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

Background

Mediator releases after high-affinity IgE receptor (FcɛRI) 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 FcɛRI activation is bell-shaped, with supraoptimal stimulation leading to reduced mediator release.

Objective

We sought to characterize the mechanisms responsible for this control of FcɛRI-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 FcɛRI⁺ 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.

Key words: Basophils, mast cells, signal transduction, allergy, IgE, Syk, p38 mitogen-activated protein kinase, extracellular signal–regulated kinase, src homology 2 domain-containing inositol 5′ phosphatase

Abbreviations used: ERK, Extracellular signal–regulated kinase, fMLP, N-formyl-methionyl-leucyl-phenylalanine, MAPK, Mitogen-activated protein kinase, PI3 kinase, Phosphatidylinositol 3-kinase, SHIP, src homology 2 domain-containing inositol 5′ phosphatase, SHP-2, src homology 2 domain-containing protein tyrosine phosphatase-2