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.

The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils. This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein. The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome. Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods. Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques. Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum. CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules. These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules. The crystalloid-containing, large, specific granules did not stain for CLC protein. Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein. The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections. The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct “primary” granule population that serves as a major intracytoplasmic repository for this enzyme.

The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils. This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein. The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome. Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods. Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques. Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum. CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules. These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules. The crystalloid-containing, large, specific granules did not stain for CLC protein. Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein. The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections. The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct “primary” granule population that serves as a major intracytoplasmic repository for this enzyme.

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 Crohn’s disease.

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