title: Hypoxia causes IL-8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid-induced apoptosis in human eosinophils.
abstract: BACKGROUND
  Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils.
  
  
  OBJECTIVE
  We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis.
  
  
  METHODS
  Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR.
  
  
  RESULTS
  Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia.
  
  
  CONCLUSIONS
  These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: L M
authors.initials: LM
authors.lastname: Porter
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: A S
authors.initials: AS
authors.lastname: Cowburn
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: N
authors.initials: N
authors.lastname: Farahi
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: J
authors.initials: J
authors.lastname: Deighton
authors.affiliation: Faculty of Life Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK.
authors.forename: S N
authors.initials: SN
authors.lastname: Farrow
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: C A
authors.initials: CA
authors.lastname: Fiddler
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: J K
authors.initials: JK
authors.lastname: Juss
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Condliffe
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK.
authors.forename: E R
authors.initials: ER
authors.lastname: Chilvers
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004804:Eosinophils / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000473:pathology; D007249:Inflammation / Q000276:immunology / Q000473:pathology; D016209:Interleukin-8 / Q000378:metabolism*
chemical_list: D000893:Anti-Inflammatory Agents; D005938:Glucocorticoids; D016209:Interleukin-8; D003907:Dexamethasone
keywords: IL-8; apoptosis; corticosteroids; eosinophils; hypoxia