Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro.

Abstract

We examined the ultrastructural localization of (a) a secondary granule matrix protein -eosinophil peroxidase (EPO) -by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMS) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhK-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMS, as well as identification of unicompartmental, MBFrich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules mroduction Mature human eosinophils are polymorphonuclear granulocytes with two large granule populations (1). One of these populations consists of primary granules (2,3), which comprise ~5 % of the cytoplasmic large granules, are unicompartmental, increase in activated eosinophils in vivo and in vitro, and are the granule storage organelle for the Charcot-Leyden crystal (CLC) protein (2). The second large granule population consists of secondary (or specific) granules, which comprise ~9 5 % of the large cytoplasmic granules, are bicompartmental, undergo quantitative decreases in number and qualitative morphological changes in activated eosinophils in vivo and in vitro, and are the storage organelle for a number of eosinophil products (reviewed in 1). These include major basic protein (MBP) (4,5), eosinophil-derived neurotoxin (EDN) (4). eosinophil cationic protein (ECP) (4,5), eosinophil peroxidase (EPO) (1,5), and tumor necrosis factor-a ( m a ) (6). MBP is confined to the

Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion

“…Secondary Ab for EM was 1.4 nm gold-conjugated goat anti-rabbit Fab fragments (1:100) (Nano-probes Immunogold EM. Agar pellets containing intact eosinophils were processed (43), and preembedding immunogold EM was performed on frozen 10-m sections. Steps, as described in ref.…”

A role for vesicles in human basophil secretion

“…An ultrastructural immunogold postembedding method was used to localize the CLC protein initially in human peripheral blood eosinophils ); later studies localized subcellular sites of the CLC protein in human eosinophils and macrophages in vitro (Dvorak et al 1991b(Dvorak et al , 1992b(Dvorak et al , 1994c, in macrophages, eosinophils and tumor cells in vivo (Dvorak et al 1990a,b), and in peripheral blood (Dvorak and Ackerman 1989) and cultured basophils (Dvorak AM 1996;Dvorak et al 1994d). The A) and full, electron-dense (arrow in B) vesicles are gold-labeled.…”

Ultrastructural Analysis of Human Eosinophils

“…We have examined the distribution of CLC-P, an eosinophil primary granule protein (see later) [30] in activated human eosinophils and their environment in vivo [31,32] and in vitro [15,20,21] with and without double imaging provided by concomitant cytochemical detection of EPO [21]. While tissue areas in eosinophil necrosis-packed lesions are rich in CLC-P [31], vesicular transport of CLC-P did not occur from eosinophils concomitantly undergoing PMD of a secondary granule matrix protein, EPO, in vitro [21].…”

“…Characteristically, eosinophilic myelocytes (EM) are large cells that undergo considerable size reduction as the large, single nucleus condenses and segments, as extensive cytoplasmic secretory organelles, such as dilated cisterns of RER and Golgi structures, are reduced in volume, as primary granules are released, and as large immature, unicompartmental, homogeneously dense specific granules are reduced in size (from several microns to 0.5-1-m structures) and shape (from round to spherical) as they become visibly bicompartmental [12][13][14]. The crystallization of central cores within these immature specific granules as well as their immunogold profile (major basic protein [MBP]-positive, Charcot-Leyden crystal protein [CLC-P]-negative) [15] unequivocally identify this large immature granule population as specific granule precursors. Small granules, lipid bodies, vesicles, tubules and glycogen are less-frequent cytoplasmic organelles in electron micrographs of immature human eosinophils [2].…”