Allergen activates peripheral blood eosinophil nuclear factor-kappaB to generate granulocyte macrophage-colony stimulating factor, tumour necrosis factor-alpha and interleukin-8

Background Allergic inflammation is characterized by the influx and activation of eosinophils. Cytokines generated by both resident and infiltrating cells are responsible for the initiation and maintenance of this pathogenesis. This study focuses on allergen‐induced activation of eosinophil NF‐κB and generation of granulocyte macrophage‐colony stimulating factor (GM‐CSF), TNF‐α, and IL‐8.Methods Peripheral blood eosinophils were enriched to &gt;99.9% by Percoll gradient sedimentation and negative magnetic affinity chromatography. NF‐κB activation by 10 μg/mL house dust mite (HDM) extract was demonstrated immunocytochemically using a monoclonal antibody against the active form of NF‐κB (NF‐κBa). The authenticity of NF‐κB was confirmed by Western blot. Cytokine production was assessed both by immuno‐staining of eosinophils and by assay of cytokines in the cell supernatant.Results Activation of peripheral blood eosinophils from atopic, but not non‐atopic, donors induced activation of NF‐κB, which peaked at 4 h and was accompanied by a decline in IκB‐α. The activation of authentic NF‐κB was confirmed in gel shift assays. Supershift assays showed p65 to be the major subunit of eosinophil NF‐κB. Immunofluorescent confocal microscopy demonstrated localization of NF‐κBa to the nucleus. Following activation, cytokine immunoreactivity was seen in a fraction of the eosinophils and cytokines were released into the supernatant. The NF‐κB inhibitors, calpain inhibitor 1 (10 μm), pentoxifylline (0.5 mm), pyrrolidine dithiocarbamate (PDTC, 10 μm) or gliotoxin (1 pg/mL) reduced the generation of GM‐CSF, TNF‐α and IL‐8 in parallel with their inhibition of NF‐κB.Conclusions HDM allergen activates human eosinophil NF‐κB leading to the production of the cytokines GM‐CSF, TNF‐α and IL‐8. We speculate that a role for eosinophil NF‐κB‐dependent cytokines is to act as an autocrine loop augmenting the survival of eosinophils in vivo.</p