Linda Letourneau

Ultrastructural identification of exocytosis of granules from human gut eosinophils in vivo.

Twenty-two percent of 117 biopsies of human intestinal tissues had ultrastructural images of classical regulated secretion from eosinophils in vivo i.e. eosinophil granule extrusion (EGE). Replicate intestinal biopsies that were positive for bacteria had EGE more often than not (p < 0.05); 77% of the isolates were Staphylococci. Some of the intestinal biopsies also had damaged nerves; all that had EGE and damaged enteric nerves also had positive bacterial cultures. The EGE that we observed could not account for all enteric nerve damage, suggesting multifactorial mechanisms for nerve damage in gut tissues. Among the possibilities are release of neurotoxic eosinophil granule proteins by an alternate secretory route, i.e., piecemeal degranulation, direct toxicity of tissue invasive bacteria and/or damaged nerves of unknown etiology such as those that are regularly present in uninvolved tissues of patients with Crohns disease.

Vesicular Transport of Charcot-Leyden Crystal Protein in f-Met Peptide-Stimulated Human Basophils

The ultrastructural localization of Charcot-Leyden crystal (CLC) protein during f-Met-peptide-induced degranulation of human basophils was analyzed at multiple times after stimulation. In this secretion model, piecemeal and anaphylactic degranulation occurred sequentially in stimulated cells and were followed by reconstitution of granule contents. This analysis showed that granule number and alteration and location of gold-labeled, formed CLCs changed over time. CLCs were extruded from granules and remained attached to plasma membranes early after stimulation. At later times, similar structures reappeared in granules in quantity. Smooth-membrane-bound vesicles, analyzed by number, by visible particle contents (or lack of contents) and by gold labeling for CLC protein, showed that empty vesicles increased at the earliest time sampled (0 time) and plunged thereafter in actively extruding and completely degranulated cells. Vesicles containing granule particles were elevated initially at 10 s and at later times. Gold-labeled CLC-protein-containing vesicles were of either empty or particle-filled varieties, and both types were involved with CLC protein transport out of cells at early times and into cells at later times as basophils recovered. Thus, vesicle transport of CLC protein is a mechanism for producing piecemeal degranulation and endocytotic recovery of released CLC protein from human basophils. This vesicular shuttle may be an effector mechanism for widespread piecemeal losses from granules in basophils in inflammatory sites in vivo in human disease.

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

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 (rhIL-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, MBP-rich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules by virtue of their lack of CLC protein. These granules occur in quantity in IL-5-activated mature human eosinophils, which have previously been shown to actively transport EPO from the matrix compartments of their secondary granules to the extracellular milieu in smooth membrane-bound cytoplasmic vesicles, a secretory process termed piecemeal degranulation, whereby eosinophils progressively empty cytoplasmic granules of their contents in the absence of classical granule extrusion.

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.

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 eosinophil- and 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 Golgi area vesicles. Individual basophil ultrastructural phenotypes similar to those associated with stimulated release and recovery reactions showed the expected variations in the gold-labeled subcellular compartments. Macrophages also were labeled for CLC protein within endocytotic-lysosomal structures; neutrophilic myelocytes did not contain CLC protein. On the basis of findings reported here, the combined ultrastructural morphology and immunogold phenotyping of cells differentiating in c-kit ligand-supplemented cultures allows accurate lineage assignment of the developing cells.

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

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.

Spontaneous Degranulation of Cultured Bone-Marrow-Derived Immature Mast Cells from X-Linked Immunodeficient (Xid) Mice

IgE-mediated, antigen-dependent stimulation of immature mouse mast cells cultured in IL-3-containing media produces secretion by granule exocytosis. Similar cultured mast cells were derived from X-linked immunodeficient (Xid) mice and examined by electron microscopy. In these cultures, Xid mast cells were also immature. In contrast to cultures obtained from control mice, 10-20% of the immature mast cells of Xid origin were undergoing secretion by granule extrusion in the absence of any secretogogue. Spontaneous secretion may be related to discordered tyrosine kinase function and/or signal transduction pathways in the Xid mouse.