title: Allergen activates peripheral blood eosinophil nuclear factor-kappaB to generate granulocyte macrophage-colony stimulating factor, tumour necrosis factor-alpha and interleukin-8.
abstract: 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-kappaB and generation of granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-alpha, and IL-8.
  
  
  METHODS
  Peripheral blood eosinophils were enriched to >99.9% by Percoll gradient sedimentation and negative magnetic affinity chromatography. NF-kappaB activation by 10 microg/mL house dust mite (HDM) extract was demonstrated immunocytochemically using a monoclonal antibody against the active form of NF-kappaB (NF-kappaBa). The authenticity of NF-kappaB 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-kappaB, which peaked at 4 h and was accompanied by a decline in IkappaB-alpha. The activation of authentic NF-kappaB was confirmed in gel shift assays. Supershift assays showed p65 to be the major subunit of eosinophil NF-kappaB. Immunofluorescent confocal microscopy demonstrated localization of NF-kappaBa to the nucleus. Following activation, cytokine immunoreactivity was seen in a fraction of the eosinophils and cytokines were released into the supernatant. The NF-kappaB inhibitors, calpain inhibitor 1 (10 microm), pentoxifylline (0.5 mm), pyrrolidine dithiocarbamate (PDTC, 10 microm) or gliotoxin (1 pg/mL) reduced the generation of GM-CSF, TNF-alpha and IL-8 in parallel with their inhibition of NF-kappaB.
  
  
  CONCLUSIONS
  HDM allergen activates human eosinophil NF-kappaB leading to the production of the cytokines GM-CSF, TNF-alpha and IL-8. We speculate that a role for eosinophil NF-kappaB-dependent cytokines is to act as an autocrine loop augmenting the survival of eosinophils in vivo.
authors.affiliation: Division of Infection, Inflammation and Repair, School of Medicine, Southampton General Hospital, Southampton UK.
authors.forename: W R
authors.initials: WR
authors.lastname: Coward
authors.forename: H
authors.initials: H
authors.lastname: Sagara
authors.forename: S J
authors.initials: SJ
authors.lastname: Wilson
authors.forename: S T
authors.initials: ST
authors.lastname: Holgate
authors.forename: M K
authors.initials: MK
authors.lastname: Church
mesh_terms: D000704:Analysis of Variance; D039741:Antigens, Dermatophagoides / Q000494:pharmacology*; D060829:Arthropod Proteins; D016022:Case-Control Studies; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D016207:Cytokines / Q000096:biosynthesis*; D004804:Eosinophils / Q000378:metabolism*; D005912:Gliotoxin / Q000494:pharmacology; D006023:Glycoproteins / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D016209:Interleukin-8 / Q000096:biosynthesis; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010431:Pentoxifylline / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D016207:Cytokines; C451163:Dermatophagoides pteronyssinus antigen p 2; D006023:Glycoproteins; D020672:I-kappa B Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D013859:Thiocarbamates; D014409:Tumor Necrosis Factor-alpha; C039304:calpain inhibitors; C066229:prolinedithiocarbamate; D005912:Gliotoxin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011392:Proline; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1; D010431:Pentoxifylline