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.     

Diverse effects of eosinophil cationic granule proteins on IMR-32 nerve cell signaling and survival

Activated eosinophils release potentially toxic cationic granular proteins, including the major basic proteins (MBP) and eosinophil-derived neurotoxin (EDN). However, in inflammatory conditions including asthma and inflammatory bowel disease, localization of eosinophils to nerves is associated with nerve plasticity, specifically remodeling. In previous in vitro studies, we have shown that eosinophil adhesion to IMR-32 nerve cells, via nerve cell intercellular adhesion molecule-1, results in an adhesion-dependent release of granule proteins. We hypothesized that released eosinophil granule proteins may affect nerve cell signaling and survival, leading to nerve cell remodeling. Culture in serum-deprived media induced apoptosis in IMR-32 cells that was dose-dependently abolished by inclusion of MBP1 but not by EDN. Both MBP1 and EDN induced phosphorylation of Akt, but with divergent time courses and intensities, and survival was independent of Akt. MBP1 induced activation of neural nuclear factor (NF)-kappaB, from 10 min to 12 h, declining by 24 h, whereas EDN induced a short-lived activation of NF-kappaB. MBP1-induced protection was dependent on phosphorylation of ERK 1/2 and was related to a phospho-ERK-dependent upregulation of the NF-kappaB-activated anti-apoptotic gene, Bfl-1. This signaling pathway was not activated by EDN. Thus, MBP1 released from eosinophils at inflammatory sites may regulate peripheral nerve plasticity by inhibiting apoptosis.     

Pitfalls in the quantitative estimation of the secretion of granule proteins by eosinophils

The secretion of preformed granule proteins by eosinophils is an important correlate of eosinophil activation. However, a review of the literature reveals large disparities in the amounts of these substances which were reportedly secreted when eosinophils were activated. In the present study we report that our attempts to quantitate the secretion of eosinophil peroxidase and eosinophil-derived neurotoxin from activated eosinophils by measuring these substances in the incubation supernatants were uniformly unsuccessful. We found that, once they were secreted, both eosinophil peroxidase and eosinophil-derived neurotoxin were promptly lost to assay and presumably destroyed. Thus the measurement of the difference in the concentration of these substances in eosinophils prior to and after activation, revealed that as much as 65% of the eosinophil-derived neurotoxin and 62% of the peroxidase in the eosinophils were lost to assay during activation of the cells whereas the largest amount of these substances which could be measured in the incubation supernatants never exceeded 2%. Evidence is presented that the destruction of eosinophil-derived neurotoxin must occur prior to the release of this substance into the medium. Attempts to inhibit the destruction of eosinophil peroxidase and of eosinophil-derived neurotoxin by incorporating various inhibitors into the incubations were unsuccessful. These results emphasize the need to monitor the overall recoveries of secreted products from activated eosinophils and suggest that meaningful estimates of the secretion of these granule proteins from activated eosinophils can only be obtained by measuring the residual content of these substances in eosinophils after they have been activated and comparing these values to the contents of eosinophils prior to activation.     

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.     

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.     

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.     

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.     

Mast cell tryptase activates peripheral blood eosinophils to release granule-associated enzymes

BACKGROUND: Mast cells and eosinophils are important effector cells in asthma. Understanding their interactions is essential for studying asthma pathophysiology. Inflammatory mediators released from mast cells, such as arachidonic acid metabolites, TNF and IL-5, are important in eosinophil biology. However, little is known about the effects of mast cell-specific mediators, such as tryptase, on eosinophils. Our objective was to investigate the effects of mast cell tryptase on human peripheral blood eosinophils. METHODS: Peripheral blood eosinophils isolated from asthmatic individuals were activated using various concentrations of tryptase- and protease-activated receptor-2 (PAR-2)-activating peptides (PAR-2 AP). Eosinophil activation was evaluated by the release of granule mediators, superoxide release, estimation of eosinophil survival, changes in intracellular Ca2+ concentration and mitogen-activated protein kinase activation. RESULTS: Tryptase induced the release of eosinophil peroxidase and beta-hexosaminidase from peripheral blood eosinophils but had no effect on RANTES release. Eosinophils isolated from two thirds of our donors responded to tryptase, while the remainder appeared not to respond. Release of granule mediators was dependent on tryptase enzymatic activity. To identify the mechanism of eosinophil activation by tryptase, we studied the expression of PAR-2 by eosinophils and its function. Using RT-PCR, we amplified PAR-2 from eosinophils. However, flow cytometry failed to detect significant PAR-2 expression on the surface of eosinophils. The PAR-2 AP SLIGRL-NH2 did not induce eosinophil activation by any of the methods we employed. CONCLUSION: Our data indicate that mast cell tryptase may affect eosinophil activation status independently of PAR-2.     

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.     

Eosinophils Induce Airway Smooth Muscle Cell Proliferation

Asthma is characterized by eosinophilic airway inflammation and remodeling of the airway wall. Features of airway remodeling include increased airway smooth muscle (ASM) mass. However, little is known about the interaction between inflammatory eosinophils and ASM cells. In this study, we investigated the effect of eosinophils on ASM cell proliferation. Eosinophils were isolated from peripheral blood of mild asthmatics and non-asthmatic subjects and co-cultured with human primary ASM cells. ASM proliferation was estimated using Ki-67 expression assay. The expression of extracellular matrix (ECM) mRNA in ASM cells was measured using quantitative real-time PCR. The role of eosinophil derived Cysteinyl Leukotrienes (CysLTs) in enhancing ASM proliferation was estimated by measuring the release of leukotrienes from eosinophils upon their direct contact with ASM cells using ELISA. This role was confirmed either by blocking eosinophil-ASM contact or co-culturing them in the presence of leukotrienes antagonist. ASM cells co-cultured with eosinophils, isolated from asthmatics, but not non-asthmatics, had a significantly higher rate of proliferation compared to controls. This increase in ASM proliferation was independent of their release of ECM proteins but dependent upon eosinophils release of CysLTs. Eosinophil-ASM cell to cell contact was required for CysLTs release. Preventing eosinophil contact with ASM cells using anti-adhesion molecules antibodies, or blocking the activity of eosinophil derived CysLTs using montelukast inhibited ASM proliferation. Our results indicated that eosinophils contribute to airway remodeling during asthma by enhancing ASM cell proliferation and hence increasing ASM mass. Direct contact of eosinophils with ASM cells triggers their release of CysLTs which enhance ASM proliferation. Eosinophils, and their binding to ASM cells, constitute a potential therapeutic target to interfere with the series of biological events leading to airway remodeling and Asthma.     

Inhibition of CCL11, CCL24, and CCL26-induced degranulation in HL-60 eosinophilic cells by specific inhibitors of MEK1/MEK2, p38 MAP kinase,and PI 3-kinase

Eosinophilic leukocytes are the cellular hallmark of allergic inflammation. Apart from being potent eosinophils chemoattractants, the eotaxins CCL11, CCL24 and CCL26 are capable of activating eosinophils to generate reactive oxygen species, lipid mediators of inflammation and degranulation of toxic granule proteins. Due to their central role in eosinophil trafficking and activation, understanding the signal transduction mechanism of the eotaxin-induced eosinophil effector functions may provide an innovative therapeutic strategy for eosinophil-associated diseases. Thus, these investigations were conducted to delineate signal transduction mechanisms of CCL11, CCL24 and CCL26-induced eosinophil peroxidase (EPO) degranulation following pretreatment of cells with or without a specific inhibitor of MEK1/MEK2 (U0126), inhibitor of p38 MAP kinase (SB203580) or a specific inhibitor of PI 3-kinase (LY294002). Results have shown that CCR3-mediated eotaxin-induced eosinophilic degranulation was concentration-dependently reduced by specific inhibitors of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. However, the rank order of U0126 with respect to inhibition of chemokine-induced degranulation was CCL11 = CCL24 > CCL26. Potentiation of eotaxin-induced EPO degranulation by IL-5 was also seen. These investigations have not only confirmed the reported co-operativity between IL-5 and the eotaxins but also showed that the eosinophil-degranulating capabilities of the eotaxin CCL11, CCL24 and CCL26 is a consequence of activation of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. Thus, these signaling molecules may provide the biochemical basis for mechanism-based therapy of allergic inflammatory diseases.     

Inhibition Of Ccl11, Ccl24, And Ccl26-induced Degranulation In Hl-60 Eosinophilic Cells By Specific Inhibitors Of Mek1/mek2, P38 Map Kinase,And Pi 3-Kinase

Eosinophilic leukocytes are the cellular hallmark of allergic inflammation. Apart from being potent eosinophils chemoattractants, the eotaxins CCL11, CCL24 and CCL26 are capable of activating eosinophils to generate reactive oxygen species, lipid mediators of inflammation and degranulation of toxic granule proteins. Due to their central role in eosinophil trafficking and activation, understanding the signal transduction mechanism of the eotaxin-induced eosinophil effector functions may provide an innovative therapeutic strategy for eosinophil-associated diseases. Thus, these investigations were conducted to delineate signal transduction mechanisms of CCL11, CCL24 and CCL26-induced eosinophil peroxidase (EPO) degranulation following pretreatment of cells with or without a specific inhibitor of MEK1/MEK2 (U0126), inhibitor of p38 MAP kinase (SB203580) or a specific inhibitor of PI 3-kinase (LY294002). Results have shown that CCR3-mediated eotaxin-induced eosinophilic degranulation was concentration-dependently reduced by specific inhibitors of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. However, the rank order of U0126 with respect to inhibition of chemokine-induced degranulation was CCL11=CCL24>CCL26. Potentiation of eotaxin-induced EPO degranulation by IL-5 was also seen. These investigations have not only confirmed the reported co-operativity between IL-5 and the eotaxins but also showed that the eosinophil-degranulating capabilities of the eotaxin CCL11, CCL24 and CCL26 is a consequence of activation of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. Thus, these signaling molecules may provide the biochemical basis for mechanism-based therapy of allergic inflammatory diseases.     

Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins.

Eosinophils, neutrophils, and mast cells have all been implicated in the pathogenesis of bullous pemphigoid (BP). To comparatively characterize the involvement of these cells in BP, 10 lesional skin biopsy specimens were identified retrospectively and studied for tissue localization of eosinophil, neutrophil, and mast cell granule proteins. Subsequently, multiple skin biopsies of lesions in various developmental stages were obtained from 3 patients with untreated BP. Involved and uninvolved skin specimens were also obtained from 2 patients. Using indirect immunofluorescence, retrospectively identified lesions showed eosinophils and extracellular granule protein deposition prominently in areas of blistering. Evolving lesions showed eosinophil granule protein deposition in all stages but was most marked in early erythematous and prebullous (urticarial) lesions and was minimal in uninvolved skin. Vascular cell adhesion molecule-1, E-selectin, and P-selectin were detected on vessels and very late activation antigen-4 was detected on mononuclear cells and eosinophils by immunoperoxidase staining of lesions. Eosinophil granule proteins were increased in the peripheral blood, urine, and blister fluid. Blister fluids caused increased eosinophil survival that was inhibited by antibodies to interleukin-5 and interleukin-3. Although neutrophil and mast cell infiltration was observed, extracellular granule protein deposition from these cells was minimal except in two specimens. These results demonstrate that eosinophils infiltrate and deposit granule proteins early in the development of BP lesions, that eosinophil-activating cytokines are present in blister fluid, and that eosinophil-selective adhesion molecules are present. These studies strongly support a role for eosinophils in blister formation in BP.     

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.     

Eosinophils function as antigen-presenting cells

Eosinophils release lipid mediators, including leukotriene C4, platelet-activating factor, and liposins, and contain four distinct granule cationic proteins, major basic protein, eosinophil peroxidase, eosinophil cationic protein, and eosinophil-derived neurotoxin, which may cause dysfunction and destruction of other cells. Eosinophils are primarily thought of as terminal effectors of allergic responses and of parasite elimination. Eosinophils are characteristically present within the airway lumina of asthmatics, and these airway eosinophils have been induced in vivo to express major histocompatibility complex II (MHC-II) complexes and costimulatory molecules, which are required for T lymphocytes to be functionally activated. In in vitro experiments, eosinophils can process antigen and express the costimulatory molecules, and after cytokine-elicited induction of MHC-II, expression can function as antigen-presenting cells in stimulating T lymphocyte responses. Airway luminal eosinophils can migrate into draining paratracheal lymph nodes, localized to T cell-rich paracortical areas, and stimulate antigen-specific T cell proliferation in vivo within paratracheal lymph nodes, which was CD80- and CD86-dependent and limited to CD4+ T cells. Furthermore, eosinophils within the lumina of airways promote expansion of T helper cell type 2 (Th2) by presenting antigen, suggesting that eosinophils actively modulate immune responses by amplifying Th2 cell responses.     

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.     

Selectivity of mediators released by eosinophils

The presence of receptors for IgE on eosinophils has drawn the attention on their direct participation in IgE-dependent hypersensitivity reactions. Surface IgE antibodies were detected on eosinophils from allergic patients. The addition of the specific allergen or anti-IgE antibodies to such purified eosinophils induced the release of eosinophil peroxidase, but not of eosinophil cationic protein. These findings associated with results obtained by using electron microscopy and immunogold staining of the various antibodies directed against the granule proteins allowed us to suggest a selectivity in the mediators released by eosinophils. In addition, preliminary results concerning the existence and the functional role of a receptor for IgA on eosinophils are reported, leading to the concept of a particular interaction of eosinophils with immunoglobulins present in the tissues and their participation in local immune responses.     

Degranulating eosinophils in human endometriosis

Degranulating eosinophils have been described in most endometrial cancers. We hypothesized that endometriosis (ectopic, nonneoplastic endometrial tissue) would be an appropriate model system for determining whether eosinophil degranulation is part of a specific immune response to endometrial cancer or if it is related to the more general phenomenon of tissue remodeling (wound healing) that is common to both disorders. To test this hypothesis, we performed immunohistochemistry and Western blotting to evaluate the presence of eosinophil peroxidase (a marker of eosinophil degranulation) in normal endometrium (n = 20) and endometriosis samples (n = 24) and to define the coexpression of three eosinophil chemoattractants: interleukin-5 (IL-5), eotaxin, and regulated on activator-normal T cell expressed and secreted (RANTES). There was focally intense deposition of eosinophil peroxidase in the fibrotic connective tissue and blood vessels of 21 of 24 human endometriosis specimens; two samples showed weak staining, and only one tissue was negative for eosinophil degranulation. None of the 10 normal proliferative endometrial specimens had evidence of eosinophil degranulation, and four of 10 secretory tissues stained only weakly for eosinophil peroxidase. The presence of degranulating eosinophils was also associated with the presence of eotaxin and IL-5 in some samples and with RANTES in others. We conclude that the abundant presence of degranulating eosinophils in the fibrous regions of endometriosis supports the interpretation that eosinophils are involved in general tissue remodeling and wound healing rather than a tissue-directed immune response.     

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.     

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.