Microaerophilic Conditions Permit to Mimic in VitroEvents Occurring during in Vivo Helicobacter pylori Infection and to Identify Rho/Ras-associated Proteins in Cellular Signaling

Abstract

Molecular dissection of the mechanisms underlyingHelicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO 2 at their basolateral surface (aerophilic conditions) and 5% CO 2 , 5% O 2 , 90% N 2 (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-␣ (growth-regulated oncogene-␣), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-B p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120RasGAP , p190 RhoGAP , p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.

Helicobacter pylori in a Korean isolate activates mitogen-activated protein kinases, AP-1, and NF-κB and induces chemokine expression in gastric epithelial AGS cells

“…20,21 Oxygen radicals were produced by the association of H. pylori with gastric epithelial cells. We previously demonstrated that lipid peroxidation, an index of oxidative membrane damage, increased by H. pylori in gastric epithelial AGS and Kato III cells, which was in parallel with a time-course stimulation of IL-8 production.…”

Potentiation of Polarized Intestinal Caco-2 Cell Responsiveness to Probiotics Complexed with Secretory IgA

“…The formation of a polarized Caco-2 cell monolayer at week 3 was established by morphology and monitoring of the transepithelial electrical resistance (TER; ohms ϫ cm 2 ) using a Millicell-ERS apparatus (Millipore, Bedford, MA). TER values of well-differentiated monolayers were in the range of 380 -450 ohms ϫ cm 2 (13). Bacterial Strains-The bacterial strains L. rhamnosus CGMCC 1.3724 and B. lactis CNCM I-3446 were obtained from the China general microbiological culture collection and Pasteur Institute microbiological culture collection, respectively.…”

“…Analysis of NF-B Nuclear Translocation and IB␣ Evaluation-Preparation of Caco-2 cell small-scale nuclear extracts and use in EMSA for the detection of NF-B was carried out as described in Cottet et al (13). The radioactivity associated with retarded oligonucleotide-NF-B complexes was quantified on an Instant Imager reader (Packard, Palo Alto, CA).…”

Reconstituted Human Polyclonal Plasma-derived Secretory-like IgM and IgA Maintain the Barrier Function of Epithelial Cells Infected with an Enteropathogen

“…Cells cultivated up to 80% confluency were seeded on polyester Snapwell filters (diameter, 12 mm; pore size, 0.4 m; Corning Costar) at a density of 0.8 ϫ 10 5 cells/cm 2 . At week 3, the Caco-2 cell monolayer integrity was checked by measuring the TER using the Millicell-electrical resistance system (ERS) device (Millipore) (34). TER values of well differentiated monolayers were in the range of 400 -500 ohm/cm 2 .…”