Localization of Charcot‐Leyden crystal protein in individual morphological phenotypes of human basophils stimulated by f‐Met peptide

Abstract

Human basophils undergo anaphylactic degranulation, characterized by extrusion of membrane-free granules, and piecemeal degranulation, characterized by progressive removal of granule contents in the absence of granule extrusion. F-Met peptide stimulates a degranulation continuum in human basophils that includes both forms of secretion. Charcot-Leyden crystal protein is stored in the granules of unstimulated human basophils. The objective of this study was to determine the subcellular localization of the Charcot-Leyden crystal protein in individual morphological basophil phenotypes that are stimulated by f-Met peptide and are associated with secretion. A post-embedding immunogold analysis was used to detect changes in the subcellular sites of Charcot-Leyden crystal protein in human basophils stimulated with f-Met peptide. Human basophils from normal donors were purified by countercurrent centrifugal elutriation and Percoll density gradients, stimulated to degranulate with 1 micron f-Met peptide (or incubated in buffer controls), and recovered for histamine assay, electron microscopy and immunogold labelling. Specificity controls included omission of the primary antibody and substitution of the primary antibody with non-immune normal rabbit IgG or with Charcot-Leyden crystal protein-Sepharose-absorbed primary antibody. The results showed new sites of labelling and different densities of labelling for Charcot-Leyden crystal protein in distinctive basophil phenotypes stimulated by f-Met peptide. New sites for Charcot-Leyden crystal protein included nucleus, cytoplasm, degranulation channel, degranulation channel membrane, plasma membrane, and a newly recognized granule population similar to primary granules in eosinophils. These new sites, as well as previously documented sites of Charcot-Leyden crystal protein (granules, intragranular Charcot-Leyden crystals, cytoplasmic vesicles) showed variable labelling when analysed by phenotype. Other sites (besides intragranular Charcot-Leyden crystals) of formed Charcot-Leyden crystals included cytoplasm, degranulation channel, extracellular space and, rarely, nucleus. Analysis of cytoplasmic vesicles, total granules and altered granules, and gold particles in subcellular compartments in seven identifiable phenotypes revealed that f-Met peptide stimulated human basophils to empty their granules by transporting Charcot-Leyden crystal protein in vesicles to the plasma membrane in the absence of granule extrusion in cells exhibiting piecemeal degranulation. In cells exhibiting anaphylactic degranulation, gold-labelled Charcot-Leyden crystals were extruded to the cells' exterior in concert with granule particles and concentric dense membranes contained within granules. Completely degranulated cells had a high density of plasma membrane gold label that was associated with numerous gold-laden endocytotic cytoplasmic vesicles. Basophils reconstituted their main granule population, within which Charcot-Leyden crystals resided, in part by endocytosis of previously released plasma membrane-bound Charcot-Leyden crystal protein. Completely recovered cells displayed decreased Charcot-Leyden crystal protein labelling of the plasma membrane and vesicle compartments, the presence of a highly labelled new granule subset that resembled Charcot-Leyden crystal protein-containing primary granules in eosinophils, and the highest density of granule and intragranular Charcot-Leyden crystal gold labelling of all phenotypes that developed after stimulation. Seven individual f-Met peptide-activated human basophil phenotypes labelled by an ultrastructural immunogold method to detect subcellular sites of Charcot-Leyden crystal protein showed changing distributions of this protein which document the capability of human basophils to undergo complex release and recovery reactions that may be pertinent to the functions of Charcot-Leyden crystal protein and the capabilit