Eosinophil granule cationic proteins: major basic protein is distinct from the smaller subunit of eosinophil peroxidase

Major basic protein and eosinophil peroxidase are predominant cationic proteins in the cytoplasmic granules of human eosinophilic leukocytes. Each of these proteins has been purified, and major basic protein and the lower molecular weight subunit of eosinophil peroxidase have been found to comigrate on polyacrylamide gel electrophoresis in sodium dodecyl sulfate with similar apparent molecular weights of about 14,700. Because previous molecular weight estimates for these proteins have not recognized the similar molecular weights of these two cationic eosinophil granule constituents, we have evaluated the possible relatedness of these proteins. Upon protein sequence analyses, it was found that the N-terminal 20 amino acid residues of each of these two purified polypeptides differed. These findings established that major basic protein and the smaller subunit of eosinophil peroxidase, although of comparable molecular weights, are two distinct proteins within the cytoplasmic granules of human eosinophils.     

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.     

Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine

The involvement of eosinophils in the host reaction to microfilariae (mf) of Onchocerca volvulus was studied by immunohistochemistry and immunoelectron microscopy. Skin biopsies were obtained from patients after transepidermal administration of the microfilaricide amocarzine. At 20–28 h after the application of amocarzine, mf were degenerated or dead and a marked eosinophil-parasite adherence (EPA) reaction was seen, with intense staining for intra- and extracellular eosinophil granule proteins such as eosinophil cationic protein (ECP) surrounding the mf. Immunoelectron microscopically the eosinophil granule matrix in intact and necrotic eosinophils was specifically labeled, whereas granules whose matrix had dissolved showed no specific gold particle binding. As specific labeling was seen on lowly electron-dense material adjacent to matrix-depleted granules, the material was regarded as released eosinophil granule matrix material. Intact and necrotic eosinophils, matrix-containing as well as matrix-depleted eosinophil granules, and released eosinophil granule matrix material were observed on the surface of damaged mf and between collagen fibers. The coincidence of mf degeneration, EPA reaction, and release of eosinophil granule matrix material on damaged mf and collagen fibers indicated a role of eosinophils and eosinophil granule matrix protein in the host reaction to mf after amocarzine application. Received: 3 March 1998 / Accepted: 13 March 1998     

Immunopathological study of eosinophils in eosinophilic granuloma of bone: Evidence for release of three cationic proteins and subsequent uptake in macrophages

Summary Eosinophils from two patients with eosinophilic granuloma of bone (EGB) were studied by combined immunohistochemical and immuno-ultrastructural methods with antibodies directed against three eosinophil granule proteins: major basic protein, eosinophil cationic protein, and eosinophil peroxidase. Immunohistostaining showed the presence and distribution of large numbers of eosinophils in the granuloma. Immuno-ultrastructural methods showed alterations of eosinophil fine structure associated with some steps in the release of granule proteins. No granule extrusion was seen, but rather cationic proteins diffused within cytoplasmic tubulo-vesicular structures. Furthermore, the three granule proteins were found within phagolysosomes of surrounding macrophages, suggesting an interaction between eosinophils and phagocytic cells at the destructive stage of EGB.     

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.     

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. 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.     

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.     

Eosinophil- and eosinophil granule-mediated pneumocyte injury

The function of the eosinophil in eosinophilic pulmonary syndromes and asthma is uncertain. To determine if eosinophils might play a harmful role in these conditions, we cocultured purified human eosinophils, eosinophil major basic protein (MBP), and chromatographically eluted eosinophil granule fractions with human A549 and rat type II pneumocytes. Damage to these target cells was measured as cell lysis and nonlethal cell detachment. We found that unstimulated intact eosinophils affected minimal lysis or detachment of either pneumocyte target, but eosinophils stimulated with phorbol myristate acetate and other activators produced time- and dose-dependent nonlytic detachment of both targets. In contrast, supernatants from activated eosinophils did not produce significant injury, suggesting that close apposition of the effector and target cells was required. Catalase and superoxide dismutase did not inhibit the detaching activity of eosinophils, suggesting that hydrogen peroxide and superoxide anion were not activity of eosinophils, suggesting that hydrogen peroxide and superoxide anion were not responsible for mediating this form of injury. In contrast to our findings with intact eosinophils, we observed that the addition of purified eosinophil MBP to pneumocytes caused marked cytolysis with little detachment. When sequential fractions of eosinophil granules separated by Sephadex G-50 chromatography were added to A549 and rat type II pneumocyte targets, it was found that different fractions produced distinct forms of injury. Higher molecular weight fractions containing lysosomal enzymes and eosinophil peroxidase produced predominantly detachment, whereas fractions enriched in MBP produced lysis. These results indicate that intact eosinophils can produce nonlytic detachment of alveolar pneumocytes that is probably not dependent on the generation of toxic oxygen radicals but rather appears to be mediated by granule-associated products, possibly lysosomal enzymes. Furthermore, although intact eosinophils are not capable of lysing alveolar epithelial cells under the conditions of our assay, MBP has the potential to do so when the protein is released in high enough concentrations. The in vivo relevance of these findings in eosinophilic lung diseases may be that eosinophils, by producing both desquamation and death of alveolar epithelium cells, may increase the permeability of the alveolus to fluid and cells. Moreover, these forms of damage might also enhance the ingress of inhaled antigens across the pulmonary epithelial barrier, thus increasing immunologic sensitization.     

Eosinophil infiltration and degranulation in oesophageal mucosa from adult patients with eosinophilic oesophagitis: a retrospective and comparative study on pathological biopsy

AIM: To examine eosinophil infiltration and degranulation in 50 oesophageal biopsy specimens from 30 patients (21 men, 9 women; mean 39 years) with eosinophilic oesophagitis, by haematoxylin and eosin staining and immunohistochemistry. METHODS: Immunohistochemistry was carried out using a monoclonal antibody for human eosinophilic major basic protein (MBP). Eosinophils were counted in three high power fields (x40) and degranulation, as quantified by extracellular MBP immunostaining, was scored on a scale of 1-4. Morphological changes (basal cell hyperplasia, elongation of papillae and dilatation of intercellular spaces) were scored on a 1-4 scale on sections stained with haematoxylin and eosin. RESULTS: Numbers of intraepithelial eosinophils were significantly higher with MBP immunostaining than with haematoxylin and eosin staining (mean 109.6 v 80.6; p<0.001), whereas numbers of eosinophils were considerably correlated (r = 0.794). Eosinophil degranulation was higher in the distal oesophagus. Additionally, basic morphological changes were markedly associated with eosinophil infiltration. Extracellular deposition of eosinophil-MBP and eosinophil infiltration in subepithelial connective tissue, present in the biopsy specimens, were detected by immunohistochemistry. CONCLUSION: Numbers of eosinophils and degranulation are underestimated by haematoxylin and eosin staining. Immunohistochemistry detected up to two times more eosinophils than routine haematoxylin and eosin staining. Moreover, eosinophil-MBP immunoreactivity in extracellular regions indicates the release of toxic eosinophil granule proteins and gives further evidence for a causative role of eosinophils with regard to structural changes in eosinophilic oesophagitis. Immunohistochemistry may serve as a useful diagnostic tool to support the morphological differential diagnosis of eosinophilic oesophagitis and gastro-oesophageal reflux disease.     

Inhibition of eosinophils degranulation by Ketotifen in a patient with milk allergy,manifested as bronchial asthma—an electron microscopic study

Electron microscopic studies provided evidence that a patient with cow's milk allergy, manifested as bronchial asthma, has prominent eosinophil granule discharge, attributable to the release of cytotoxic major basic protein (MBP). This finding illustrates a critical role of eosiniphil MBP in anaphylactic injury induced by food allergen. Patient white blood cells pretreated with Ketotifen revealed intact ultrastructure of eosinophils granules and cytoplasm. Inhibition of eosinophils degranulation by Ketotifen demonstrates a new pharmacologic mode of action of this anti-allergic, anti-anaphylactic agent.     

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.     

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.     

Allergen extracts directly mobilize and activate human eosinophils

Allergic diseases are characterized by the presence of eosinophils, which are recruited to the affected tissues by chemoattractants produced by T cells, mast cells and epithelium. Our objective was to evaluate if allergens can directly activate human eosinophils. The capacity of purified allergen extracts to elicit eosinophil chemotaxis, respiratory burst, degranulation and up-regulation of the adhesion molecule complement receptor 3 (CR3) was determined in eosinophils isolated from healthy blood donors. Eosinophils stimulated with an extract from house dust mite (HDM) released the granule protein major basic protein (MBP) and up-regulated the surface expression of CR3. Cat allergen extracts also induced the up-regulation of CR3, but not the release of MBP; instead cat, as well as birch and grass allergens, elicited the release of eosinophil peroxidase (EPO). In addition, grass pollen extract caused the secretion of MBP. None of the allergens stimulated eosinophilic cationic protein release, nor production of free oxygen radicals. Both HDM and birch extracts were chemotactic for eosinophils. These findings establish that common aeroallergens can directly activate eosinophils in vitro. We propose that eosinophil activation in vivo is not exclusively mediated by cytokines and chemokines of the allergic inflammatory reaction, but could partly be the result of direct interaction between allergens and eosinophils.     

Localization of human eosinophil granule major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin by immunoelectron microscopy

By utilizing the colloidal gold particle technique, we localized eosinophil granule major basic protein, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) in human nasal polyp sections by immunoelectron microscopy. Sections stained with affinity chromatography purified rabbit anti-human major basic protein, and subsequently with gold colloidal particle-goat anti-rabbit IgG, showed gold particles predominantly within granule cores, and not within other eosinophil organelles, plasma cells, mast cells, lymphocytes, or neutrophils. Sections stained with anti-ECP or anti-EDN showed gold particles concentrated over the granule matrix with fewer particles centrally. Control sections treated with preimmunization sera showed no staining of cells or organelles. These results verify the localization of major basic protein to the crystalloid core of the human eosinophil granule and show that ECP and EDN reside in the granule matrix. This technique provides a means of accurately locating the sites of major basic protein, ECP, and EDN deposition and thus of identifying eosinophil degranulation patterns in human disease.     

Eosinophil-mediated bile duct stricture. A case studied by immunohistochemistry.

Major basic protein accounts for the majority of the protein within the eosinophilic granule. Utilizing immunohistochemical staining for major basic protein, we have demonstrated the dominant role of the eosinophil in a reversible bile duct stricture.     

Antimicrobial activity of human eosinophil granule proteins: involvement in host defence against pathogens

Eosinophils have been associated with the pathophysiology of various allergic diseases and asthma. Eosinophils secrete a number of granule proteins that have been identified as effector molecules responsible for many of the actions of eosinophils. The four major eosinophil granule proteins, major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase have been shown to be involved in a number of eosinophil associated functions. EDN possesses antiviral activity against single stranded RNA viruses like respiratory syncytial virus, Hepatitis and HIV, whereas ECP and MBP have antibacterial and antiparasitic properties. This review summarizes the studies on antipathogenic activities of eosinophil granule proteins against bacteria, viruses, protozoans and helminths.     

Localization and content of nerve growth factor in peripheral blood eosinophils of atopic dermatitis patients

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing eczematous skin disorder characterized by eosinophilia. Nerve growth factor (NGF) modulates the allergic response through interactions with immune-inflammatory cells. Eosinophils have been reported to store NGF as a preformed mediator. OBJECTIVE: To gain further insight into the significance of eosinophils in association with NGF in the pathogenesis of AD, the localization of NGF within eosinophils and the difference of the eosinophil-derived NGF content in the peripheral blood of normal volunteers vs. AD patients were investigated. METHODS: We examined the localization of NGF within human eosinophils using the post-embedding immunoelectron microscopy and compared NGF content in freshly isolated eosinophil sonicates from the peripheral blood of 31 normal volunteers vs. 42 AD patients by immunoenzymatic assay. A possible correlation between the levels of NGF and major basic protein was also examined. RESULTS: Immunoelectron microscopic studies revealed that NGF was localized in the central core of normal eosinophil granules, where major basic protein is also present as a preformed mediator, in homogeneous granules and in intergranular ductal or vesicular structures adjacent to specific granules of eosinophils. NGF content in eosinophils was significantly increased in AD patients. Furthermore, there was a significant correlation between levels of NGF and major basic protein in eosinophils of AD patients. CONCLUSIONS: Increased levels of NGF contained in eosinophils of the peripheral blood from AD patients, when released with other mediators such as basic proteins, could promote inflammation and local tissue damage.     

Immunofluorescence analysis of cytokine and granule protein expression during eosinophil maturation from cord blood–derived CD34 progenitors

Background: In allergic inflammation and asthma, eosinophils are major effector cells. They have been shown to synthesize at least 23 cytokines, some of which are stored intracellularly in their unique crystalloid granules together with cationic granule protein. Little is known about the synthesis and storage of cytokines relative to cationic granule proteins in maturing eosinophils during eosinophilopoiesis. Objective: Our purpose was to analyze the expression of eosinophil-derived mediators, major basic protein (MBP), eosinophil cationic protein (ECP), IL-6, and RANTES, during early stages of eosinophil maturation in CD34⁺ cell-derived colonies. Methods: Purified human cord blood CD34⁺ cells were grown in methylcellulose cultures in the presence of recombinant human IL-3 and IL-5. By confocal laser scanning microscopy, the coexpression of eosinophil granular proteins MBP and ECP was determined concurrently with IL-6 and RANTES during eosinophil maturation on days 16, 19, 23, and 28 of culture. Results: Immunoreactivity against MBP, ECP, IL-6, and RANTES was not detectable in freshly purified CD34⁺ cells. Maturing eosinophils (>95%) exhibited positive immunostaining for all these proteins between days 16 and 28 of culture. At early stages of culture, discrete immunostaining was observed around the periphery but not in the center of granular structures. By day 28 cultured eosinophil-like cells showed evidence of the acquisition of crystalloid granule-like structures, analogous to those observed in mature peripheral blood eosinophils. Conclusions: Eosinophils express and store cytokines simultaneously with cationic granule proteins during the process of maturation. We propose that the storage of cytokines during the development of eosinophils is an early event and it may be integral to inflammatory responses involving these cells. The results of this study suggest a potential immunoregulatory function for maturing eosinophils. (J Allergy Clin Immunol 2000;105:1178-84.)     

Arginine-rich cationic proteins of human eosinophil granules: comparison of the constituents of eosinophilic and neutrophilic leukocytes.

Several arginine-rich cationic proteins previously isolated from granules of leukemic myeloid cells have been found to reside primarily in human eosinophil leukocytes. The major component has a molecular weight of 21,000 and it contains approximately 2.6 moles of zinc per mole of protein. Velocity centrifugation of cytoplasm from leukocytes of patients with marked eosinophilia showed that this group of proteins is packaged in the crystalloid-containing large eosinophil granules. Approximately 30% of the protein content of eosinophil granules belonged to this group of cationic proteins. Bactericidal or esterolytic activities of the cationic proteins were not detected, nor did they inhibit guinea pig anaphylatoxin or histamine-induced contraction. The basic protein previously demonstrated in guinea pig eosinophils may be analogous to the group of basic proteins of human eosinophils but great differences are found for molecular weight and amino acid composition.