title: Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood.
abstract: BACKGROUND
  Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring.
  
  
  METHODS
  With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population.
  
  
  RESULTS
  Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation.
  
  
  CONCLUSIONS
  This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.
authors.affiliation: Department of Molecular Medicine, The Rayne Institute, King's College School of Medicine and Dentistry, London, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Mendes
authors.forename: K V
authors.initials: KV
authors.lastname: Bromelow
authors.forename: M
authors.initials: M
authors.lastname: Westby
authors.forename: J
authors.initials: J
authors.lastname: Galea-Lauri
authors.forename: I E
authors.initials: IE
authors.lastname: Smith
authors.forename: M E
authors.initials: ME
authors.lastname: O'Brien
authors.forename: B E
authors.initials: BE
authors.lastname: Souberbielle
mesh_terms: D020126:Brefeldin A / Q000494:pharmacology; D017252:CD3 Complex / Q000032:analysis; D019002:CD56 Antigen / Q000032:analysis; D016207:Cytokines / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000187:drug effects / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*
chemical_list: D017252:CD3 Complex; D019002:CD56 Antigen; D016207:Cytokines; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D008985:Monensin