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

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-α, natural IFN-γ, rIL-18, and anti-human IL-18 mAb 2-10C (mouse IgG1) were kindly provided by Hayashibara Biochemical Laboratories (Okayama, Japan). Human rIFN-β 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 α-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 × 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 ×10⁴ 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-γ-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 α₁-antitrypsin (α₁-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 × g for 5 min at 4°C twice to remove nuclei. Supernatants were centrifuged at 15,000 × 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 × g at 4°C and used as cytoplasm fractions.

Immunoblot analysis

Cell pellets (3 × 10⁵ cells each) and vacuum-dried supernatants (equivalent to 3 × 10⁵ cells each) were used for the detection of IL-18 and caspase-1, and cell membrane and cytoplasm fractions (equivalent to 5 × 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 M_r of the proteins was estimated by comparison with the position of a standard (Bio-Rad).

Assay for IFN-γ-inducing activity of IL-18

Assay for IFN-γ-inducing activity of IL-18 was performed as described previously (35). Briefly, human PBMCs (3 × 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-γ in the supernatant was determined with a human IFN-γ 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)] ×100.

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 α₁-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.

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. 1⇓A). 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. 1⇓B). 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-1β, 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. 2⇓A) and to constitutively express the 24-kDa proIL-18 but not 18-kDa active form by immunoblot analysis (Fig. 2⇓B).

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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 × 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-γ 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. 3⇓A). IFN-γ (1000 IU/ml) treatment for 3 days was also ineffective in generating IL-18. Following the IFN-γ 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. 3⇓A). This priming by IFN-γ was the most effective in inducing IL-18, and neither IFN-α nor IFN-β 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-γ by PBMCs in the presence of rIL-12, the principal activity of IL-18 (1). As shown in Fig. 3⇓B, the supernatant induced production of IFN-γ along with rIL-12 from PBMCs, and the IFN-γ-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-γ by PBMCs, while stimulation with rIL-18 (0.5 and 1 ng/ml) along with rIL-12 did (Fig. 3⇓C), as previously described (1, 2, 35), indicating that the IFN-γ-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-γ priming (Fig. 3⇓D), indicating that the IL-18-inducing activity was specific to PR3. The results obtained using HSC-2 are depicted in Fig. 3⇓D and in additional experiments, except those for Fig. 4⇓A, 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-γ 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-γ 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-γ in the supernatants were analyzed by ELISA. D, Subconfluent HSC-2 cells were incubated in the presence or absence of IFN-γ (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-γ priming. A, LDH activity in the supernatants as in Fig. 3⇑A 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-γ (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 × 10⁵ cells each) and vacuum-dried supernatants (equivalent to 3 × 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-γ 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-γ priming (Fig. 4⇑A), 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 M_r 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 M_r as rIL-18 in addition to 24-kDa proIL-18 only on costimulation with PR3 and LPS after IFN-γ priming (Fig. 4⇑B). 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. 5⇓A), and the generation of IL-18 in the supernatant was completely inhibited by the treatment with CHX (Fig. 5⇓B). 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. 5⇓A). 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-γ (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-γ-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-γ 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, α₁-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 α₁-AT. As shown in Fig. 6⇓A, PR3 showed substantial enzymatic activity compared with the reference HLE (9.7 and 9.5 U/mg protein, respectively), and both Pefabloc SC and α₁-AT markedly inhibited the activity. Concurrently, both inhibitors completely inhibited the induction of IL-18 by PR3 (Fig. 6⇓B), 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 α₁-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-γ (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 α₁-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. 7⇓, lane 1). After IFN-γ priming of HSC-2 cells, PR3 was detected weakly with PR3 treatment alone (Fig. 7⇓, lane 3), and a strong PR3 signal was detected with both PR3 and LPS treatment for 24 h (Fig. 7⇓, 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. 7⇓, lanes 5–8). These findings indicate that PR3 can bind efficiently to the membrane of IFN-γ-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-γ-treated oral epithelial cells with PR3. Subconfluent HSC-2 cells were incubated with or without IFN-γ (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 × 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-γ priming as determined by immunoblot analysis (Fig. 8⇓A). 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. 8⇓B). 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-γ-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 × 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 × 10⁵ cells) was loaded as a control. B, The IFN-γ-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.