Effect of Helicobacter pylori on polymorphonuclear leukocyte migration across polarized T84 epithelial cell monolayers: role of vacuolating toxin VacA and cag pathogenicity island

Abstract

Helicobacter pylori infection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis. The mechanisms underlying this process are poorly documented. The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced by H. pylori. In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains of H. pylori. We have evaluated the role of the vacuolating cytotoxin VacA and of the cag pathogenicity island (PAI) of H. pylori in PMNL migration via their effects on T84 epithelial cells. We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positive H. pylori strains. In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells with cag PAI-positive and cagE-positive H. pylori strains. Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL. These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that the cag PAI, with a pivotal role for the cagE gene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response.

FIG. 1

(A to D) Effect of the vacuolating cytotoxin VacA (BCF from the VacA⁺ 60190 strain) on the actin cytoskeleton of confluent T84 cell monolayers. Cells were stained by fluorescein isothiocyanate-phalloidin and observed by epifluorescence. VacA does not modify the perijunctional F-actin rings in control cells (A) and VacA-treated T84 monolayers (B) and does not alter the basolateral actin filament network of the T84 epithelial monolayers in control cells (C) and VacA-treated T84 monolayers (D). Bars, 20 μm. (E) Measurements of TER in T84 cell monolayers after 2, 12, and 24 h of incubation with either the BCF from the VacA⁺ 60190 strain or 2 × 10⁻³ M EDTA. Note the lack of effect of VacA (solid squares) on TER during the course of incubation compared to data for control cells (open squares). As expected, monolayers responded to EDTA (solid diamond) by decreasing junctional resistances. Results are means ± SEM for six experiments.

FIG. 2

VacA does not modify the rate of PMNL transmigration across the T84 cell monolayers. T84 monolayers were incubated for 4 h with bacterial suspensions or for 24 h with BCFs from different H. pylori strains. Shown is an MPO assay indicating total numbers of PMNL (PMNL-associated monolayers plus PMNL that transmigrated to the lower reservoirs) after a 2-h transmigration, induced by f-MLP (10⁻⁷ M) or not induced. Results are means ± SEM for 6 to 12 monolayers.

FIG. 3

The effects of H. pylori strain interactions with polarized T84 monolayers on apical membrane structure. (A to C) H. pylori strain 60190. (A) A bacterium (arrowhead) is associated with the brush border, with adherence pedestal formation (arrow). (B) The bacterial membrane (arrowhead) is closely apposed to the epithelial cell membrane, and the brush border architecture disappears at the contact site. (C) Some bacteria are exceptionally internalized (arrowheads). (D and E) 60190:M22 (D) and 60190:C⁻ (E) H. pylori strains are adherent to the brush border. (F) G21 H. pylori strain. No adherence pedestal or association of bacteria (arrowhead) with the epithelial membrane is noted. Bars, 0.5 μm (A to E) and 3 μm (F).

FIG. 4

PMNL transmigration across the T84 cell monolayers is dependent on cag PAI expression. Epithelial cells were incubated for 4 h with bacterial suspensions from different H. pylori strains. The MPO assay indicates total numbers of PMNL (PMNL-associated monolayers plus PMNL that migrated to the lower reservoirs) after a 2-h transmigration. Data are pooled from 6 to 12 individual monolayers for each condition, and results are means ± standard errors. ∗, P < 0.01.

FIG. 5

Colonization of the apical membrane with cag⁺ H. pylori strains imprints a directional chemotactic signal on the underlying matrix. Epithelial cells were incubated for 4 h with bacterial suspensions from different H. pylori strains. The MPO assay indicates total numbers of PMNL (PMNL-associated matrices plus PMNL that migrated to the lower reservoirs) after a 2-h transmigration. Data are pooled from 6 to 12 individual monolayers for each condition, and results are means ± standard errors. ∗, P < 0.01.

FIG. 6

The effect of neutralizing anti-IL-8 antibodies on PMNL migration across T84-derived matrices. The MPO assay indicates total numbers of PMNL (PMNL-associated matrices plus PMNL that migrated to the lower reservoirs) after a 2-h transmigration. Solid bars, absence of IL-8 antibody; hatched bars, presence of IL-8 antibody. Data are pooled from 6 to 12 individual monolayers for each condition, and results are means ± standard errors. ∗, P < 0.01. CE, cell equivalents.