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Proteolysis of ICAM-1 on Human Oral Epithelial Cells by Gingipains

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;

View larger version (29K): [in a new window]   Figure 1. Kinetics of ICAM-1 reduction on KB cells treated with purified gingipains. (A) KB cells were treated with the given concentrations of purified HRgpA, RgpB, and Kgp for the period indicated at 37°C. After being washed with PBS, the cells were stained with anti-ICAM-1 mAb or matched-isotype mAb and analyzed by flow cytometry. (B) Representative FACS profile of ICAM-1 expression on KB cells after treatment with 1 and 0.3 µmol/L HRgpA for 1 hr at 37°C. Isotype-matched IgG1 was used as a control. (C) KB cells were treated with the given concentrations of HLE for 1 hr at 37°C, and then stained with anti-ICAM-1 mAb or matched-isotype mAb and analyzed by flow cytometry. Representative findings of three independent experiments are expressed as the mean of the MFI (% of control). *P < 0.05 and **P < 0.01 vs. respective untreated cells at each time point.

View larger version (23K): [in a new window]   Figure 2. Proteolysis of ICAM-1 on KB cells by Rgp. (A) KB cells were fixed with 3% paraformaldehyde for 3 min at 4°C. After being washed with PBS, unfixed or fixed cells were incubated with 0.3 µmol/L HRgpA for 1 hr at 37°C, and then expression of ICAM-1 on KB cells was assessed by flow cytometry. Representative findings of three independent experiments are expressed as the mean ± SD of the MFI. **P < 0.01 vs. respective control. (B) Purified cell membrane of KB cells was untreated or treated with 0.3 µmol/L RgpB for the period indicated at 37°C, and then the expression of ICAM-1 was analyzed by Western blotting with anti-ICAM-1polyclonal Ab. (C) Purified cell membrane of KB was untreated (lane 1) or treated with 0.3 µmol/L RgpB for 1 hr without (lane 2) or with 3 µmol/L FPR-cmk (lane 3), and 3 µmol/L Z-FK-cmk (lane 4) pre-treatments for 10 min at room temperature. Then, the expression of ICAM-1 was analyzed by Western blotting with anti-ICAM-1polyclonal Ab. Molecular-mass markers (kDa) are shown on the left. Findings are representative of three independent experiments.

View larger version (20K): [in a new window]   Figure 3. Preferential reduction of ICAM-1 on oral epithelial cells by Rgp, and effect of serum on the ICAM-1 proteolysis. (A) KB cells were treated with or without 0.3 µmol/L HRgpA for 1 hr at 37°C. After being washed with PBS, cells were stained with anti-ICAM-1, CD29, CD48, CD49b, CD49e, CD58, CD13, and MHC class I or matched-isotype mAb and analyzed by flow cytometry. (B) KB cells were treated with 0.3 µmol/L HRgpA for 1 hr in the presence or absence of 3 µmol/L FPR-cmk or 3 µmol/L Z-FK-cmk, or treated with 1 µmol/L HRgpA for 1 hr in the presence or absence of the indicated concentrations of freshly isolated human serum. After a wash with PBS, the expression of ICAM-1 was analyzed by flow cytometry. Representative findings of three independent experiments are expressed as the mean of the MFI (% of control). *P < 0.05 and **P < 0.01 vs. respective control.

View larger version (12K): [in a new window]   Figure 4. Inhibition of ICAM-1-dependent PMN adhesion to oral epithelial cells by Rgp. (A) HSC-2 cells were pre-treated with or without 103 U/mL IFN- for 3 days at 37°C. Cells were then treated with or without 0.3 µmol/L RgpB for 30 min at 37°C. After cells were washed with PBS, the expression of ICAM-1 was analyzed by flow cytometry. Representative findings of three independent experiments are expressed as the means of the MFI. **P < 0.01 vs. respective control. HSC-2 cells (B) and primary oral (gingival) epithelial cells (C) were pre-treated with or without 103 U/mL IFN- for 3 days at 37°C. Cells were then treated with 0.3 µmol/L RgpB in the presence of 3 µmol/L FPR-cmk, anti-ICAM-1 mAb (10 µg/mL), or isotype-matched control IgG1 (10 µg/mL) for 30 min at 37°C. After the treatment, they were washed with warmed medium three times, and co-cultured with calcein-labeled PMNs (5 x 105 cells/well) for 30 min at 37°C. At the end of the incubation, non-adherent PMNs were washed away with warmed PBS three times, and adherent PMNs were evaluated by means of a spectrophotofluorometer. We used serial dilutions of calcein-labeled PMNs as a standard to calculate the number of adherent PMNs. Results are expressed as the number of adherent cells. Representative findings of three independent experiments are expressed as the mean ± SD of the binding of PMNs. **P < 0.01 vs. respective control.