Charcot-Leyden crystal protein distribution in basophils and its absence in mast cells that differentiate from human umbilical cord blood precursor cells cultured in murine fibroblast culture supernatants or in recombinant human c-kit ligand.

Abstract

Suspension cultures of human umbilical cord blood mononuclear cells supplemented with c-kit ligand-containing additives give rise to a mixture of cells belonging to several lineages. Among those that differentiate in quantity are mature basophils, immature mast cells, and neutrophilic myelocytes. We used an ultrastructural immunogold method to detect the Charcot-Leyden crystal (CLC) protein, an eosinophiland basophil-specific protein, to study cells that were obtained at sequential times from 3 to 14 weeks in culture. Basophils (and eosinophils, which were present in smaller numbers) labeled for the CLC protein; mast cells did not. The labeled basophil subcellular sites included formed intragranular, cytoplasmic and nuclear CLCs, cytoplasmic particle-filled and homogeneously dense granules, cytoplasm, nucleus, plasma membrane, and cytoplasmic and IntroductionHuman basophils and mast cells can now be reliably induced to develop in vitro from their agranular precursors which are present in cord blood (1-6). Initially, these studies were done with supplements of various cell-conditioned media now known to contain a basophil growth factor, interleukin-3 (IL3) (l), or a mast cell growth factor, the c-kit ligand [also called stem cell factor (SCF)] (5,6). Some of these studies were also done with cord blood cells cocultured with mouse 3T3 fibroblasts (3,4 repeated with these reagents. In all of these cultures, ultrastructural criteria were used to assign immature and mature cells to the appropriate lineages (1-5) (AM Dvorak et al., unpublished data).Charcot-Leyden crystals (CLCs) are hexagonal, bipyramidal structures originally described in eosinophil-rich disorders (7,8). CLC protein is the sole protein constituent of CLCs purified from human eosinophils; it expresses lysophospholipase activity, forms these characteristic crystals, and comprises an estimated 7-10% of total cellular protein in the eosinophil (9-12). CLCs and the CLC protein, once considered unique to the eosinophil lineage, have also been found in human basophils (2,13,14). Using an ultrastructural immunogold method, we have defined the subcellular locations of the CLC protein in quiescent peripheral blood eosinophils (15) and basophils (16) (17-19). Although the cellular function(s) of this lysophospholipase is still obscure, the presence of such large quantities of this enzyme in eosinophils and basophils suggests an important role in normal or pathological events associated with these lineages. The recent cloning of the gene for the CLC protein (20) and expression of the protein product (12) should aid future studies of the biological properties of this unique basophil and eosinophil constituent.Human mast cells, in contrast to basophils, do not stain for the CLC protein by light microscopy (21). Since we noted an admixture of lineages in suspension cultures of cord blood supplemented with c-kit ligand-containing additives, we examined these cells for CLC protein using an immunogold method to detect this protein.We found that mature basophils that ...

Ultrastructural Analysis of Human Eosinophils

“…2 Frequently seen, contains matrix proteins only, and cannot be distinguished from oblique cuts of mature secondary granules. [32]; and to peripheral blood basophils [33][34][35][36], activated basophils [34][35][36] and to basophils and mast cells which developed in vitro from human cord blood cells [74]. These studies showed that CLC-P in immature eosinophils was also a cytosolic and nuclear protein but that plasma membranes, cisterns of RER and mature and immature secondary granules did not label for CLC-P [15,20].…”

“…Rarely, cytoplasmic and nuclear CLCs stained for CLC-P in eosinophils; CLCs were absent from any granule populations in eosinophils [2]. CLC-P was absent from human mast cells [74] but peripheral blood [33], activated [34][35][36] and culture-derived [74] human basophils displayed multiple nuclear, cytoplasmic, granular, plasma membrane, CLC and vesicular sites of CLC-P. As in human eosinophils, a CLC-rich primary granule population was apparent in developing [74] and recovering [35] basophils. The CLC-P-rich a b Fig.…”

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.…”

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

“…Formed CLCs were regularly but variably present in the main particle granules of mature basophiis in situ or developing in vitro [17,32] (Fig. 3C).…”