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Notes: Background 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 tbe granules of unstimulated human basopbils.Objective 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.Methods 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 nonnal donors were purified by countercurrcnt centrifugal elutriation and Percoll density gradients, stimulated to degranulate with 1 μm 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.Results 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 variahic 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 exbibiting 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 Correspondence: Ann M. Dvorak MD, Department of Pathology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, USA. 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 tbat developed after stimulation.Conclusion 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 basopbils to undergo complex release and recovery reactions that may be pertinent to the functions of Charcot-Leyden crystal protein and the capabilities of these cells in vivo in a variety of diseases.