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RESEARCH REPORT |
1 Department of Microbiology and Immunology and
2 Department of Periodontics and Endodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan;
3 Division of Molecular Pathology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Science, Kumamoto, Japan;
4 Department of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, Cracow, Poland; and
5 Department of Biochemistry, University of Georgia, Athens, GA, USA;
*corresponding author, sugawars{at}mail.cc.tohoku.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSDISCUSSIONREFERENCES |
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KEY WORDS: gingipains • proteolysis • ICAM-1 • oral epithelial cells • neutrophil adhesion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSDISCUSSIONREFERENCES |
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P. gingivalis produces two trypsin-like cysteine proteinases specific for Arg-X (50 and 95 kDa) or Lys-X (105 kDa) bonds, referred to as arginine-specific gingipain (Rgp) and lysine-specific gingipain (Kgp), respectively (Chen et al., 1992; Pike et al., 1994). The 95-kDa high-molecular-mass Rgp (HRgpA) differs from the 50-kDa Rgp (RgpB) in that the protein non-covalently complexes with the hemagglutinin/adhesin domain in the same manner as Kgp. It has been shown that gingipains play a critical role in the onset of inflammation through a wide variety of biological activities, including host immune evasion (Potempa et al., 2000), which leads to the question of whether P. gingivalis evades immune surveillance by attenuating PMN-epithelial cell interaction by means of the bacterial proteinases. The present study clearly showed that purified gingipains cleaved ICAM-1 on oral epithelial cells, consequently inhibiting PMN-oral epithelial cell interaction.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSDISCUSSIONREFERENCES |
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Purification and Activation of Gingipains
Three forms of gingipains—95-kDa HRgpA, 50-kDa RgpB, and 105-kDa Kgp—were purified from P. gingivalis HG66 culture supernatant, as described previously (Pike et al., 1994). The amount of active enzyme in each purified preparation was determined by active site titration with Phe-Pro-Arg-chloromethyl ketone (FPR-cmk) and benzyloxycarbonyl-Phe-Lys-cholomethyl ketone (Z-FK-cmk) (Bachem Bioscience, King of Prussia, PA, USA) for Rgps and Kgp, respectively (Potempa et al., 1997). To activate gingipains, we diluted gingipains to 10 µmol/L in 0.2 mol/L HEPES, 5 mmol/L CaCl2, and 10 mmol/L cysteine, pH 8.0, and incubated them at 37°C for 10 min. To block the enzymic activity of gingipains, we incubated activated gingipains with FPR-cmk, Z-FK-cmk, or freshly isolated human serum for 10 min at room temperature before use.
Cell Culture and Treatment
The human oral epithelial cell lines KB (Eagle, 1955) and HSC-2 (Momose et al., 1989) were obtained from the American Type Culture Collection (Rockville, MD, USA), and the Cancer Cell Repository, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan), respectively. KB was grown in alpha-minimum essential medium with 10% heat-inactivated fetal calf serum (FCS; Life Technologies, Auckland, New Zealand). HSC-2 was grown in RPMI 1640 medium with 10% FCS. Cells were treated with given concentrations of gingipains and human leukocyte elastase (HLE; Calbiochem-Novabiochem, La Jolla, CA, USA) at 105 cells/50 µL at 37°C. Cells were also fixed with 3% paraformaldehyde, as described previously (Nemoto et al., 2000). Primary human gingival epithelial cells were prepared from explants of normal human gingival tissues obtained with informed consent from donors, as described previously (Sugawara et al., 2001). PMNs from heparinized peripheral venous blood of healthy adult donor, obtained with informed consent, were isolated by density-gradient centrifugation on Mono-Poly resolving medium® (ICN Biomedical, Costa Mesa, CA, USA), as described (Nemoto et al., 2000). The experimental procedures were approved by the Ethical Review Board of Tohoku University Graduate School of Dentistry (Sendai, Japan).
Flow Cytometry
Cells were collected by trypsinization, washed with PBS, and stained with mAbs. Staining was then analyzed on a FACScan® (BD Biosciences, Mountain View, CA, USA), as described previously (Sugawara et al., 2000). The arithmetic mean was used in the computation of the mean fluorescence intensity (MFI).
ICAM-1 Detection by Western Blotting
The cell membrane fraction of KB was prepared by Dounce homogenization, as described previously (Sugawara et al., 2001). The cell membranes in a 10-cm2 area were suspended in 20 µL of PBS containing 0.3 µmol/L RgpB for 1 hr at 37°C. ICAM-1 protein in the membrane pellets was then detected by Western blotting (Nemoto et al., 2002). Briefly, samples were mixed with Laemmli sample buffer, subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and transferred to a polyvinylidene difluoride membrane. The membrane was probed with goat anti-human ICAM-1 polyclonal antibody (Ab) (R&D Systems Inc., McKinley Place, MN, USA) at 1:3000, with the use of an ECL Western blotting detection system (Amersham Pharmacia Biotech Inc., Piscataway NJ, USA). The molecular weight of the proteins was estimated by comparison with the positions of standards (Bio-Rad Laboratories, Hercules, CA, USA).
Adhesion Assay
HSC-2 cells (105 cells/well) were treated with 103 U/mL of human natural interferon-
(IFN-) (Hayashibara Biochemical Laboratories, Okayama, Japan) in wells of a 96-well plate coated with collagen I (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA) for 3 days at 37°C. Cells were washed with warmed medium three times, treated with RgpB, 10 µg/mL of anti-CD54 mAb or isotype-matched control mAb (Immunotch, Marceille, Cedex, France) for 30 min at 37°C, and then washed with warmed medium three times. RgpB at 0.3 µmol/L was also pre-treated with 3 µmol/L of FPR-cmk for 10 min at room temperature before use. PMNs (5x106 cells/mL) were labeled with 5 µmol/L of calcein acetoxymethyl (Molecular Probes, Eugene, OR, USA) in RPMI 1640 medium for 30 min at 37°C. The labeled PMNs (5 x 105 cells/well) were then added to cell monolayers, and incubated for 30 min at 37°C. At the end of incubation, cells were gently washed three times with warmed PBS, and adherent PMNs were evaluated by means of a Versa Fluor® spectrophotofluorometer (Bio-Rad Laboratories) at excitation 494 nm and emission 510 nm.
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 ± standard deviations (SD) of triplicate assays. We examined the statistical significance of differences between two means by a one-way analysis of variance, using the Bonferroni or Dunnett method, and P values less than 0.05 were considered significant.
RESULTS
We first examined the effects of purified gingipains (HRgpA, RgpB, and Kgp) on the expression of ICAM-1 by oral epithelial cells by flow cytometry. When KB cells were treated with 0.03 to 0.3 µmol/L of HRgpA, RgpB, and Kgp for 30 min, the expression of ICAM-1 on the cell surface was significantly (P < 0.01) reduced (Fig. 1A
). The expression was rapidly abolished by 0.1 and 0.3 µmol/L of both HRgpA and RgpB. Kgp exhibited slightly less activity for the reduction as compared with Rgps, indicating that Rgps rather than Kgp efficiently reduced the expression. Therefore, Rgps were mainly used in subsequent experiments. Fig. 1B shows a representative FACS profile of ICAM-1 expression on KB cells after HRgpA treatment at the dose indicated for 60 min. It is reported that HLE also cleaved ICAM-1 on human monocytic cells (Champagne et al., 1998). However, HLE was ineffective in down-regulating the expression of ICAM-1 on KB cells (Fig. 1C).
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). The cell membrane was then treated with RgpB, followed by Western blot analysis with anti-ICAM-1 polyclonal Ab. A strong 90-kDa ICAM-1 band was detected in the untreated cell membrane, and the band disappeared in a time-dependent manner after treatment with RgpB (Fig. 2B). The reduction of ICAM-1 caused by RgpB was abolished by the pre-treatment of RgpB with FPR-cmk, an Rgp-inhibitor, but not with Z-FK-cmk, a Kgp-inhibitor (Fig. 2C). These results indicate that the reduction in ICAM-1 resulted from direct proteolysis, and that ICAM-1 could be degraded into fragments.
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shows that the elimination of surface molecules on KB cells by HRgpA was a preference for ICAM-1, because the expression of CD29 (ß1 integrin), CD48, CD49b (
2 integrin), CD49e (5 integrin), CD13 (aminopeptidase N), and MHC class I was unchanged, or only slightly decreased, after treatment with HRgpA. The expression of CD58 (leukocyte-function-associated antigen-3) was decreased to 50% by the treatment. The reduction in ICAM-1 caused by HRgpA was inhibited by an Rgp inhibitor, FPR-cmk, but not by a Kgp inhibitor, Z-FK-cmk (Fig. 3B), confirming that enzymic activity was required for the reduction. HRgpA at 1.0 µmol/L still effectively reduced ICAM-1 expression, even in the presence of 10% freshly isolated human serum, which is known as a source of naturally occurring protease inhibitors (Grøn et al., 1997). The reduction was completely inhibited by 80% serum.
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-primed HSC-2 cells with PMNs in the absence or presence of RgpB. The HSC-2 line was used in this study because the cells adhere more tightly to the culture plates than do KB cells. Since HSC-2 cells normally express low levels of ICAM-1, they were pre-treated with IFN-. The treatment up-regulates the expression of ICAM-1 on the cells, and the expression was also down-regulated by RgpB (Fig. 4A). PMNs efficiently bound IFN--primed HSC-2 cells for 30 min as compared with unprimed cells, the binding being significantly inhibited by RgpB to the level caused by anti-ICAM-1 mAb (Fig. 4B). Pre-treatment of RgpB with FPR-cmk for 10 min recovered PMN adhesion. Furthermore, the finding obtained with HSC-2 cells was reproduced with the use of freshly isolated primary oral epithelial cells (Fig. 4C). These results indicate that the proteolytic activity of RgpB was required for the inhibition of ICAM-1-dependent PMN adhesion to oral epithelial cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSDISCUSSIONREFERENCES |
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). Rgps down-regulated the expression of ICAM-1 on the fixed cells (Fig. 2A) and degraded ICAM-1 in the cell membrane into fragments (Figs. 2B, 2C), indicating that the reduction resulted from direct proteolysis, not from shedding or internalization after cell activation by gingipains. Consequently, purified gingipains down-regulated ICAM-1-dependent PMN-oral epithelial cell interaction (Fig. 4).
It was reported that ICAM-1 expression on oral epithelial cells was decreased at 2-4 hrs after P. gingivalis infection (Madianos et al., 1997; Huang et al., 1998), in contrast to the up-regulation of ICAM-1 mRNA expression (Huang et al., 2001), although the underlying mechanism was unclear. The present study clearly showed that the purified gingipains proteolytically cleave ICAM-1 within 2 hrs on oral epithelial cells in culture, which may partially account for the phenomenon. HLE at 680 nmol/L (20 µg/mL) efficiently reduced ICAM-1 expression on human monocytic cells (Champagne et al., 1998). However, the present study showed that HLE was ineffective against ICAM-1 expressed on human oral epithelial cells (Fig. 1C). Human oral epithelial cells express and secrete secretory leukocyte protease inhibitor (Sumi et al., 2000), an effective reversible inhibitor of HLE and cathepsin G, which may be involved in the attenuation of HLE activity on oral epithelial cells. It also indicates that neutrophil serine proteinases do not influence the PMN-oral epithelial cell interaction.
In the present study, we showed that the very late activation antigen family (CD29, CD49b, and CD49e), immunoglobulin superfamily (CD48 and CD58), CD13 (aminopeptidase N, a membrane-bound metalloprotease), and MHC class I were not eliminated as efficiently as ICAM-1, which belongs to the immunoglobulin superfamily, by HRgpA (Fig. 3A). The results excluded the possibility that gingipains preferentially cleave the molecules belonging to the immunoglobulin superfamily. We previously demonstrated that CD18 on human monocytes is also only slightly affected by gingipains (Sugawara et al., 2000), suggesting that gingipains preferentially cleave ICAM-1 expressed on oral epithelial cells, probably due to the structural accessibility of gingipains to ICAM-1, compared with the other molecules.
HRgpA at 1 µmol/L still effectively reduced ICAM-1 expression in the presence of 20% freshly isolated human serum, and the reduction was completely inhibited by 80% serum (Fig. 3B). It is also reported that human serum (plasma) is ineffective in preventing the activation of pre-kallikrein (Imamura et al., 1994), factor X (Imamura et al., 1997), and protein C (Hosotaki et al., 1999) by Rgp. These observations indicate that high doses of gingipains are resistant to inhibitors in serum. A previous report (Eley and Cox, 1996) showed that the mean value of gingipain activity in the gingival crevicular fluids from periodontitis patients with attachment loss was 40-90 µU/µL, as determined by Z-Val-Lys-Lys-Arg-AFC. According to our estimation of ICAM-1-cleaving activity using the culture supernatants of P. gingivalis W83 and ATCC 33277, as determined by N--benzyloxycarbonyl-L-Arg-p-nitroanilide, one µU/µL of gingipain activity was equivalent to about 0.1 µmol/L of purified Rgp (data not shown). It is conceivable that the local concentration of gingipains around P. gingivalis was much higher than that in the GCF. Therefore, the down-regulation of ICAM-1 on human gingival epithelial cells caused by gingipains is likely to occur in vivo.
The present study showed that the mechanism of PMN adhesion to HSC-2 cells is ICAM-1-dependent, because the adhesion was completely inhibited by anti-ICAM-1 mAb (Fig. 4B). The observation was also confirmed by the use of freshly isolated primary oral (gingival) epithelial cells (Fig. 4C). Furthermore, PMN adhesion to HSC-2 cells was inhibited by RgpB to the level caused by anti-ICAM-1 mAb, and this activity was completely neutralized by FPR-cmk, an Rgp-specific inhibitor. These results clearly indicate that gingipains inhibited ICAM-1-dependent PMN adhesion to oral epithelial cells. It is reported that P. gingivalis infection of oral epithelial cells inhibited PMN transmigration induced by N-formylmethionyl leucyl phenylalanine and IL-8, and that ICAM-1 is partially involved in the transmigration process with use of the Transwell system in vitro (Madianos et al., 1997). It has also been reported that the level of expression of ICAM-1 increases from basal cells toward the surface of the junctional epithelium, which is topographically correlated with PMN accumulation (Tonetti et al., 1998), suggesting that the gradient is important for directing the migration of PMN. Oral epithelial cells have the ability to produce IL-8 (Lundqvist et al., 1994), and gingipains modulate IL-8 activity (Mikolajczyk-Pawlinska et al., 1998). Gingipains hydrolyze epithelial junctional proteins (Katz et al., 2002), indicating that P. gingivalis can invade periodontal connective tissues as well as epithelium and produce gingipains in the tissues. Therefore, these observations and the present study suggest that gingipains are involved in the environment of PMN/junctional epithelium interaction by cleaving ICAM-1 and inhibiting ICAM-1-dependent adhesion between PMN/junctional epithelium at the surface and/or basal area of the epithelium. Consequently, gingipains could indirectly attenuate the transmigration.
Oral epithelial cells produce inflammatory cytokines such as IL-8 upon activation and are thought to participate actively in the host defense mechanism (Sugawara et al., 2001). It has been reported that endothelial cells can be activated by the direct surface interaction of ICAM-1 with its ligands on inflammatory cells (Clayton et al., 1998). The results also indicate that direct ICAM-1-mediated interaction of junctional epithelial cells with PMN may cause activation of the epithelial cells, and that gingipains may attenuate the activation, although the importance of PMN adherence to the epithelial cells in controlling the biofilms is not known.
Gingipains are reported to exhibit a wide variety of pathophysiological properties during the onset of periodontitis, including immune evasion (Potempa et al., 2000). The present study reveals a novel role for gingipains in PMN-oral epithelial cell interaction, and further confirmed that the manipulation of gingipains is important for the control of periodontitis.
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ACKNOWLEDGMENTS |
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Received October 1, 2002; Last revision June 16, 2003; Accepted June 27, 2003
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSDISCUSSIONREFERENCES |
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