Initial FcεRI-mediated signal strength plays a key role in regulating basophil signaling and deactivation

Background: Mediator releases after high-affinity IgE receptor (FcεRI) cross-linking in basophils and mast cells crucially govern the symptoms of allergic disease and amplify underlying T _H2-type responses. Interestingly, the dose-response curve for FcεRI activation is bell-shaped, with supraoptimal stimulation leading to reduced mediator release. Objective: We sought to characterize the mechanisms responsible for this control of FcεRI-triggered basophil activation. Methods: Human basophils were purified by means of Ficoll density centrifugation, elutriation, and negative selection with immunomagnetic beads. Various intracellular signal protein activities were assessed by means of Western blotting, and mediator releases were analyzed either spectrofluorometrically (histamine) or by means of ELISA (IL-4 and IL-13). Results: Supraoptimal anti-IgE concentrations led to lower mediator release than optimal concentrations but simultaneously to considerably faster histamine release kinetics. In parallel, basophil signaling proteins (Syk, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinases 1 and 2) were more rapidly phosphorylated at higher anti-IgE concentrations but more transiently activated in the supraoptimal range. This endogenous regulation most likely involved src homology 2 domain-containing inositol 5′ phosphatase (SHIP), which was highly phosphorylated after supraoptimal anti-IgE triggering compared with lower stimulus concentrations. Conversely, N-formyl-methionyl-leucyl-phenylalanine-stimulated basophils failed to phosphorylate SHIP in the supraoptimal concentration range and did not display a bell-shaped dose-response curve. Conclusion: The kinetics of IgE-mediated signaling and mediator release in primary human FcεRI ⁺ cells varies substantially according to the magnitude of stimulation, and SHIP most likely plays an important role in terminating these events. Clinical implications: The speed of allergic symptom generation depends on the degree of IgE receptor triggering, which is downregulated by SHIP, a potential target for allergy therapy.