 was used (Fig. 5GoA), and the generation of IL-18 in the supernatant was completely inhibited by the treatment with CHX (Fig. 5GoB). PR3 at 1 µg/ml had only a marginal effect, and at 0.1 µg/ml, no effect, on the generation of IL-18 from the cells (Fig. 5GoA). PR3 at 3 µg/ml showed variations in the induction of IL-18 (data not shown). In addition, cell-bound proIL-18 on oral epithelial cells was not detected by flow cytometry. These results suggest that the active 18-kDa form of IL-18 was generated in the cells and secreted after the treatment.



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  		FIGURE 5. Time kinetics of IL-18 generation in culture supernatant of oral epithelial cells in response to PR3 and LPS, and inhibition of the IL-18 generation by CHX. A, Subconfluent HSC-2 cells were incubated with IFN-{gamma} (1000 IU/ml) for 3 days. After being washed, cells were stimulated with given concentrations of PR3 and LPS (100 ng/ml) for the time indicated at 37°C. B, The IFN-{gamma}-primed HSC-2 cells were washed and pretreated with or without 1 µg/ml CHX for 6 h at 37°C. Then the cells were stimulated with or without PR3 (10 µg/ml) and LPS (100 ng/ml) for 24 h in the presence or absence of 1 µg/ml CHX. The supernatants were collected and the amount of IL-18 in each supernatant was measured by ELISA. The results are representative of three different experiments with similar results. ND, Not detected.

 
Requirement of PR3 enzymatic activity for PR3-induced secretion of IL-18 by oral epithelial cells

We next examined whether the secretion of IL-18 from the cells induced by PR3 and LPS after IFN-{gamma} priming was due to the enzymatic activity of PR3. It has been reported that the enzymatic activity of PR3 was inhibited by sulfonyl fluoride-type serine proteinase inhibitors such as PMSF and diisopropyl fluorophosphate, and by a naturally occurring serine proteinase inhibitor, {alpha}1-AT (17, 28). Therefore, we first measured the enzymatic activity of PR3 using Boc-Ala-ONp as a substrate with or without a low toxic sulfonyl fluoride-type serine proteinase inhibitor, Pefabloc SC, and {alpha}1-AT. As shown in Fig. 6GoA, PR3 showed substantial enzymatic activity compared with the reference HLE (9.7 and 9.5 U/mg protein, respectively), and both Pefabloc SC and {alpha}1-AT markedly inhibited the activity. Concurrently, both inhibitors completely inhibited the induction of IL-18 by PR3 (Fig. 6GoB), indicating that the enzymatic activity of PR3 is critical to induce secretion of bioactive IL-18 from oral epithelial cells.



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  		FIGURE 6. Requirement of enzymatic activity for PR3-induced secretion of IL-18 by oral epithelial cells. A, PR3 and HLE (1 µg/ml each) were pretreated with or without Pefabloc SC or {alpha}1-AT at the doses indicated for 30 min. Enzymatic activity was quantified using Boc-Ala-ONp, as described in Materials and Methods, and the results are expressed as percent activity. The activities of PR3 and HLE were 9.7 and 9.5 U/mg protein, respectively. **, p < 0.01 compared with enzyme alone. B, Subconfluent HSC-2 cells were incubated with IFN-{gamma} (1000 IU/ml) for 3 days. After being washed, cells were stimulated with PR3 (10 µg/ml) and LPS (100 ng/ml) for 24 h at 37°C. PR3 (10 µg/ml) was pretreated with or without Pefabloc SC (4 mM) or {alpha}1-AT (100 µg/ml) for 30 min at 37°C before use. The supernatants were collected and the amount of IL-18 was measured by ELISA. The results are representative of three different experiments with similar results. ND, Not detected.

 
Localization of PR3 on the epithelial cell surface after PR3 treatment

The observation above leads to the possibility that PR3 can penetrate cells and is available intracellularly to convert proIL-18 to the active form. To examine this, we focused on the localization of PR3 after the treatments. Immunoblot analysis showed that PR3 was not detected following treatment of HSC-2 cells with PR3 for 24 h in the cell membrane fraction (Fig. 7Go, lane 1). After IFN-{gamma} priming of HSC-2 cells, PR3 was detected weakly with PR3 treatment alone (Fig. 7Go, lane 3), and a strong PR3 signal was detected with both PR3 and LPS treatment for 24 h (Fig. 7Go, lane 4), which was the condition of active IL-18 secretion from the cells in the cell membrane fraction. In contrast, no PR3 was detected following any of the treatments used in the cytoplasm fractions (Fig. 7Go, lanes 5–8). These findings indicate that PR3 can bind efficiently to the membrane of IFN-{gamma}-primed oral epithelial cells with LPS costimulation, and that PR3 acts on the cell surface and not in the cells, probably generating signals to activate the cells.



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  		FIGURE 7. Localization of PR3 after treatment of IFN-{gamma}-treated oral epithelial cells with PR3. Subconfluent HSC-2 cells were incubated with or without IFN-{gamma} (1000 IU/ml) for 3 days. After being washed, cells were stimulated with LPS (100 ng/ml) and/or PR3 (10 µg/ml) for 24 h at 37°C. After treatment, cell membrane and cytoplasm fractions were separated from the cells and mixed with Laemmli sample buffer. Samples (equivalent to 5 x 105 cells each) were then subjected to 15% SDS-PAGE and transferred to a PVDF membrane. The blots were probed with anti-PR3 Ab. PR3 (2 ng) was loaded as a control (lane 9). The results are representative of three different experiments with similar results.

 
Caspase-1-independent secretion of IL-18 from PR3-stimulated oral epithelial cells

Next, the possible involvement of caspase-1 and the caspase family in inducing the secretion of active IL-18 from the cells was examined. Although PBMCs express 45-kDa precursor, 30-kDa intermediate, and 10-kDa mature forms of caspase-1, no form of caspase-1 was detected in the cells stimulated with PR3 and LPS after IFN-{gamma} priming as determined by immunoblot analysis (Fig. 8GoA). Consistent with this, a caspase-1-specific inhibitor, YVAD-CHO, had no inhibitory effect on the secretion of IL-18 at any doses used in this study (Fig. 8GoB). A caspase family inhibitor, ZVAD-FMK, showed only a marginal inhibitory effect at 20 µM. These results suggest that PR3 is involved in induction of bioactive IL-18 secretion in combination with LPS from oral epithelial cells via a caspase-1-independent pathway.



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  		FIGURE 8. Absence of caspase-1 protein in oral epithelial cells and no influence of caspase inhibitors on PR3-induced IL-18 secretion by the cells. A, IFN-{gamma}-treated HSC-2 cells were stimulated with LPS (100 ng/ml) and/or PR3 (10 µg/ml) for 24 h. After the treatment, cells were collected by trypsinization. The cell pellets (3 x 105 cells each) were then solubilized with Laemmli sample buffer, subjected to 15% SDS-PAGE, and transferred to PVDF membrane. The blot was probed with anti-caspase-1 Ab. PBMC lysate (3 x 105 cells) was loaded as a control. B, The IFN-{gamma}-treated HSC-2 cells were pretreated with the indicated concentrations of YVAD-CHO (YVAD) or ZVAD-FMK (ZVAD) for 1 h at 37°C. Then cells were stimulated with PR3 (10 µg/ml) and LPS (100 ng/ml) in the presence of YVAD or ZVAD for 24 h. The amount of IL-18 in the supernatant was measured by ELISA. The percentage of inhibition was evaluated on the basis of the value obtained without inhibitors. The amount in the supernatant without inhibitors (vehicle control, 0.1% DMSO) was 24.6 ± 1.0 ng/ml. The results are representative of three different experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated whether oral epithelial cells express IL-18 and the mechanism of generation of active IL-18 by the cells. The results showed for the first time that oral epithelial cells constitutively express proIL-18 and IL-18 mRNA, and that the cells secreted bioactive IL-18 on combined stimulation with PR3 and LPS after IFN-{gamma} priming through a caspase-1-independent pathway. The possibility that the IFN-{gamma}-inducing activity was due to LPS and PR3 carried over from the epithelial cell culture into PBMC culture could be ruled out by the following reasons: 1) IL-18 ELISA detects only active IL-18, 2) the epithelial cells were washed extensively after IFN-{gamma} priming to remove residual IFN-{gamma}, and 3) LPS and/or PR3 at the same doses as in the supernatants in PBMCs even in the presence of rIL-12 did not induce production of IFN-{gamma} in PBMCs (Fig. 3GoC). Human intestinal mucosa also expresses proIL-18, and active IL-18 expression was up-regulated in individuals with Crohn’s disease, a chronic inflammation of the gastrointestinal tract (14, 15), but the underlying mechanism was unclear. Thus, the mechanism behind the generation of active IL-18 by PR3 presented in this study may be unique to human oral epithelial cells, though it may be applicable to other IL-18-expressing nonimmune cells.

In the present study, we obtained evidence that, in addition to LPS treatment, costimulation with PR3 is needed to induce the generation of bioactive IL-18. One possible mechanism of action of PR3 is that it cleaves proIL-18 in the supernatant or membrane-bound proIL-18 to the active form via proteolytic activity as proposed by Fantuzzi and Dinarello (38). But this does not seem to be the case, because 1) LDH activity was not detected in the supernatant of the epithelial cell culture after the LPS and/or PR3 treatment (Fig. 4GoA), which indicates that the cell membrane was not damaged by the treatment; 2) proIL-18 in the supernatant of the epithelial cells and cell-bound proIL-18 on the cells could not be detected with any of the treatments used, as determined by immunoblot analysis and flow cytometry, respectively (Fig. 4GoB and data not shown); 3) the active 18-kDa form of IL-18 was present only in the cell lysate and supernatant of the PR3- and LPS-treated cells after IFN-{gamma} priming (Fig. 4GoB); and 4) it took >18 h to induce IL-18 to form in the supernatant of the cells using PR3, a process which was completely inhibited by CHX, suggesting the requirement of de novo synthesis of proIL-18 (Fig. 5Go). Therefore, another possibility is that PR3 directly binds to the cell membrane or molecule(s) on the oral epithelial cell surface where it generates signals to cleave proIL-18 in the cells. PR3 is cationic and this enables it to bind to negatively charged plasma membrane constituent(s) (17). But among the three neutrophil serine proteinases, HLE and Cat G are more cationic than PR3 (isoelectric points for PR3, HLE, and Cat G are 9.1, 10.5, and >11, respectively) (17). Fig. 3GoD showed that HLE and Cat G had no effect on the induction of active IL-18 secretion, which excludes the possibility that PR3 binds to the plasma membrane due to its hydrophobicity, and as a consequence generates signals to cleave proIL-18. Rather, it is possible that PR3 specifically binds to a molecule on the cell surface and elicits signaling. This possibility is supported by the evidence that PR3 is present only in the cell membrane and not in the cytoplasm fractions after treatment of the IFN-{gamma}-primed epithelial cells with PR3 and LPS (Fig. 7Go), and by the recent finding that PR3 directly interacts with a 111-kDa membrane molecule, which was comprised of two subunits of endothelial cells (27). The property of the PR3-binding molecule is unclear, but protease-activated receptors are suggested to be candidate receptors for PR3 (27). Experiments are under way to clarify this point.

It has been identified that Toll-like receptor (TLR)2 is a peptidoglycan- and lipoprotein-signaling receptor, TLR4 is an LPS-signaling receptor, and myeloid differentiation factor 88 (MyD88) is an adaptor molecule for TLR-mediated-signaling (39). Faure et al. (40) recently demonstrated that IFN-{gamma} and LPS up-regulate TLR2 and TLR4 gene expression in human endothelial cells in a NF-{kappa}B-dependent manner. We obtained the similar result that oral epithelial cells express TLR2, TLR4, and MyD88 mRNA, and the gene expressions were increased by IFN-{gamma} (A. Uehara, S. Sugawara, and H. Takada, manuscript in preparation). Our present study showed that stimulation with a high dose of LPS alone (up to 100 µg/ml) was ineffective in inducing IL-18 in the epithelial culture supernatants even after IFN-{gamma} priming (Fig. 3GoA), indicating the requirement of an additional signaling pathway to TLR4/MyD88 for the active IL-18 induction. It is also reported that IFN-{gamma} and LPS up-regulate IL-18 gene expression via IFN consensus sequence-binding protein and AP-1 elements in macrophages (41), which may be the mechanism of the IFN-{gamma} and LPS effects in this study. In support of this, the expression of 24-kDa proIL-18 protein was not reduced in the lysate of the cells treated with LPS and PR3 after IFN-{gamma} priming, even though 18-kDa IL-18 was markedly generated in the cell lysate and supernatant by the treatment (Fig. 4GoB).

The intracellular cysteine proteinases caspase-1, -4, and -3 are known to cleave proIL-18, and caspase-3 produces degraded inactive forms of IL-18 (7, 42). The present study showed that caspase-1 protein was not expressed in oral epithelial cells on induction of active IL-18 as determined by immunoblot analysis (Fig. 7GoA), and that a caspase-1-specific inhibitor, YVAD-CHO, had no effect on the PR3-induced secretion of IL-18 from HSC-2 and only partial inhibition was achieved by a caspase family inhibitor, ZVAD-FMK (Fig. 7GoB). These results suggest that PR3-induced secretion of IL-18 from oral epithelial cells was caspase-1-independent, and caspase 4 might be partially involved. Recently, it was reported that caspase-1-independent secretion of IL-18 from macrophages caused acute liver injury in caspase-1-deficient mice (43). Therefore, the priming of oral epithelial cells with IFN-{gamma} for 3 days, followed by stimulation with PR3 and LPS, may elicit undefined signals to activate other proteinases, which cleave proIL-18. Although the mediator of this caspase-1-independent proIL-18 processing is unknown, MMPs may be involved because they contribute to generate biologically active IL-1{beta} independently of caspase-1 (44). Further study is under way to clarify the mechanism behind the caspase-1-independent induction of active IL-18 by PR3 from oral epithelial cells.

Active PR3 is secreted as a soluble form by activated PMNs and also exists as a membrane-bound form on PMNs. Previous study demonstrated that each azurophil granule of PMNs contains PR3 at 13.4 mM, and activation of PMNs resulted in an ~10-fold increase in membrane-bound PR3 (17). In addition, although serum contains abundant naturally occurring proteinase inhibitors, membrane-bound PR3 is substantially resistant to inhibition by naturally occurring inhibitors including {alpha}1-AT and elafin, even when these inhibitors are used at a 100- to 300-fold molar excess over the enzyme (17). Therefore, even though the present study showed that the induction of active IL-18 by soluble PR3 is inhibited by serine proteinase inhibitors (Fig. 6Go), the induction is likely to occur in vivo.

It is conceivable that active IL-18 is secreted from oral epithelial cells after their exposure to IFN-{gamma} produced by T and NK cells in direct or indirect response to pathogenic organisms and after infiltration by active PMNs of the inflamed site in the course of inflammation induced by pathogens such as LPS. Although similar IL-18 expression was found in normal and inflamed gingival epithelium (Fig. 1Go), many inflammatory cells were infiltrated into lamina propria of inflamed gingiva, suggesting that bioactive IL-18 is secreted from epithelium at the inflamed site. Recently, it was reported that IL-18 possesses proinflammatory properties: it directly induces secretion of TNF-{alpha} from T and NK cells with subsequent production of IL-1{beta} and IL-8 from monocytes (10). Therefore, these findings suggest that the PR3-induced secretion of active IL-18 from oral epithelial cells plays an important role in the regulation of inflammation by development of Th1 responses in the epithelium and lamina propria in periodontal diseases. It is reported that numerous Th1 cells infiltrate the gingival epithelium and lamina propria in inflamed gingival tissues (45). It is also possible that PR3-induced IL-18 induces Th2 responses in the lamina propria at the site of inflammation because IL-18 by itself enhances production of IL-4 and IL-13 by basophils, mast cells, and CD4+ T cells (6). Collectively, the present findings provide an insight into the potential involvement of IL-18-expressing nonimmune cells in addition to activated macrophages in the regulation of tissue destruction, remodeling, and inflammation by producing bioactive IL-18 interacting with active PMNs.


   Acknowledgments
 
We thank C. Galanos for providing an LPS preparation.


   Footnotes
 
1 This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (11671796, 12470380, and 13671894). Back

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

3 Abbreviations used in this paper: proIL-18, precursor form of IL-18; PR3, proteinase 3; HLE, human leukocyte elastase; Cat G, cathepsin G; PMN, polymorphonuclear neutrophil; YVAD-CHO, Tyr-Val-Ala-Asp-aldehyde; CHX, cycloheximide; ZVAD-FMK, Z-Val-Ala-Asp-fluoromethyl ketone; Boc-Ala-ONp, Boc-Ala-p-nitrophenyl ester; {alpha}1-AT, {alpha}1-antitrypsin; TLR, Toll-like receptor; MyD88, myeloid differentiation factor 88; PVDF, polyvinylidene difluoride; LDH, lactate dehydrogenase. Back

Received for publication May 7, 2001. Accepted for publication October 1, 2001.


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