title: Cytokine-induced protein tyrosine phosphorylation is essential for cytokine priming of human eosinophils.
abstract: BACKGROUND
  Human eosinophils are strongly modulated by the eosinophilotrophic cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). A clear intracellular effect of these cytokines is the induction of tyrosine phosphorylation of multiple cellular substrates. However, the relevance of tyrosine phosphorylation for eosinophil functioning has not been established.
  
  
  OBJECTIVE
  In this study we have investigated dose-response and time curves of IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation in eosinophils. Moreover, we have evaluated the importance of IL-5-induced tyrosine phosphorylation for priming of human eosinophils.
  
  
  METHODS
  Cytokine-induced tyrosine phosphorylation was monitored on western blot with an antiphosphotyrosine antibody (4G10). To probe the relevance of tyrosine phosphorylation for priming, eosinophils were primed with IL-5 in the presence of the tyrosine kinase inhibitor herbimycin A. Platelet activating factor (PAF) was used as a control priming agent. Subsequently, the eosinophils were incubated with serum-treated zymosan (STZ) to activate the respiratory burst. Binding of STZ was determined by FACS analysis.
  
  
  RESULTS
  IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation was found at concentrations that primed eosinophil effector mechanism (median effective dose values: approximately 5.10(-11) mol/L, approximately 5.10(-10) mol/L, and approximately 5.10(-12) mol/L for IL-5, IL-3, and GM-CSF, respectively). Cytokine-induced tyrosine phosphorylation was transient with an optimum value at 15 minutes. IL-5 priming of STZ-induced activation of the respiratory burst was blocked by herbimycin A, whereas PAF still primed this response. In fact, herbimycin A inhibited IL-5 priming of STZ binding to human eosinophils. On the other hand, PAF priming of STZ binding was not affected by herbimycin A. Both IL-5-induced and PAF-induced tyrosine phosphorylation were inhibited by herbimycin A.
  
  
  CONCLUSIONS
  These data demonstrate for the first time that IL-5 priming of opsonized particle-induced responses is mediated by tyrosine kinase activity in human eosinophils.
authors.affiliation: Department of Pulmonary Diseases, University Hospital Utrecht, The Netherlands.
authors.forename: T
authors.initials: T
authors.lastname: van der Bruggen
authors.forename: D
authors.initials: D
authors.lastname: Kanters
authors.forename: A T
authors.initials: AT
authors.lastname: Tool
authors.forename: J A
authors.initials: JA
authors.lastname: Raaijmakers
authors.forename: J W
authors.initials: JW
authors.lastname: Lammers
authors.forename: A J
authors.initials: AJ
authors.lastname: Verhoeven
authors.forename: L
authors.initials: L
authors.lastname: Koenderman
mesh_terms: D016227:Benzoquinones; D016207:Cytokines / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004804:Eosinophils / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000494:pharmacology; D015848:Interleukin-5 / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D016897:Respiratory Burst / Q000187:drug effects; D017828:Rifabutin / Q000031:analogs & derivatives; D014443:Tyrosine / Q000378:metabolism*; D015054:Zymosan / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D016207:Cytokines; D004791:Enzyme Inhibitors; D007377:Interleukin-3; D015848:Interleukin-5; D047029:Lactams, Macrocyclic; D010972:Platelet Activating Factor; D011506:Proteins; D011809:Quinones; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015054:Zymosan; D011505:Protein-Tyrosine Kinases