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1.
Eur J Immunol ; 36(6): 1374-85, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16708405

ABSTRACT

Repetitive antigen stimulation induces peripheral T cell tolerance in vivo. It is not known, however, whether multiple stimulations merely suppress T cell activation or, alternatively, change the transcriptional program to a distinct, tolerant state. In this study, we have discovered that STAT3 and STAT5 were activated in response to antigen stimulation in vivo, in marked contrast to the suppression of AP-1, NF-kappaB and NFAT. In addition, a number of transcription factors were induced in tolerant T cells following antigen challenge in vivo, including T-bet, Irf-1 and Egr-2. The altered transcription program in tolerant cells associates closely with the suppression of cell cycle progression and IL-2 production, as well as with the induction of IL-10. Studies of T-bet and Egr-2 show that the function of T-bet in peptide treatment-induced regulatory T cells is not associated with Th1 differentiation, but correlates with the suppression of IL-2, whereas expression of Egr-2 led to an up-regulation of the cell cycle inhibitors p21(cip1) and p27(kip). Our results demonstrate a balanced transcription program regulated by different transcription factors for T cell activation and/or tolerance during antigen-induced T cell responses. Persistent antigen stimulation can induce T cell tolerance by changing the balance of transcription factors.


Subject(s)
Epitopes, T-Lymphocyte/immunology , T-Lymphocytes, Regulatory/immunology , Transcription, Genetic/immunology , Animals , Cell Cycle/immunology , Cell Nucleus/immunology , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/immunology , Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/immunology , Early Growth Response Protein 2/biosynthesis , Early Growth Response Protein 2/genetics , Early Growth Response Protein 2/immunology , Gene Expression Profiling , Gene Expression Regulation/immunology , Immune Tolerance/immunology , Interleukin-2/biosynthesis , Interleukin-2/immunology , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , RNA/chemistry , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/immunology , T-Box Domain Proteins , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolism , Transfection
2.
J Biol Chem ; 281(17): 11637-48, 2006 Apr 28.
Article in English | MEDLINE | ID: mdl-16513653

ABSTRACT

Host-pathogen interactions that allow Helicobacter pylori to survive and persist in the stomach of susceptible individuals remain unclear. Human beta-defensins (hBDs), epithelial-derived antimicrobial peptides are critical components of host-defense at mucosal surfaces. The role of H. pylori-mediated NF-kappaB and epidermal growth factor receptor (EGFR) activation on beta-defensin expression was investigated. Transient transfection studies utilizing beta-defensin promoter constructs were conducted in gastric cells with contribution of individual signaling events evaluated by the addition of specific inhibitors, small interference nucleotide-binding oligomerization domain 1 (NOD1) RNA or plasmids encoding Vaccinia virus proteins that interrupt interleukin-1 and Toll-like receptor signaling. The role of individual MAPK pathways was further delineated in HEK-293 cells expressing conditional MAPK mutants. We found hBD2 expression exclusively dependent on the presence of the bacterial cag pathogenicity island, with NOD1 a critical host sensor. Impairment of murinebeta-defensin 4 (an orthologue of hBD2) expression in NOD1-deficient mice 7-days post-infection further confirmed the role of this cytoplasmic pattern-recognition receptor in eliciting host innate immunity. In contrast to hBD2, hBD3 expression was NOD1-independent but EGFR and ERK pathway-dependent. Importantly, Toll-like receptor signaling was not implicated in H. pylori-mediated hBD2 and hBD3 gene expression. The divergent signaling events governing hBD2 and hBD3 expression suggest temporal functional variation, such that hBD2 may contribute to antimicrobial barrier function during the inflammatory phase with hBD3 playing a greater role during the repair, wound healing phase of infection.


Subject(s)
Carrier Proteins/physiology , ErbB Receptors/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori/pathogenicity , beta-Defensins/genetics , Animals , Carrier Proteins/genetics , Cells, Cultured , ErbB Receptors/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gastric Mucosa/cytology , Gastric Mucosa/metabolism , Helicobacter Infections/pathology , Humans , Interleukin-1/antagonists & inhibitors , Interleukin-1/genetics , Interleukin-1/metabolism , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Promoter Regions, Genetic/genetics , RNA, Small Interfering/pharmacology , Signal Transduction , Toll-Like Receptors/antagonists & inhibitors , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism , Transcriptional Activation , Transfection , beta-Defensins/metabolism
3.
Clin Cancer Res ; 9(6): 2015-21, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12796363

ABSTRACT

PURPOSE: Helicobacter pylori causes gastric damage and is involved in gastric carcinogenesis. Vascular endothelial growth factor (VEGF) plays a major role in gastric mucosa repair and is overexpressed in gastric cancer. We investigated: (a) whether H. pylori, and in particular H. pylori VacA toxin, affected VEGF expression in gastric epithelial cells in culture; and (b) the signal transduction pathway involved in any effect exerted by H. pylori. EXPERIMENTAL DESIGN: MKN-28 cells were incubated with uninoculated BCF (control) or with BCF obtained from VacA-producing wild-type H. pylori 60190 strain or from its isogenic mutant 60190:v1, specifically lacking vacA gene in the presence or absence of ZD 1839, a selective inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, PD098059, a selective inhibitor of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase, the kinase responsible for ERK phosphorylation, or SC-236, a selective inhibitor of cyclooxygenase (COX)-2 for 24-48 h. RESULTS: (a) Toxigenic H. pylori up-regulated VEGF mRNA and protein expression and caused a 2.5-fold increase in VEGF release compared with control, whereas nontoxigenic H. pylori did not; (b) H. pylori VacA toxin-induced up-regulation of VEGF was counteracted by selective inhibition of EGFR tyrosine kinase; (c) toxigenic H. pylori activated the ERK/MAP kinase cascade, and inhibition of MAP kinase activation counteracted H. pylori-induced VEGF up-regulation; (d) toxigenic H. pylori up-regulated COX-2 expression, and this effect was counteracted by blockade of EGFR tyrosine kinase; and (e) COX-2 selective inhibition counteracted H. pylori-induced up-regulation of VEGF. CONCLUSION: (a) H. pylori up-regulates VEGF expression in gastric epithelial cells; and (b) this effect is specifically related to VacA toxin and seems to depend on the activation of an EGFR-, MAP kinase-, and COX-2-mediated pathway.


Subject(s)
Bacterial Proteins/physiology , ErbB Receptors/physiology , Helicobacter pylori/pathogenicity , Isoenzymes/physiology , Prostaglandin-Endoperoxide Synthases/physiology , Stomach Neoplasms/metabolism , Stomach Neoplasms/microbiology , Vascular Endothelial Growth Factor A/biosynthesis , Cyclooxygenase 2 , Gefitinib , Humans , MAP Kinase Signaling System/physiology , Membrane Proteins , Quinazolines/pharmacology , RNA, Messenger/analysis , Signal Transduction , Up-Regulation , Vascular Endothelial Growth Factor A/genetics
4.
Am J Physiol Gastrointest Liver Physiol ; 283(3): G511-20, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12181162

ABSTRACT

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Bacterial Proteins/pharmacology , Gastric Mucosa/drug effects , Gastric Mucosa/physiology , Vacuoles/physiology , Ammonia/pharmacology , Cell Division/drug effects , Cells, Cultured , Chloride Channels/antagonists & inhibitors , Diphtheria Toxin/pharmacology , Gastric Mucosa/cytology , Gastric Mucosa/ultrastructure , Humans , Nitrobenzoates/pharmacology , Prostaglandin Antagonists/pharmacology , Ricin/pharmacology , Vacuoles/drug effects , Vacuoles/ultrastructure
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