Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation.

The mechanism of piecemeal degranulation by human eosinophils was investigated. Mature eosinophils that developed in rhIL-5-containing conditioned media from cultured human cord blood mononuclear cells were prepared for ultrastructural studies using a combined technique to image eosinophil peroxidase by cytochemistry in the same sections on which postembedding immunogold was used to demonstrate Charcot-Leyden crystal protein. Vesicular transport of eosinophil peroxidase from the specific granule matrix compartment to the cell surface was associated with piecemeal degranulation. This process involved budding of eosinophil peroxidase-loaded vesicles and tubules from specific granules. Some eosinophil peroxidase that was released from eosinophils remained bound to the cell surface; some was free among the cultured cells. Macrophages and basophils bound the released eosinophil peroxidase to their plasma membranes, internalized it in endocytotic vesicles, and stored it in their respective phagolysosomes and secretory granules. Charcot-Leyden crystal protein was diffusely present in the nucleus and cytoplasm of IL-5-stimulated mature eosinophils. Extensive amounts were generally present in granule-poor and subplasma membrane areas of the cytoplasm in contrast to eosinophil peroxidase, which was secreted and bound to the external surface of eosinophil plasma membranes. These studies establish vesicular transport as a mechanism for emptying the specific eosinophil granule matrix compartment during IL-5-associated piecemeal degranulation.     

Ultrastructural localization of Charcot-Leyden crystal protein (lysophospholipase) and peroxidase in macrophages, eosinophils, and extracellular matrix of the skin in the hypereosinophilic syndrome

Electron microscopy, ultrastructural cytochemistry, and postembedding immunogold ultrastructural immunocytochemistry were used to study a papular cutaneous lesion from a patient with the hypereosinophilic syndrome. Peroxidase activity was detected cytochemically in 40-microns sections of skin utilizing the substrate diaminobenzidine; Charcot-Leyden crystal (CLC) protein was detected immunocytochemically in skin utilizing a postembedding immunogold technique; and a combined method was used where postembedding immunogold staining of CLC protein was performed on sections previously prepared to detect peroxidase activity. We describe a unique, eosinophil-rich inflammatory process in involved skin which contained extraordinary numbers of morphologically activated macrophages. Electron microscopy demonstrated (a) widespread eosinophil necrosis, (b) interstitial CLC formation, (c) macrophage activation, endocytosis, and phagocytosis, and (d) CLC formation in phagosomes of activated macrophages. Peroxidase activity was present as follows: (a) in the matrix of eosinophil specific granules in eosinophil cytoplasm, in membrane-bound specific granules released into interstitial tissues from dying eosinophils, being phagocytized by activated macrophages, and within macrophage phagosomes; (b) as amorphous interstitial debris; (c) in cytoplasm and nuclei of damaged eosinophils in the dermal tissues as well as in macrophage phagosomes; and (d) in endocytotic vesicles and vacuoles of macrophages and in CLC-containing phagosomes of macrophages. CLC protein was localized by immunocytochemistry to (a) eosinophil primary granules, (b) cytoplasm and nuclei of damaged eosinophils located in the interstitial tissues or within macrophage phagosomes, (c) CLC located in interstitial tissues adjacent to necrotic eosinophils and in macrophage phagosomes, and (d) aggregates of amorphous protein bound to macrophage surfaces; endocytotic vesicles and vacuoles of macrophages; amorphous protein aggregates in macrophage lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Eosinophilic leucocytes in nasal allergy—movement of enzymes

The changes in peroxidase of the granules of eosinophils were examined in cells in the nasal mucous membranes of persons allergic to house dust. There was a slight leakage of peroxidase from eosinophil granules immediately after placing an antigen-treated disc (house dust, Torii, 2560 μg/disc) on the nasal mucous membrane. Leakage of peroxidase from the eosinophil granules into the cytoplasm, and extracellular release from some eosinophils, was greater 20 min after provocation, than at 30 sec. There were two forms of peroxidase release from the plasma membrane. In one, the granule membrane fused with the plasma membrane and the peroxidase was released from the cell through destruction of the two fused membranes. This was considered to be active secretion. In the other form, the plasma membrane was damaged by peroxidase released from the granules into the cytoplasm. This was considered to be passive release following degeneration of the cell.     

Degranulation of human eosinophils in nasal allergy]

The localization of eosinophil peroxidase (EPO), one of the basic proteins in eosinophil grunules, was studied in the nasal mucosae of nasal allergy patients. EPO in granules was initially induced into the cytoplasm via finetubules and then diffused into the extracellular space through micro-destruction of the plasma membrane. When the eosinophil lysis was advanced a large amount of granule countents was released into the extracellular space. The total number of eosinophils which migrated to epithelial region was not significantly different between in the damaged epithelium and in the intact one. But the rate of cytolytic eosinophils was significantly higher in the area of severely shedded epithelium than in the area of intact epithelium. In conclusion, the degranulation of eosinopils in nasal allergy was induced by lysis rather than by exocytosis and many cytolytic eosinophils caused epithelial shedding.     

Eosinophilic gastroenteritis: ultrastructural evidence for a selective release of eosinophil major basic protein.

Ultrastructural study of mucosal eosinophils in a case of eosinophilic gastroenteritis involving stomach, duodenum and ileum showed an altered structure in ulcerated duodenal areas. The electron core density of eosinophil granules was inverted or disappeared and tubulovesicular structures occurred. Using immunogold staining with specific antibodies, major basic protein was detected diffusely in the matrix of eosinophil granules and out of the granules in tight association with extragranular membrane formations. In contrast, eosinophil cationic protein and eosinophil peroxidase were normally distributed in the granule matrix. When compared with the eosinophils in macroscopically normal duodenal mucosa in the same patient, these changes support a role for major basic protein in tissue damage in eosinophilic gastroenteritis. The diffusion of one granule protein from the granules to the exterior of the cells favours the view of a selective release of eosinophil mediators.     

Circulating eosinophils in asthma, allergic rhinitis, and atopic dermatitis lack morphological signs of degranulation

BACKGROUND: In allergic diseases, eosinophils in affected tissues release granule proteins with cytotoxic, immunoregulatory, and remodelling-promoting properties. From recent observations, it may be assumed that eosinophils degranulate already in circulating blood. If degranulation occurs in the circulation, this could contribute to widespread systemic effects and provide an important marker of disease. OBJECTIVE: To determine the degranulation status of circulating eosinophils in common allergic diseases. METHODS: Using a novel approach of whole blood fixation and leucocyte preparation, the granule morphology of blood eosinophils from healthy subjects, non-symptomatic patients, symptomatic patients with asthma, asthma and Churg-Strauss syndrome, allergic rhinitis, and atopic dermatitis was evaluated by transmission electron microscopy (TEM) and eosinophil peroxidase (TEM) histochemistry. Plasma and serum levels of eosinophil cationic protein were measured by fluoroenzymeimmunoassay. Selected tissue biopsies were examined by TEM. RESULTS: Regardless of symptoms, circulating eosinophils from allergic patients showed the same granule morphology as cells from healthy subjects. The majority of eosinophil-specific granules had preserved intact electron-density (96%; range: 89-98%), while the remaining granules typically exhibited marginal coarsening or mild lucency of the matrix structure. Abnormalities of the crystalline granule core were rarely detected. Furthermore, granule matrix alterations were not associated with any re-localization of intracellular EPO or increase in plasma eosinophil cationic protein. By contrast, eosinophils in diseased tissues exhibited cytolysis (granule release through membrane rupture) and piecemeal degranulation (loss of granule matrix and core structures). CONCLUSION: In symptomatic eosinophilic diseases, circulating blood eosinophils retain their granule contents until they have reached their target organ.     

Pivotal Advance: eosinophil infiltration of solid tumors is an early and persistent inflammatory host response

Tumor-associated eosinophilia has been observed in numerous human cancers and several tumor models in animals; however, the details surrounding this eosinophilia remain largely undefined and anecdotal. We used a B16-F10 melanoma cell injection model to demonstrate that eosinophil infiltration of tumors occurred from the earliest palpable stages with significant accumulations only in the necrotic and capsule regions. Furthermore, the presence of diffuse extracellular matrix staining for eosinophil major basic protein was restricted to the necrotic areas of tumors, indicating that eosinophil degranulation was limited to this region. Antibody-mediated depletion of CD4+ T cells and adoptive transfer of eosinophils suggested, respectively, that the accumulation of eosinophils is not associated with T helper cell type 2-dependent immune responses and that recruitment is a dynamic, ongoing process, occurring throughout tumor growth. Ex vivo migration studies have identified what appears to be a novel chemotactic factor(s) released by stressed/dying melanoma cells, suggesting that the accumulation of eosinophils in tumors occurs, in part, through a unique mechanism dependent on a signal(s) released from areas of necrosis. Collectively, these studies demonstrate that the infiltration of tumors by eosinophils is an early and persistent response that is spatial-restricted. It is more important that these data also show that the mechanism(s) that elicit this host response occur, independent of immune surveillance, suggesting that eosinophils are part of an early inflammatory reaction at the site of tumorigenesis.     

Structural, cytochemical, immunocytochemical and ultrastructural characterization of blood granulocytes of the roadside hawk Buteo magnirostris (Gmelin, 1788) (Avian, Falconiform)

In the peripheral blood of the roadside hawk, Buteo magnirostris, the following types of granulocytic leucocytes were identified: heterophil, eosinophil and basophil. The heterophils presented acidophilic and spindle shaped granules, the eosinophils possess spherical eosinophilic granules and the basophils showed spherical and basophilic granules. The heterophils and eosinophils presented positive cytochemical reaction to glycogen and basic polyaminoacid, while the eosinophils presented sudanophilic granules, which were also positive for myeloperoxidase. The heterophils, alone, presented positivity for acid phosphatase in some granules and immunoreactivity to TGF-beta1 was observed only in the cytoplasm of the eosinophils. Electron microscopy demonstrated the heterophil granules as predominantly spindle shaped, being strongly electron-dense, while the eosinophils had numerous uniformly electron-dense spherical granules and the basophils presented three different types of granules identified according to their electron-density and the aspect of their matrix.     

Discontinuous Percoll gradient centrifugation combined with immunomagnetic separation obviates the need for erythrocyte lysis and yields isolated eosinophils with minimal granule abnormalities

Isolated blood eosinophils are routinely used to study eosinophil activation mechanisms. However, as revealed by ultrastructural analysis, different isolation protocols may yield purified eosinophils with marked variability in granule electron density. In this study, using eosinophil peroxidase (EPO) histochemistry and transmission electron microscopy (TEM), we have compared the morphology of eosinophils in immediately fixed whole blood (to represent a morphological baseline) with isolated eosinophils purified by a number of protocols. Eosinophils in whole blood contained intact specific secondary granules of which a few exhibited marginal coarsening of matrix electron density (4% (95% CI: 2 to 7) altered granules per eosinophil). By contrast, eosinophils purified according to standard protocols, which included erythrocyte lysis with either ammonium chloride or distilled water, showed moderate to extensive loss in density of secondary granule core and/or matrix (NH4Cl: 62% (95% CI: 58 to 66); dH2O: 37% (95% CI: 30 to 44) altered granules). Stepwise analysis of eosinophils during the cell separation processes indicated that the granule abnormalities seen following erythrocyte lysis were further increased following immunomagnetic separation. However, when erythrocyte lysis was omitted, by use of a two-layered Percoll gradient (1.076 g/ml/1.088 g/ml) to which diluted whole blood was applied directly, eosinophils with minimal granule abnormalities (11% (95%CI: 9 to 13) altered granules) could be obtained after immunomagnetic separation. In conclusion, to obtain eosinophils with granule morphology more closely resembling the whole blood baseline phenotype, erythrocyte lysis should be avoided when separating eosinophils from human blood. Thus it will be possible to study in vitro the early transformation of resting eosinophils into the degranulating phenotype found in diseased tissues.     

Danger signals derived from stressed and necrotic epithelial cells activate human eosinophils

Eosinophilic granulocytes are found in tissues with an interface with the external environment, such as the gastrointestinal, genitourinary and respiratory tracts. These leucocytes have been associated with tissue damage in a variety of diseases. The aim of this study was to evaluate whether necrotic epithelial cells can activate eosinophils. The danger theory postulates that cells of the innate immune system primarily recognize substances that signal danger to the host. We damaged epithelial cell lines derived from the genital (HeLa cells), respiratory (HEp-2 cells) and intestinal tracts (HT29 cells) and assessed their capacity to cause eosinophilic migration, release of putative tissue-damaging factors, such as eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP), as well as secretion of tissue-healing factors, e.g. fibroblast growth factors (FGF)-1 and -2 and transforming growth factor (TGF)-beta1. We found that necrotic intestinal cells induced chemotaxis in human eosinophils. EPO release was elicited in eosinophils stimulated with necrotic cells derived from all cell lines, as well as from viable HEp-2 and HT29 cells. Release of ECP was only seen in eosinophils incubated with necrotic intestinal or genital cells, not viable ones. Both necrotic intestinal and genital cells elicited FGF-2 secretion from eosinophils. TGF-beta1 was released from eosinophils exposed to viable and necrotic HT29 cells. These findings indicate that eosinophils are able to recognize and be activated by danger signals released from damaged epithelial cells, which may be of importance in understanding the role of eosinophils in the various inflammatory conditions in which they are involved.     

Ultrastructural morphology, cytochemistry, and morphometry of eosinophil granules in Chédiak-Higashi syndrome.

Lysosomal enlargement in Chédiak-Higashi Syndrome (CHS) occurs to varying degrees in different cell types and has provided insight into the pathophysiology of lysosomal granules. This study was undertaken to determine the extent of involvement of eosinophil crystalloid granules (CGs) and smaller non-crystalloid granules (NCGs) in giant granule formation. Eosinophils from two CHS patients were evaluated after glutaraldehyde fixation and staining for morphologic examination, peroxidase, and complex carbohydrate using uranyl acetate-lead citrate, diaminobenzidine-lead citrate, and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) methods, respectively. Although many CGs appeared normal in shape and size, several CGs appeared enlarged and a few measured over 5 microns in diameter, consistent with giant granule formation in CHS. These giant granules either occasionally contained a single large crystalloid or, more frequently, contained numerous normal-size crystalloids. Enlargement of granules was also observed in some precursor CGs of bone marrow early eosinophils, indicating that giant granule formation was initiated during granule genesis. Almost all NCGs in late eosinophils were small granules and stained strongly with PA-TCH-SP in contrast to CGs. Most, but not all small granules were peroxidase-positive in eosinophil precursors, whereas the percentage of peroxidase-negative small granules increased in late eosinophils. This indicated the presence of at least two small granule populations. Morphometric studies indicated CHS selectively involved CGs and demonstrated that neither the average size nor numbers of NCGs were significantly different from normal eosinophils. Thus, these studies indicate that CHS selectively involves CGs, and demonstrate preservation of normal granule size and heterogeneity for NCGs in late eosinophils. These observations suggest that the underlying CHS pathophysiology does not involve all lysosomal subpopulations.     

Fibroblasts and eosinophils in normal fibroplasia of ferret vulval skin, shown by light and electron microscopy; with special reference to eosinophil degranulation

In order to compare aspects of normal fibroplasia with reports for pathologically fibrotic tissue, light and electron microscopic observations were made on ferret vulval skin, which proliferates markedly with the onset of estrus and regresses equally fast after mating. Estrous tissue, which contained hypertrophied fibroblasts, new collagen, elastic fibers and ground substance, was invaded by numerous eosinophils with extensive release of granules. Eosinophil degranulation was both extra- and intracellular; some cells, completely disintegrated, had released all contents into the extracellular matrix. Extruded granules reacted positively for major basic protein. In regression, diminished fibroblasts incorporated collagen fibrils. Many of the features seen in this normal fibroplasia in ferret vulval skin are similar to those in pathologic situations. Thus, in particular, the presence of degranulating eosinophils in pathologic fibrosis does not necessarily indicate that they are the cause of the disease. Our information and that from other sources implies a role for eosinophils in connective tissue change.     

Extracellular deposition of eosinophil and neutrophil granule proteins in the IgE-mediated cutaneous late phase reaction

Intradermal injection of allergens in sensitive subjects produces an IgE-dependent prolonged inflammatory reaction, the late phase reaction (LPR). Histologically, eosinophils are present in the LPR but are not as numerous as neutrophils or mononuclear cells. We determined whether extracellular deposition of eosinophil and neutrophil granule proteins occurs in the LPR by immunofluorescent localization of eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and neutrophil elastase. Before intradermal challenge, eosinophils and neutrophils were present only in blood vessels, and MBP, EDN, and elastase were localized to cells. At 15 minutes, small amounts of MBP, EDN and elastase were found outside of cells in focal areas. By 1 to 3 hours, MBP, EDN and elastase were extensively deposited throughout the dermis in a granular and diffuse manner; these deposits persisted up to 56 hours. Both actively and passively sensitized subjects showed similar MBP and elastase deposition. Skin sites passively sensitized by sera depleted of IgE showed essentially no MBP or elastase deposition. Electron microscopy showed degenerating eosinophils and free eosinophil granules in the dermis. Mast cell numbers diminished during the LPR when extracellular eosinophil and neutrophil granule protein deposition was maximal. These results demonstrate that striking dermal eosinophil and neutrophil granule protein deposits are prominent features of the cutaneous LPR, are IgE-dependent and precede the maximal clinical expression of the LPR. The possible significance of these findings in the pathophysiology of the LPR is discussed.     

Eosinophil crystalloid granules: structure, function, and beyond

Eosinophils are granulocytes associated with host defense against parasitic helminths with allergic conditions and more recently, with immunoregulatory responses. Eosinophils are distinguished from leukocytes by their dominant population of cytoplasmic crystalloid (also termed secretory, specific, or secondary) granules that contain robust stores of diverse, preformed cationic proteins. Here, we provide an update on our knowledge about the unique and complex structure of human eosinophil crystalloid granules. We discuss their significance as rich sites of a variety of receptors and review our own recent research findings and those of others that highlight discoveries concerning the function of intracellular receptors and their potential implications in cell signaling. Special focus is provided on how eosinophils might use these intracellular receptors as mechanisms to secrete, selectively and rapidly, cytokines or chemokines and enable cell-free extracellular eosinophil granules to function as independent secretory structures. Potential roles of cell-free eosinophil granules as immune players in the absence of intact eosinophils will also be discussed.     

Electron microscopic studies on granulocytopoiesis in the slender salamander

The morphology of differentiating heterophils and eosinophils of the slender salamander, Batrachoseps attenuatus, was studied with electron microscopy. Enzymes of the granules of both cell types appear to arise from the Golgi cisternae. An agranular stem cell was not observed and the least differentiated cell type encountered was an “early” promyelocyte having small granules which are morphologically distinct from mature granules of either the heterophil or eosinophil series. Heterophil myelocytes and later stages contain only one population of granule which is fibrous in content. Eosinophils likewise possess but one type of granule; the granules are larger than those of heterophils, have a homogeneous content, and lack the crystallin core so characteristic of mammalian eosinophils.     

Piecemeal degranulation of peripheral blood eosinophils: a study of allergic subjects during and out of the pollen season

The variability of serum and plasma levels of eosinophil granule proteins in different clinical conditions, interpreted as the result of different patterns of cytokine priming, suggests a selective mobilization of granule proteins. Inasmuch as piecemeal degranulation (PM) is the mechanism proposed for the differential release of eosinophil granule proteins, we decided to investigate whether blood eosinophils from allergic subjects show characteristics of PM during natural allergen challenge. Eosinophils from three birch-sensitive subjects were studied before and during the pollen season. Electron microscopy analysis showed that during the season, eosinophils presented morphologic features of PM. By immunogold labeling, eosinophil cationic protein (ECP) was detected not only in normal specific granules but also in the cytoplasm, in the vicinity of partially lucent specific granules. These results were confirmed by subcellular fractionation, where the amount of ECP associated with compartments containing small vesicles increased 2-fold during the pollen season. A study of the distribution of ECP, eosinophil peroxidase, and hexosaminidase in eosinophils of different densities showed that the profile of each of these proteins differed depending on cell density. All of these proteins decreased in the specific granule of hypodense cells and increased in other cell compartments. We conclude that allergen exposure causes PM of the peripheral blood eosinophils of allergic subjects, and that the density of these cells reflects the degree of degranulation. Our results provide novel information for the understanding of the selective mobilization of granule proteins into the circulation.     

The stimulus-dependent release of eosinophil cationic protein and eosinophil protein x increases in apoptotic eosinophils

Apoptotic cells are regarded as inert bodies that turn off intracellular processes and functional abilities. To study the changes in the ability of eosinophils to release their granule proteins while undergoing apoptosis. Eosinophils were cultured for up to 72 h. Living cells were separated from the apoptotic cells and their release of eosinophil cationic protein (ECP) and eosinophil protein X (EPX) was measured in response to serum-opsonized sephadex particles and phorbol 12-myristate 12-acetate (PMA). Changes in cell structure were examined by electron microscopy, and surface receptor expression of beta1- and beta2-integrins was investigated by flow cytometry. Stimulus-dependent release of the granule proteins ECP and EPX was found to increase in apoptotic eosinophils, whereas surface expression of beta1- and beta2-integrins was downregulated. Ultrastructural examination revealed that the granules of apoptotic eosinophils were translocated to the periphery of the cell, just beneath the plasma membrane. Apoptotic eosinophils are able to release their toxic granule proteins, which is probably because of the rearrangement of the cytoskeleton and spontaneous translocation of granules to the membrane. Our results suggest that apoptotic eosinophils are potentially harmful cells that have retained their ability to react to certain extracellular stimuli. The findings point to unexpected consequences of eosinophil apoptosis.     

Dermal deposition of eosinophil-granule major basic protein in atopic dermatitis. Comparison with onchocerciasis

Although blood eosinophilia is commonly present in atopic dermatitis, accumulation of tissue eosinophils is not prominent. To determine whether eosinophil degranulation occurs in lesions of atopic dermatitis, we analyzed tissues by immunofluorescence for the presence of the eosinophil-granule major basic protein. Twenty biopsy specimens from 18 patients with atopic dermatitis were studied, and all showed major basic protein staining outside eosinophils. In 18 specimens, the staining was fibrillar, was located in the upper half of the dermis, and was similar to the distribution of elastic fibers. Twelve specimens with fibrillar staining also showed major basic protein staining in the form of extracellular granules. One specimen from unaffected skin showed minimal faint, fine, fluorescing fibrils, but there was marked deposition of the protein in affected skin. The fibrillar pattern of major basic protein staining in atopic dermatitis was very similar to that seen in lichenified lesions of untreated onchocerciasis. These results suggest that eosinophils commonly release granule proteins in the dermis and that assessment of eosinophil involvement in disease cannot be based simply on numbers of eosinophils in tissue.     

Mature eosinophils stimulated to develop in human cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. Part I. Piecemeal degranulation of specific granules and distribution of Charcot-Leyden crystal protein.

Human cord blood mononuclear cells were cultured for 35 days in media containing recombinant human interleukin 5 (rhIL-5) supplemented with a fraction of the culture supernatant of phytohemagglutinin (PHA)-stimulated human T lymphocytes from which interleukin 2 (IL-2) was eliminated. Cultured cells were studied by electron microscopy and an immunogold procedure to detect subcellular site(s) of Charcot-Leyden crystal (CLC) protein. The majority of cells (greater than 70%) developing in this system were mature eosinophils, with descending frequency of other cells, including macrophages, mature basophils, eosinophilic myelocytes, and mature neutrophils. Mature eosinophils were characterized by increased numbers of primary granules, small granules, tubulovesicular structures, and decreased secondary granules. These eosinophils showed extensive piecemeal degranulation (PMD) characterized by partially empty and empty secondary granule chambers in the cytoplasm. Small, smooth vesicles were evident within empty granule chambers as well as adjacent to them. Eosinophils formed close associations with phagocytic macrophages that contained both standard-shaped and irregularly shaped CLC within phagolysosomes. Subcellular sites of CLC protein were demonstrated by immunogold in eosinophils and macrophages arising in these cultures. Charcot-Leyden crystal protein was present in the nuclear matrix and extraorganellar cytoplasm of eosinophils. Primary granules and some cytoplasmic vesicles were labeled for CLC protein, but full and empty secondary granules and the extensive network of tubulovesicles were not. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals within phagosomes of macrophages were labeled by the immunogold procedure for CLC protein. These results demonstrate that rhIL-5-supplemented, PHA-stimulated, T-cell-conditioned media induced the development of mature human eosinophils from cord blood cells. These eosinophils underwent PMD of secondary granule contents. Immunogold analysis showed eosinophil CLC protein in the cytoplasm, nucleus, and primary granules of eosinophils. Macrophages also were present in these cultures. They contained CLC protein-containing crystals in their phagosomes, suggesting active sequestration of eosinophil CLC protein by macrophages in vitro.     

Ultrastructure of monkey peripheral blood basophils stimulated to develop in vivo by recombinant human interleukin 3

Ultrastructural and cytochemical studies of peripheral blood samples from a monkey continuously infused with recombinant human interleukin 3 were performed. Recombinant human interleukin 3 stimulated a delayed granulocytosis primarily characterized by numerous mature basophils and fewer eosinophils and neutrophils. Basophilic leukocytes were identified by ultrastructural analysis. They were found to be typical granulocytes with polylobed nuclei containing condensed chromatin and numerous cytoplasmic granules. Basophil secretory granules were filled with homogeneous dense contents and were larger than eosinophil and neutrophil secretory granules. Evidence of increased basophil production was accompanied by interleukin 3-associated activation morphologies. These included increased numbers of cytoplasmic and granule-associated vesicles, as are routinely present in a non-IgE-mediated basophil release reaction, termed piecemeal degranulation, and focal perigranular matrix swelling and granule membrane fusion which accompanies anaphylactic degranulation of basophils in other species. Monkey basophils were shown to have a different ultrastructural morphology than that published for monkey mast cells, but exhibited general morphologic criteria for the identification of circulating mature basophils in a number of species. Like human and guinea pig basophils, monkey basophils did not display endogenous peroxidase or peroxidatic activity in a cytochemical assay which simultaneously identified peroxidase-positive granules in neutrophils and eosinophils as well as in synthetic structures in eosinophils. In summary, these studies have identified monkey basophils in an in vivo recombinant human interleukin 3-stimulated model. Interleukin-3 induction of basophilia clearly allowed differentiation of activated mature basophils from eosinophils and neutrophils and mast cells in this species using ultrastructural morphologic criteria.