Elucidation of developing mechanisms for infectious endocarditis using a proteomic technique

Research Project

Project/Area Number	14571953
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	矯正・小児・社会系歯学
Research Institution	Kyushu University
Principal Investigator	YAMAGUCHI Noboru Kyushu University, Faculty of Dental Science, Preventive Dentistry, Instructor, 歯学研究院, 助手 (00230368)
Co-Investigator(Kenkyū-buntansha)	MASUHIRO Yoshikazu Instructor, Faculty of Dental Science, Oral Infectious Diseases & Immunolgy, Instructor, 歯学研究院, 助手 (00336083)
Project Period (FY)	2002 – 2003
Project Status	Completed (Fiscal Year 2003)
Budget Amount *help	¥3,400,000 (Direct Cost: ¥3,400,000) Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000) Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
Keywords	periodontopathic bacteria / human endothelial cell / proteomics / inflammatory cytokine / leukotoxin / apoptosis / integrin / caspase
Research Abstract	First of all, to elucidate the developing mechanisms for infectious endocarditis, we analyzed some proteins which are induced and produced from human endothelial cells (EA.hy926) infected with Acanobacillus acdnornycetemcomians (Aa) strain SUNY465 using a proteomic technique. This result suggested that several inflammatory cytokines might be induced after the bacterial invasion to the cell. We demonstrated previously that Aa leukotoxin (Ltx) is strongly able to induce apoptotic signal of cells that are positive for lymphocyte function-associated antigen-1 (LFA-1), a cell receptor of Ltx. So we next investigated whether inflammatory cytokines regulate apoptosis of human leukemic HL-60 cells induced by the Ltx. Of these cytokines tested, TNF-α, IL-1α and IL-1β significantly enhanced the Ltx-induced cell apoptosis. Northern and Western blotting analyses showed that TNF-a enhanced the expression of CD11a in the cells at mRNA and protein level, but did not for CD18. TNF-α also enhanced the binding of Ltx to the cells. We also observed by measuring mitochondrial transmembrane potential and the generation of superoxide. anion that the cytokine enhanced Ltx-induced HL-60 cell apoptosis. The enhancing action was strongly neutralized by anti-TNF-receptor 1 antibody, though anti-TNF-receptor 2 antibody did not so. In addition, IL-1α and β enhanced the cell apoptosis as the same mode as TNF-α. Furthermore, caspase-3 intracellular activity assay (PhiPhiLux^<TM>G_1D_2) showed that the Ltx-induced caspase-3 activation was completely neutralized by CD18 antibody treatment, though the significant neutralization was also observed by CD11a antibody one. Taken together, the present study indicates that TNF-α acts as a potent stimulator of the Ltx-induced HL-60 apoptosis via TNF-receptor 1-mediated upregulation for LEA-1 expression.