Helicobacter pylori outer membrane vesicle proteins induce human eosinophil degranulation via a β2 Integrin CD11/CD18- and ICAM-1-dependent mechanism

Abstract

Eosinophil cationic protein (ECP), a cytotoxic protein contained in eosinophils granules, can contribute to various inflammatory responses. Although Helicobacter pylori infection increases infiltration of eosinophils, the mechanisms of eosinophil degranulation by H. pylori infection are largely unknown. The goal of this study was to investigate the role of H. pylori outer membrane vesicles (OMVs) in modulating eosinophil degranulation. We found that eosinophils treated with H. pylori OMVs released significantly more ECP compared with untreated controls. In addition, eosinophils cocultured with OMV-preexposed primary gastric epithelial cells exhibited significantly increased ECP release. Similarly, eosinophils cocultured with culture supernatant (CM) from primary gastric epithelial cells exposed to OMVs (OMV-CM) released significantly higher amounts of ECP compared with eosinophils cocultured with CM from unexposed control cells. Furthermore, OMVs and OMV-CM both induced the upregulation of ICAM-1 on gastric epithelial cells and β2 integrin CD11b on eosinophils. In addition, both transduction of ICAM-1 shRNA into gastric epithelial cells and treatment with neutralizing mAbs to CD18 significantly decreased OMV-mediated or OMV-CM-mediated release of ECP. These results suggest that the eosinophil degranulation response to H. pylori OMVs occurs via a mechanism that is dependent on both β2 integrin CD11/CD18 and ICAM-1.

Figure 1

Degranulation of eosinophils stimulated with H. pylori OMVs. (a) Freshly isolated human eosinophils were stimulated with H. pylori OMVs (200 μg/mL) for 24 h. Cells were analyzed by TEM (×12,000). OMV-treated cells exhibited cytoplasmic degranulation (arrowhead). (b) Human eosinophils were stimulated with OMVs (200 μg/mL) obtained from wild-type H. pylori or the indicated isogenic mutants for 24 h. The concentrations of ECP in the culture supernatants were measured by ELISA (mean ± SEM, n = 5). ^* P < 0.05 control; NS, statistically nonsignificant.

Figure 2

ECP release from eosinophils stimulated with intact H. pylori OMVs or individual components of OMVs. (a) ECP release is induced in a dose-dependent manner by H. pylori OMVs. Freshly isolated human eosinophils were treated with the indicated concentrations of H. pylori OMVs for 24 h. (b) Human eosinophils were treated with H. pylori LPS (1,000 ng/mL), heat-treated OMVs (200 μg/mL), intact OMVs (200 μg/mL), or purified VacA (1,000 ng/mL) for 24 h. The concentration of ECP in each culture supernatant was determined by ELISA (mean ± SEM, n = 5). ^* P < 0.05; NS, statistically nonsignificant.

Figure 3

Effects of H. pylori OMV-exposed gastric epithelial cells on ECP released from eosinophils. (a) Primary human gastric epithelial cells (GECs) were exposed to either OMVs (200 μg/mL) or heat-treated OMVs (200 μg/mL) for 24 h and then washed twice in PBS. Human eosinophils were cocultured with either OMV-exposed or -unexposed GECs for 24 h. The concentrations of ECP in the culture supernatants were measured by ELISA (mean ± SEM, n = 5). ^* P < 0.05; NS, statistically nonsignificant. (b) Primary human GECs were cultured for 24 h in either the presence or absence of OMVs (200 μg/mL). Culture supernatants were then collected as described in Materials and Methods. GECs were stimulated with control-CM (50% v/v) or OMV-CM (50% v/v) for 24 h and then washed twice in PBS. Finally, control-CM-exposed or OMV-CM-exposed GECs were cocultured with human eosinophils for 24 h. The concentrations of ECP in the culture supernatants were measured by ELISA (mean ± SEM, n = 5). ^* P < 0.05.

Figure 4

ICAM-1 expression in gastric epithelial cells stimulated with H. pylori OMVs or OMV-induced culture supernatant. (a) OMV-CM and control-CM were prepared as described in Materials and Methods. Primary human gastric epithelial cells were stimulated with OMVs (200 μg/mL), OMV-CM (50% v/v), or control CM (50% v/v) for 24 h. Cells were stained with a mAb against ICAM-1 and then analyzed using flow cytometry. Results are representative of more than five independent experiments. (b) Time course of ICAM-1 mRNA expression in gastric epithelial cells after stimulation with OMVs, OMV-CM, or control-CM. Cells were stimulated with OMVs (200 μg/mL), OMV-CM (50% v/v), or control-CM (50% v/v) for the indicated periods of time. The expression levels of ICAM-1 and β-actin mRNA were analyzed by quantitative RT-PCR using standard RNAs. Values are expressed as mean ± SD (n = 5). Asterisks indicate statistical significance after comparison with unstimulated controls (P < 0.05).

Figure 5

Increased CD11b expression in eosinophils stimulated with H. pylori OMVs or OMV-CM. Freshly isolated human eosinophils were stimulated with OMVs (200 μg/mL), OMV-CM (50% v/v), or control-CM (50% v/v) for 24 h. Cells were stained with mAb against CD11b (a), CD11a (b), or CD11c (c) and then analyzed using flow cytometry. Results are representative of more than five independent experiments.

Figure 6

Relationship between ICAM-1 suppression and ECP release from eosinophils. (a) Primary human gastric epithelial cells were transduced with lentivirus harboring either shRNA directed against ICAM-1 or control shRNA. Transduced cells were then stimulated with TNF-α (20 ng/mL) for 1 h. The cellular levels of ICAM-1 and actin were determined by immunoblot analysis. Results are representative of more than three independent experiments. (b) Transduced or nontransduced gastric epithelial cells were stimulated with OMVs (200 μg/mL), OMV-CM (50% v/v), or control-CM (50% v/v) for 24 h. Cells were stained with a mAb against ICAM-1 and then analyzed by flow cytometry. Data are represented as MFI ± SEM (n = 5). (c) Transduced or nontransduced gastric epithelial cells were exposed to OMVs (200 μg/mL) for 24 h and then washed twice in PBS. OMV-exposed gastric epithelial cells were cocultured with human eosinophils for 24 h (left panel). Transduced or nontransduced gastric epithelial cells were stimulated with control-CM (50% v/v) or OMV-CM (50% v/v) for 24 h and then washed twice in PBS. Finally, human eosinophils were cocultured with either control-CM-stimulated or OMV-CM-stimulated epithelial cells for 24 h (right panel). The concentration of ECP in each culture supernatant was measured by ELISA (mean ± SEM, n = 5). ^* P < 0.05.

Figure 7

Effects of anti-CD18 mAb on ECP released from eosinophils. (a) Eosinophils were cocultured either with unexposed control cells or with OMV-exposed primary human gastric epithelial cells (GECs) for 24 h in the presence of either anti-CD18 mAb or mouse IgG isotype control Ab. The concentration of ECP in each culture supernatant was measured by ELISA (mean ± SEM, n = 5). (b) Human eosinophils were cocultured either with OMV-CM-exposed or control CM-exposed GECs for 24 h in the presence of either anti-CD18 mAb or mouse IgG isotype control Ab. The concentration of ECP in each culture supernatant was measured by ELISA (mean ± SEM, n = 5). ^* P < 0.05 compared with unstimulated control; NS, statistically nonsignificant.