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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.

Notes: Backgrottnd Basophils arc circulaling, secretory grunulocytes that are generally considered to be end-stage cells. In one species of guinea-pigs, basophilic leucocytes have been shown to recover from stimulated secretion in short-term cultures. Similar studies have not been done using human basophils.Objective The purpose of this study was to examine human hasophils in short-term recovery intervals following stimulation of secretion to determine whether visual evidence of recovery occurred.Methods We examined the ultrastrucural morphology of early recovery (l0 min-6h) of human basophils following secretion stimulated by formyl-mcthionyl-lcucyl-phenyl-alanine (FMLP). A combined technique for electron microscopy consisted of postfixation exposure to cationizcd ferritin and reduced osmium, providing maximum quality images and allowing identification of intracellular spaces/organelles that opened to the cell surface, often out of the plane of section.Results The ultrastructural evaluation revealed that control basophils (0 time–6h) did not undergo regulated secretion or develop the morphologies associated with recovery following secretion. FMLP-stimulated basophils underwent an overlapping continuum of piecemeal degranulation - anaphylactic degranutation (0 time- I min), producing vesicle- and granule-free, completely degranulated, viable, mature basophils with polylobed nuclei. The early recovery period (10 min-h) following FMLP stimulation was characterized by reconstitution of granules. Morphological mechanisms for granule reconstitution included a mixture of conservation, condensation, and synthetic events.Coticlttsion Human basophils, like guinea pig basophils, have the polential to recover from regulated secretion.

Notes: Summary. Fibroblasts have been implicated as culture-competent cells for the mast cell lineage in several species. In man, fibroblast monolayers sustain the ultrastructural phenotype and function of isolated human lung mast cells and permit the differentiation and full maturation of human mast cells from their agranular precursors in cord blood cells. We examined whether development and maturation of the mast cell lineage in man can be achieved by a supply of the soluble products present in fibroblast culture supernatants. Suspension cultures of cord blood cells were supplemented with culture supernatants derived from two different murine fibroblast lines; controls were not supplemented. The cultures were sampled for light and electron microscopy at 6, 7 and 8 weeks. Human mast cells developed in quantity when cultures were supplemented with the supernatants from BALB/c 3T3 fibroblasts, in reduced numbers when supplemented with Swiss Albino 3T3 fibroblast supernatants, and not at all in culture media alone. By ultrastructural criteria, the newly developed mast cells did not achieve full maturity; they did continue to synthesize new granules and to undergo intragranular maturational events. Small numbers of mature basophils persisted in suspension cultures, and many were undergoing piecemeal degranulation. Other cell lineages noted included eosinophils, neutrophils, macrophages and endothelial cells. We conclude that a factor(s) of fibroblast origin permits the differentiation and partial maturation of human mast cells from their agranular precursors in cord blood, but that fibroblasts must be physically present for complete maturation of these lineages to occur.

Notes: We performed ultrastructural cytochemistry to detect peroxidase in developmentally arrested human eosinophilic myelocytes. Human umbilical cord blood mononuclear cells were cultured for 21 days in the presence of murine-derived conditioned media, resulting in the development of eosinophilic myelocytes. Unlike normally developing eosinophilic myelocytes, which contain peroxidase in synthetic organelles (i.e. cisterns surrounding the nucleus and hounded by the rough endoplasmic reticulum and Golgi structures) and in immature and mature granules, the developmentally arrested cells showed ultrastructural evidence of decreased synthesis and secretor transport of peroxidase. Thus. peroxidase was generally absent in the perinuclear and rough endoplasmic cisterns, in Golgi structures, in immature granules and in the matrix-compartment of most mature granules. Rather, bicompartmental specific granules displayed empty, peroxidase-negative matrix and central, peroxidase-negative core material. Peroxidase was present in perigranular vesicles, some of which were attached to granules. Such peroxidase-loaded transport vesicles are similar to those that effect piecemeal degranulation of mature human eosinophils cultured in rhIL-5-containing media [1]. These findings establish vesicle-mediated piecemeal degranulation in the secretory repertoire of immature human eosinophils and suggest the possibility that eosinophilic myelocytes may participate in vivo in important physiological and/or pathological events that require selective secretion from the specific granule matrix compartment.