The Treponema denticola Surface Protease Dentilisin Degrades Interleukin-1 (IL-1 ), IL-6, and Tumor Necrosis Factor Alpha

Chronic periodontitis is an infectious disease characterized by the accumulation of inflammatory cells in extravascular gingival connective tissue, which finally causes the breakdown of periodontal tissue and tooth loss (21, 28, 35, 36, 38). Treponema denticola has been identified as a major pathogen in human periodontal disease (1, 18, 20, 25). Previous studies have revealed that a number of inflammatory cytokines are synthesized in response to periodontopathic bacteria and their products, thus inducing and maintaining an inflammatory response in the periodontium (3, 29). Inflammatory cytokines, such as interleukin-1 (IL-1 ), IL-6, and tumor necrosis factor alpha (TNF), are closely associated with the development of periodontal lesions (9, 16, 23, 30–32, 34, 40). It has been shown that T. denticola induces the production of various cytokines, including IL-1 , IL-6, IL-8, and TNFfrom various cell types (2, 6, 15, 26, 33). On the other hand, it has also been suggested that T. denticola hydrolyzes IL-8 (2, 26). This microorganism possesses a surface protease, dentilisin, which hydrolyzes host proteins and is believed to be a major pathogen of this microorganism (12, 13, 37). Proteases from periodontopathic bacteria such as Porphyromonas gingivalis have been reported to modulate immunoresponses by hydrolyzing inflammatory mediators (3, 5). The aim of this study was to verify the ability of dentilisin to degrade inflammatory cytokines, including IL-1 , IL-6, and TNF. In this study, we used T. denticola ATCC 35405 and dentilisin-deficient mutant K1 (12). T. denticola was propagated in TYGVS medium (27) and incubated at 37°C for 4 days under anaerobic conditions as described previously (10). In order to estimate the effect of T. denticola on the production of inflammatory cytokines, we determined the amount of cytokines produced in human peripheral blood mononuclear cells (PBMCs) after exposure to T. denticola. Peripheral blood was obtained from three healthy adult volunteers by venipuncture after informed consents were obtained. The PBMCs were separated by density gradient centrifugation using a Lymphoprep tube (Axis-Shield, Oslo, Norway) according to the manufacturer’s instructions. The cells obtained were washed with phosphatebuffered saline (PBS [pH 7.4]; Nissui, Tokyo, Japan) and resuspended in RPMI 1640 medium containing 2 mM L-glutamine (Nissui) and 10% fetal bovine serum (Bio-Whittaker, Maryland). The PBMCs (5 10 cells/ml) were then seeded in 96-well microtiter plates at 200 l/well, and a 20l T. denticola cell suspension was then added at 1 10 cells/well. Cells were incubated at 37°C for 24 h in humidified air containing 5% CO2. After incubation, the amounts of cytokines in the supernatants were measured with enzyme-linked immunosorbent assay (ELISA) systems (IL-6 and TNF; ENDOGEN, Rockford, IL; IL-1 , R & D systems, Minneapolis, MN) according to the manufacturer’s instructions. Statistical significance was determined with an analysis of variance (ANOVA) followed by the Student-Newman-Keuls test to obtain multiple comparisons of cytokine levels and cytokine mRNA expression. Figure 1 shows the amounts of IL-1 , IL-6, and TNFproduced by the PBMCs after exposure to T. denticola ATCC 35405 and K1. All the targeted cytokines in the PBMC supernatants showed significantly larger amounts after exposure to K1 cells than after exposure to ATCC 35405 (P 0.01). The inactivation of a gene sometimes induces pleiotropic effects. In T. denticola K1, this has been shown to result in the ability to organize oligomeric proteins being affected (11). To investigate proteolytic activity by agents other than dentilisin, endoacting proline-specific oligopeptidase (22) and trypsin-like peptidase (8) activities in both T. denticola ATCC 35405 and K1 were evaluated by using synthetic substrata, Z-Gly-Pro-4nitroanilide (Sigma-Aldrich, Milwaukee, MI) and N -benzoylDL-arginin 4-nitroanilide (Sigma), respectively. The activities of both enzymes were found to be at almost the same level. These results indicated that these other peptidase activities were not affected by inactivation of dentilisin. This suggests that dentilisin is involved in the reduction of inflammatory cytokines. To evaluate the degradation of cytokines by dentilisin, T. denticola and each cytokine were incubated at 37°C in PBS at a ratio of 1 10 cells of T. denticola/pg cytokines for 12 h. The remaining IL-1 , IL-6, and TNFlevels (average percent standard deviation) after incubation with T. denticola ATCC 35405 were 0.48 1.35, 69.2 5.45, and 25.6 0.35, respectively. The reduction rates of IL-1 , IL-6, and TNFafter incubation with T. denticola K1 were 10.2 3.12, 125.8 4.56, and 91.7 2.79, respectively. These results also indicated degradation of IL-1 and TNFby dentilisin. IL-6, however, showed a small decrease after exposure to dentilisin relative to * Corresponding author. Mailing address: Department of Microbiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 2618502, Japan. Phone: 81-043-270-3742. Fax: 81-043-270-3744. E-mail: [email protected].