title: Analyzing histamine release by flow cytometry (HistaFlow): a novel instrument to study the degranulation patterns of basophils.
abstract: BACKGROUND
  Stimulated human basophils exhibit different degranulation patterns with release of mediators and appearance of activation markers such as CD63 and CD203c. Traditionally, released mediators are quantified in the supernatant of activated cells, whereas the expression of activation markers by individual cells is analyzed by flow cytometry. Alternatively, intracellular histamine and its release by basophils and mast cells have been repeatedly studied applying an enzyme-affinity-gold method based on the affinity of the histaminase diamine oxidase for its substrate histamine.
  
  
  OBJECTIVE
  To develop a flow cytometric technique enabling to study histamine release by individual basophils in combination with the expression of activation markers. To elucidate the principles of basophil degranulation on a single cell level.
  
  
  METHODS
  Intracellular histamine and its release is analyzed flow cytometrically by an enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells implied flow cytometric quantification of CD63 and CD203c. Stimuli such as allergen, anti-IgE, N-formyl-met-leu-phe (fMLP), phorbol 12-myristate 13-acetate (PMA), ionomycin and interleukin (IL-)3 are applied to obtain different degranulation profiles.
  
  
  RESULTS
  Stimulation with anti-IgE, allergen, fMLP and PMA±ionomycin induces a rapid release of histamine that can be analyzed flow cytometrically. Analyses on a single cell level reveal that histamine release is not restricted to cells showing significant up-regulation of CD63. Alternatively, up-regulation of CD203c does not per se indicate histamine release. In some patients, priming of cells with IL-3 not only facilitates basophil responsiveness but also implies an increased ability of DAO to label the cells.
  
  
  CONCLUSIONS
  This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation.
authors.affiliation: Immunology-Allergology-Rheumatology, University of Antwerp, Antwerpen, Belgium. immuno@ua.ac.be
authors.forename: Didier G
authors.initials: DG
authors.lastname: Ebo
authors.forename: Chris H
authors.initials: CH
authors.lastname: Bridts
authors.forename: Christel H
authors.initials: CH
authors.lastname: Mertens
authors.forename: Margo M
authors.initials: MM
authors.lastname: Hagendorens
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Stevens
authors.forename: Luc S
authors.initials: LS
authors.lastname: De Clerck
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000494:pharmacology; D006631:Amine Oxidase (Copper-Containing) / Q000378:metabolism; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D015549:Basophil Degranulation Test / Q000295:instrumentation* / Q000379:methods; D001491:Basophils / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D002648:Child; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D006632:Histamine / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D006636:Histamine Release / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D007377:Interleukin-3 / Q000276:immunology / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011755:Pyrophosphatases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D060149:Tetraspanin 30 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D055815:Young Adult
chemical_list: D000485:Allergens; D000888:Antibodies, Anti-Idiotypic; C554586:CD63 protein, human; C427944:ENPP3 protein, human; C508596:IL3 protein, human; D007377:Interleukin-3; D010703:Phorbol Esters; D060149:Tetraspanin 30; C026408:anti-IgE antibodies; C119222:phorbol-12-myristate; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; C042205:methionyl-leucyl-phenylalanine; D006632:Histamine; D006631:Amine Oxidase (Copper-Containing); D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases