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Abstract
Helicobacter pylori-infected gastric mucosa is characterized by infiltration of various inflammatory cells such as neutrophils and eosinophils. Although several mechanisms for neutrophil infiltration are well known, there has been little known the role of eotaxin, which is a potent chemoattractant for eosinophils, on the inflammatory process of H. pylori infection. The present study was to investigate the mechanisms of eotaxin expression in gastric epithelial cells stimulated with H. pylori vacuolating cytotoxin (VacA). Stimulation with VacA purified from VacA+ H. pylori slightly increased eotaxin expression in MKN-45 gastric epithelial cells. In contrast, the combined stimulation with VacA and IL-4 synergistically increased the eotaxin expression as determined by quantitative RT-PCR and ELISA. In MKN-45 cells transfected with an eotaxin promoter-luciferase reporter plasmid, costimulation with VacA and IL-4 induced more luciferase activity than either VacA or IL-4 alone did. However, such up-regulation was significantly decreased in the cells transfected with luciferase reporter plasmid bearing an eotaxin promoter which has a mutation at STAT6 binding site. These results suggest that the up-regulation of eotaxin in VacA-stimulated gastric epithelial cells may be synergistically facilitated by IL-4 via a STAT6-dependent mechanism.
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Figure 1.
Purification of H. pylori vacuolating cytotoxin. (A) Immunoblot analysis of VacA preparations electrophoresed on a 10% acrylamide gel, transferred to nitrocellulose, and reacted with goat anti-VacA antibody. (B) Vacuolation in HeLa cells treated with H. pylori vacuolating cytotoxin. HeLa cells in a 96 well plate were treated with H. pylori vacuolating cytotoxin (20 μg/ml) for 24 h. Vacuolation was observed by an inverted microscopy (× 300).
Figure 2.
RT-PCR for eotaxin expression in MKN-45 cells treated with H. pylori vacuolating cytotoxin and/or IL-4. Confluent monolayers of MKN-45 cells were treated with H. pylori vacuolating cytotoxin (5 μg/ml) and/or IL-4 (50 ng/ml) for the indicated hours, after which cellular RNA was extracted. In this experiment, tissue culture medium was also supplemented with 5 mM NH4Cl. The (+) and (–) represent positive and negative controls, respectively.
Figure 3.
Quantification of eotaxin mRNA molecules in MKN-5 cells treated with H. pylori vacuolating cytotoxin and/or IL-4. Confluent monolayers of MKN-45 cells were treated with H. pylori vacuolating cytotoxin (5 μg/ml, VacA) and/or IL-4 (50 ng/ml) for the indicated hours, after which cellular RNA was extracted. Quantification of eotaxin and β-actin mRNA molecules was performed as described by Materials and Methods. In this experiment, tissue culture medium was also supplemented with 5 mM NH4Cl. The values are expressed as the mean of five different experiments.
Figure 4.
Eotaxin production in MKN-45 cells treated with H. pylori vacuolating cytotoxin and/or IL-4. Culture supernatants of MKN-45 cells were collected 24 h after stimulation with H. pylori vacuolating cytotoxin (5 μg/ml, VacA) and/or IL-4 (50 ng/ml). Tissue culture medium was also supplemented with 5 mM NH4Cl. Protein levels of eotaxin in culture supernatants were determined by ELISA. Data are mean ± SEM (n=5). ∗p<0.05 versus MKN-45 cells in medium without VacA or IL-4. ∗∗p<0.01 versus MKN-45 cells in medium with VacA alone or IL-4 alone.
Figure 5.
The effects of live H. pylori on the eotaxin production in MKN-45 cells treated with IL-4. MKN-45 cells were incubated with IL-4 (50 ng/ml) in the presence of live H. pylori or purified VacA (5 μg/ml). Tissue culture medium was also supplemented with 5 mM NH4Cl. After 24 h of incubation, the levels of eotaxin protein were measured by ELISA. Results are expressed as the mean ± SEM of five experiments. ∗p<0.05 versus MKN-45 cells in medium with IL-4 alone.
Figure 6.
Eotaxin production in IL-4-stimulated MKN-45 cells after incubation with acidified H. pylori vacuolating cytotoxin in the presence or absence of ammonium chloride. MKN-45 cells were incubated with IL-4 (50 ng/ml) in the presence of purified VacA (either acid pH or neutral pH, 5 μg/ml), or in the presence of buffer controls. Tissue culture medium was also either supplemented with 5 mM NH4Cl or not supplemented. After 24 h of incubation, eotaxin production was measured by ELISA. Results are expressed as the mean ± SEM of five experiments. ∗p<0.05 versus MKN-45 cells in medium without NH4Cl.
Figure 7.
Activation of eotaxin reporter gene in MKN-45 cells stimulated with H. pylori vacuolating cytotoxin and IL-4. MKN-45 cells were transfected with pEotaxin-luciferase transcriptional reporter (pEotx.1363). Forty-eight hours after transfection, cells were stimulated with the indicated concentrations of H. pylori vacuolating cytotoxin (VacA) and 50 ng/ml of IL-4 for 6 h. Tissue culture medium was also supplemented with 5 mM NH4Cl. Data are the mean fold induction in luciferase activity relative to unstimulated control. Values are the mean ± SEM of five experiments.
Figure 8.
Activation of the eotaxin promoter by STAT6. MKN-45 cells were transfected with either a wild-type eotaxin promotor luciferase reporter plasmid (pEotx.1363) or a plasmid mutated at binding site for STAT6 (pEotx.M1). Forty-eight hours after transfection, cells were incubated with or without VacA (5 μg/ml) and/or IL-4 (50 ng/ml) for 6 h and then harvested. Tissue culture medium was also supplemented with 5 mM NH4Cl. The luciferase activity normalized to protein concentration and calculated as the fold induction compared with the control vector. The data are presented as the mean ± SEM of five independent experiments. ∗, p<0.05 compared with pEotx.1363
Table 1.
Sequences of oligonucleotide primers for RT-PCR analysis of human eotaxin and β-actin
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