Abstract
Our previous studies have shown an association between Helicobacter pylori infection, the strong up-regulation of cathepsin X (CTSX, also called cathepsin Z/P), and the development of gastric cancer. In the present study, we analyzed primary and conventional gastric epithelial cell lines to establish an optimal in vitro mouse model system for the examination of H. pylori-induced overexpression of Ctsx in a functional way. Gastric epithelial cells were isolated from stomachs of wild-type C57BL6/N and Ctsx(-/-) mice and compared with the gastric cancer cell line CLS103. Indirect co-cultures of epithelial cells and macrophages were infected with H. pylori strain SS1 and analyzed for the expression of cathepsins, cytokines, and adhesion factors. Cellular interactions, migration capability, and adherence of H. pylori were assessed using time-lapse video microscopy and colony-forming assays. Isolated primary cells from wild-type and transgenic mice revealed qualities and expression profiles similar to those of corresponding tissue samples. Adherence of H. pylori was significantly higher in primary compared with commercially cells. Thus, induction of cathepsins, cytokines, and adhesion proteins was detected solely in primary cells and co-cultured macrophages. Microarray and migration experiments indicated that Ctsx is involved in B/T-cell proliferation/migration and adhesion of macrophages. Primary epithelial cells from stomach of Ctsx(-/-) mice represent an excellent model of H. pylori gastritis to elaborate the special functions of Ctsx in regulating the immune response to H. pylori.
Highlights
Our previous studies have shown an association between Helicobacter pylori infection, the strong up-regulation of cathepsin X (CTSX, called cathepsin Z/P), and the development of gastric cancer
Expression of MIF-1 in WT and CtsxϪ/Ϫ Epithelial Cells Cocultured with Macrophages—Wong et al demonstrated that the macrophage migration inhibitory factor (MIF)2 is a critical mediator of both the innate and the Th1 immune response in gastritis induced by H. pylori infection in a transgenic mouse model [32]
CTSB/L/K and X are differentially expressed in normal and chronically inflamed gastric mucosa, but only CTSX shows a significant induction in H. pylori-infected gastric epithelial cells and macrophages [12,13,14]
Summary
Animals—Mice with deficiencies in cathepsin B (mutant allele Ctsbtm1Jde, designated here as CtsbϪ/Ϫ) and cathepsin X (mutant allele Ctsztm1Thre; designated here as CtsxϪ/Ϫ) were generated by gene targeting in mouse embryonic stem cells as described [22, 23]. After pelleting dispersed cells and washing with PBS (PAA, Linz, Austria), the cells were resuspended in epithelial cell culture medium Quantum 286 (PAA) supplemented with gentamycin (10 g/ml), penicillin/streptomycin (5 g/ml, Invitrogen) and seeded into Matrigel-coated six-well-plates (Biochrom, Berlin, Germany) containing Quantum 286 medium (PAA) with antibiotics penicillin/streptomycin/gentamycin (Invitrogen) and incubated at 37 °C for at least 24 h. The cell line was cultured in high glucose DMEM supplemented with 10% fetal calf serum and 1% antibiotics (penicillin/ streptomycin, 10 mg/ml) and incubated at 37 °C under standard conditions of 5% CO2. PCR reactions were done using the quantitative PCR Mastermix kit (Quantace, London) according to the man-
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