Identification of the Fc epsilonRI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils

J Allergy Clin Immunol. 1998 Aug;102(2):304-15. doi: 10.1016/s0091-6749(98)70100-9.

Abstract

Background: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production.

Methods and results: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion).

Conclusions: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Basophil Degranulation Test
  • Basophils / enzymology*
  • Blotting, Western
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Fluorescent Antibody Technique, Indirect
  • Histamine Release
  • Humans
  • Phosphorylation
  • Protein-Tyrosine Kinases / metabolism*
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Receptors, IgE / metabolism*
  • Stilbenes / pharmacology
  • ZAP-70 Protein-Tyrosine Kinase
  • src-Family Kinases / metabolism*

Substances

  • Enzyme Inhibitors
  • Receptors, IgE
  • Stilbenes
  • 3,3',4,5'-tetrahydroxystilbene
  • Protein-Tyrosine Kinases
  • Receptor Protein-Tyrosine Kinases
  • TYRO3 protein, human
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human
  • lyn protein-tyrosine kinase
  • src-Family Kinases