Helicobacter pylori

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Microbiology (2004), 150, 3913–3922

DOI 10.1099/mic.0.27527-0

Helicobacter pylori heat-shock protein 60 induces inflammatory responses through the Toll-like receptor-triggered pathway in cultured human gastric epithelial cells
Ryuta Takenaka,1 Kenji Yokota,2 Kiyoshi Ayada,2 Motowo Mizuno,1 Ying Zhao,2 Yoshihito Fujinami,2 Song-Nan Lin,2 Tatsuya Toyokawa,1 Hiroyuki Okada,1 Yasushi Shiratori1and Keiji Oguma2
Correspondence Kenji Yokota yokochan@md.okayama-u.ac.jp

Department of Medicine and Medical Science (Medicine 1)1 and Department of Bacteriology2, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Contact between Helicobacter pylori and gastric epithelial cells results in activation of NF-kB followed by secretion ofinterleukin (IL)-8. However, host-cell receptor(s) and their ligands involved in H. pylori-related IL-8 production have yet to be fully defined. In this study, the interaction between Toll-like receptors (TLRs), which are host receptors for pathogens involved in the innate immune response, and heat-shock protein (HSP) 60, an immune-potent antigen of H. pylori, was examined during H. pylori-induced IL-8secretion in vitro. Recombinant H. pylori HSP60 (rHpHSP60) was prepared and added to cultured KATO III human gastric epithelial cells with or without pre-incubation with mouse monoclonal anti-TLR2 or anti-TLR4 antibodies. IL-8 mRNA expression and IL-8 protein release were analysed by Northern blotting and immunoassay. Involvement of NF-kB activation was analysed immunocytochemically by anti-NF-kBp65 antibody and ammonium pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-kB-mediated transcriptional activation. rHpHSP60 induced IL-8 mRNA expression and IL-8 secretion in a dose-dependent manner in KATO III cells. Anti-TLR2 antibody inhibited rHpHSP60-induced IL-8 secretion by 75 %, and anti-TLR4 antibody inhibited it by 30 %. rHpHSP60 induced nuclear translocation of NF-kB p65, which wasinhibited by pretreatment with anti-TLR2 antibody. Treatment with PDTC significantly decreased the secretion of IL-8 induced by rHpHSP60. These findings suggest that H. pylori HSP60 activates NF-kB and induces IL-8 production through TLR-triggered pathways in gastric epithelial cells. Thus, it is possible that H. pylori HSP60 and TLR interaction in host cells contributes to the development ofgastric inflammation caused by H. pylori infection.

Received 29 July 2004 Accepted 2 September 2004

INTRODUCTION
Chronic infection with Helicobacter pylori is recognized as the cause of chronic active gastritis, peptic ulcer diseases and mucosa-associated lymphoid tissue lymphoma, and is an important risk factor in gastric carcinoma development (Blaser, 1990; Parsonnet et al., 1991, 1994). Bindingof H. pylori to gastric epithelial cells, in particular through the blood group antigen-binding adhesin, and the binding of strains expressing the cag pathogenicity island results in the production of proinflammatory chemokines such as
Abbreviations: FCS, fetal calf serum; G3PDH, glyceraldehyde-3phosphate dehydrogenase; HSP, heat-shock protein; IL, interleukin; PDTC, ammoniumpyrrolidinedithiocarbamate; PMB, polymyxin B; rHpHSP60, recombinant H. pylori HSP60; TLR, toll-like receptor; TNF-a, tumour necrosis factor a.

interleukin (IL)-8 (Crabtree et al., 1995a; Rad et al., 2002). NF-kB and AP-1 function as intracellular messengers in this process (Meyer-ter-Vehn et al., 2000; Sharma et al., 1998). The 60 kDa heat-shock protein (HSP60), an immunepotent antigen of H. pylori (Sharma et al.,1997), induces IL-8 secretion from human gastric epithelial cells (Yamaguchi et al., 1999). We have reported that humoral immune responses to HSP60 are closely associated with gastric inflammation and play a role in the pathogenesis of mucosa-associated lymphoid tissue lymphoma (Hayashi et al., 1998; Kawahara et al., 1999). With regard to host-cell receptors for HSP60, sulfated glycolipids are...
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