Eosinophils maintain their capacity to signal and release eosinophil cationic protein upon repetitive stimulation with the same agonist

Eosinophils contain in their granules eosinophil cationic protein (ECP) and other basic proteins that have been implicated in immunity to parasites an d pathophysiology of chronic allergic responses. In a model of eosinophil d egranulation, we show that eosinophils release ECP upon short-term GM-CSF p riming and stimulation with either platelet-activating factor (PAF) or the anaphylatoxin C5a, but not eotaxin, Restimulation with the same agonist (PA F or C5a) was unsuccessful as assessed by monitoring intracellular calcium concentration and ECP release. In contrast, upon an intermediate washing st ep, eosinophils rapidly transduced PAF and C5a signals followed by signific ant ECP releases. Ligand-binding studies demonstrated that only a proportio n of PAF receptors is internalized upon cell stimulation land that washing of the cells removes the agonist from the cell surface. Upon repetitive sti mulation, eosinophils with less than 50% of the original ECP content were o btained. Such eosinophils did not increase cellular ECP levels even in the presence of the eosinophil survival factor GM-CSF in overnight cultures. In vivo studies revealed that eosinophils always express detectable amounts o f ECP under chronic inflammatory conditions. In conclusion, we have shown t hat eosinophils maintain their capacity to degranulate upon repetitive stim ulation with the same agonist as long as the receptor is not occupied from a previous stimulation. The cellular content of ECP appears to be a no limi ting factor in the case of repetitive stimulation, implying that mature eos inophils may mot require a significant ECP resynthesis.