Regulation of RANTES Promoter Activation in Gastric Epithelial Cells Infected withHelicobacter pylori

Abstract

RANTES, a CC chemokine, plays an important role in the inflammatory response associated with Helicobacter pylori infection. However, the mechanism by which H. pylori induces RANTES expression in the gastric mucosa is unknown. We cocultured gastric epithelial cells with wild-type H. pylori, isogenic oipA mutants, cag pathogenicity island (PAI) mutants, or double knockout mutants. Reverse transcriptase PCR showed that RANTES mRNA was induced by H. pylori and that the expression was both OipA and cag PAI dependent. Luciferase reporter gene assays and electrophoretic mobility shift assays showed that maximal H. pyloriinduced RANTES gene transcription required the presence of the interferon-stimulated responsive element (ISRE), the cyclic AMP-responsive element (CRE), nuclear factor-interleukin 6 (NF-IL-6), and two NF-B sites. OipA-and cag PAI-dependent pathways included NF-B3NF-B/NF-IL-6/ISRE pathways, and cag PAI-dependent pathways additionally included Jun N-terminal kinase3CRE/NF-B pathways. The OipAdependent pathways additionally included p383CRE/ISRE pathways. We confirmed the in vitro effects in vivo by examining RANTES mRNA levels in biopsy specimens from human gastric antral mucosa. RANTES mRNA levels in the antral mucosa were significantly higher for patients infected with cag PAI/OipA-positive H. pylori than for those infected with cag PAI/OipA-negative H. pylori or uninfected patients. The mucosal inflammatory response to H. pylori infection involves different signaling pathways for activation of the RANTES promoter, with both OipA and the cag PAI being required for full activation of the RANTES promoter. Gastric epithelial damage inHelicobacter pylori infection is thought to be related to the cellular and humoral inflammatory response to the infection. The cellular immune response to the organism initially consists of the recruitment of neutrophils, followed by T and B lymphocytes, plasma cells, and macrophages. Members of the chemokine supergene family, particularly the CXC and CC chemokine subfamilies, are thought to be responsible for recruitment of these inflammatory cells into the gastric mucosa (7,8,14,28,31,(35)(36)(37). RANTES (regulated on activation normal T cell expressed and secreted) is a CC chemokine produced by epithelial cells, CD8 ϩ T cells, fibroblasts, and platelets that mediates the trafficking and homing of classical lymphoid cells such as T cells and monocytes. RANTES also acts on a range of other cells, including basophils, eosinophils, natural killer cells, dendritic cells, and mast cells (2, 30).Increased RANTES production is a feature of H. pyloriinduced gastric inflammation (7,14,28,31,37). RANTES mRNA expression is also thought to play an important role in maintaining residual memory T lymphocytes and eosinophils in gastric mucosa following H. pylori eradication (14). The mechanisms responsible for inducible RANTES gene transcription associated with H. pylori infections have not been fully elucidated. Mori et al. (23) recently reported that the regulation of RANTES gene trans...

Pattern of Transcription Factor Activation in Helicobacter pylori–Infected Mongolian Gerbils

“…Phosphorylation of JNK and IκB was induced in a time-dependent manner through 18 months, a pattern similar to the activation of NF-κB and ISRE binding. These data agree with results obtained from human gastric cells infected with H pylori, showing that JNK and IκB are involved in upstream NF-κB signaling and that p38 and IκB are upstream of ISRE 4,15,22,26 Immunoblots were used to examine mucosal samples from the 3 groups at 18 months. Levels of phosphorylated ERK were highest in the ulcer group, followed by the gastritis and the atrophy groups (see Supplementary Figure 3A online at www.gastrojournal.org).…”

“…In previous in vitro studies using human gastric cancer cell lines, we showed that both p50 and p65 were components of NF-κB binding complexes. 15,22,26 However, because the accuracy of commercially available antibodies against p50 in Mongolian gerbils is unknown, we cannot conclude that p50 was not present.…”

Regulation of IL-18 inHelicobacter pyloriInfection

“…This result also is consistent with prior studies showing that in gastric epithelial cells, OipA, but not the cag PAI induced the p38 pathway (23,26). To test the hypothesis that OipA plays a role in p38 induction in THP-1 cells, we preincubated cells with chemical inhibitors or DMSO at 37°C for 1 h followed by infection with the oipA mutants.…”

“…Importantly, the behavior of gastric epithelial cells and monocytes differed markedly in response to the virulent factors of H. pylori; cag PAI and OipA. Previous studies (23,25,26) have suggested that both cag PAI and OipA were involved in the induction of various cytokines and chemokines in the gastric mucosa, such as IL-6, IL-8, and RANTES cells. Although the relationship between OipA and IL-8 remains unclear in epithelial cells (36), there is agreement that the cag PAI does not play a role in the induction of these cytokines in THP-1 cells (37).…”

“…H. pylori strain TN2GF4 was isolated from a Japanese gastric ulcer patient and was reported to induce gastric cancer in Mongolian gerbils (24). We also used its isogenic oipA mutants (25) and the whole cag PAI deleted mutants (26). H. pylori were cultured on brain heart infusion agar plates containing 7% horse blood in a microaerobic atmosphere at 37°C.…”