Purification of eosinophils from normal human blood, preparation of eosinoplasts and characterization of their functional response to various stimuli

Abstract

Eosinophils from the blood of normal individuals were purified by centrifugation over discontinuous Percoll gradients. Eosinophil suspensions were obtained with a mean purity of 96% and a mean recovery of 64% (n = 19). When incubated with phorbol-myristate acetate, eosinophils consumed twice as much oxygen as did neutrophils from the same donors. With serum-treated zymosan, 70% and 100% of the maximal oxidative response (i.e. the response to phorbol-myristate acetate) was obtained with eosinophils and neutrophils, respectively. The calcium ionophore A23187 is a weak stimulus that triggered only 2.5% of the eosinophil and 10% of the neutrophil oxidative capacity. The response of both cell types to formyl-methionyl-leucyl-phenylalanine (fMLP) was rapid, with a maximum after 3 min. The magnitude of this eosinophil reaction was half that of neutrophils. Although the activities of the granule enzymes beta-glucuronidase and arylsulphatase were 2.5 and 6 times higher in eosinophils than in neutrophils, respectively, the exocytosis of these enzymes in response to various stimuli was lower in eosinophils. The high yield of eosinophils from our separation method enabled us to prepare eosinoplasts by centrifugation of eosinophils over discontinuous Ficoll gradients that contained cytochalasin B. Eosinoplasts are plasma membrane vesicles derived from eosinophils, filled with cytoplasm but devoid of granules and nucleus. The eosinoplasts contained 30% of the cytoplasm and plasma membrane present in intact eosinophils. Eosinoplasts still possessed a functionally intact oxidase enzyme that could be stimulated with various stimuli. Therefore, eosinoplasts may provide a valuable tool to study separately the role of the oxidase products and that of the granule contents in eosinophil functions.