Helicobacter pylori-infected gastric mucosa is characterized by infiltration of inflammatory cells such as neutrophils and eosinophils. However, little information is available on the relationship between H. pylori virulence factors and chemokine expression in eosinophils. This study investigates the role of vacuolating cytotoxin (VacA) in chemokine expression from human eosinophils. Eosinophils were isolated from the peripheral blood of healthy volunteers using a magnetic cell separation system. VacA(+) H. pylori water-soluble proteins (WSP) induced higher expression of interleukin-8, growth-related oncogene alpha, monocyte chemotactic protein 1, and RANTES (regulated on activation, normal, T-cell expressed and secreted) than Vac(-) WSP in human eosinophils, as assessed by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. Purified VacA not only increased chemokine expression but also activated p65/p50 NF-kappaB heterodimers and phosphorylated IkappaB kinase (IKK) alpha/beta signals in human eosinophils. Inhibition of NF-kappaB and IKK significantly decreased the chemokine expression in VacA-stimulated eosinophils. Furthermore, VacA-induced NF-kappaB activation and chemokine release from eosinophils were dependent on Ca(2+) influx and mitochondrial generation of reactive oxygen intermediates (ROI). These results suggest that NF-kappaB and IKK signals via Ca(2+) influx and mitochondrial ROI play a role in the up-regulation of chemokine expression in eosinophils stimulated with H. pylori VacA.