Simultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy

Abstract

Background: Phosphorylation of p38 MAPK is a crucial step in IgE-receptor signaling in basophils. The relation of p38 MAPK to the well-validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model. Methods: Expression of CD63 and phosphorylation of p38 MAPK were analyzed flow cytometrically in anti-IgE-gated basophils from 18 birch pollen allergic patients, five grass pollen allergic patients, and five healthy individuals, after 3 and 20 min of stimulation with recombinant major birch pollen allergen (rBet v 1). Additional time points and the influence of p38 MAPK inhibitor SB203580 were studied in birch pollen allergic patients. Results: Phospho-p38 MAPK and CD63 were expressed dose-dependently in birch pollen allergic patient basophils within 1 minute of rBet v 1 stimulation. P38 MAPK phosphorylation was fastest and subsided gradually while CD63 expression remained elevated for at least 20 min. Inhibition of p38 MAPK significantly inhibited CD63 upregulation. With optimal stimulation of the cells (1 μg/mL), sensitivity and specificity for the discrimination between patients and a group of control individuals (grass pollen allergic patients and healthy controls) were 94% and 100% for CD63 at 3 and 20 min and for phospho-p38 MAPK at 3 min. Conclusion: Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation. It is a sensitive and specific intracellular marker for allergy diagnosis and offers new insight into the mechanisms of basophil activation. © 2008 Clinical Cytometry Society.