12 research outputs found
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 allergenâinduced activation of eosinophil NFâÎșB and generation of granulocyte macrophageâcolony stimulating factor (GMâCSF), TNFâα, and ILâ8.Methods Peripheral blood eosinophils were enriched to >99.9% by Percoll gradient sedimentation and negative magnetic affinity chromatography. NFâÎșB activation by 10âÎŒg/mL house dust mite (HDM) extract was demonstrated immunocytochemically using a monoclonal antibody against the active form of NFâÎșB (NFâÎșBa). The authenticity of NFâÎșB was confirmed by Western blot. Cytokine production was assessed both by immunoâstaining of eosinophils and by assay of cytokines in the cell supernatant.Results Activation of peripheral blood eosinophils from atopic, but not nonâatopic, donors induced activation of NFâÎșB, which peaked at 4âh and was accompanied by a decline in IÎșBâα. The activation of authentic NFâÎșB was confirmed in gel shift assays. Supershift assays showed p65 to be the major subunit of eosinophil NFâÎșB. Immunofluorescent confocal microscopy demonstrated localization of NFâÎșBa to the nucleus. Following activation, cytokine immunoreactivity was seen in a fraction of the eosinophils and cytokines were released into the supernatant. The NFâÎșB inhibitors, calpain inhibitor 1 (10âÎŒm), pentoxifylline (0.5âmm), pyrrolidine dithiocarbamate (PDTC, 10âÎŒm) or gliotoxin (1âpg/mL) reduced the generation of GMâCSF, TNFâα and ILâ8 in parallel with their inhibition of NFâÎșB.Conclusions HDM allergen activates human eosinophil NFâÎșB leading to the production of the cytokines GMâCSF, TNFâα and ILâ8. We speculate that a role for eosinophil NFâÎșBâdependent cytokines is to act as an autocrine loop augmenting the survival of eosinophils in vivo.</p