Flow cytometric detection and microscopic observation of activated eosinophils in peripheral blood.

Eosinophils possess highly electron-dense granules with crystal-like structures and are characterized as high side scatter (SSC) areas by flow cytometry analysis. Eosinophils with low SSC features have been noted in extremely rare cases; however, the underlying cause remains unclear. Eosinophils in the low SSC area were analyzed using microscopy. A transmission electron microscope revealed the loss of crystal-like structures in granules with low electron density and piecemeal degranulation, which was undetectable by May-Grünwald-Giemsa staining. Based on the results of flow cytometry, May-Grünwald-Giemsa staining and transmission electron microscopy, SSC values could help potentially detect crystal-like structures and piecemeal degranulation eosinophils.

Eosinophil exocytosis in a poorly differentiated tubular gastric adenocarcinoma: case report.

A case of poorly differentiated tubular gastric adenocarcinoma with tumor-associated tissue eosinophilia (TATE) is studied by light and electron microscopy, focusing on membrane interactions between eosinophils and tumor cells. 29.2% of the eosinophils in contact with tumor cells showed intact granules, 28.3% exhibited piecemeal degranulation (PMD), 40% were characterized by coexistence of PMD and compound exocytosis in the same granulocyte, whereas classical exocytosis was found in 2.5% of the eosinophils with PMD. Eosinophil Sombrero Vesicles (EoSVs), important tubulovesicular carriers for delivery of cytotoxic proteins from the specific granules during PMD, were also studied at the ultrastructural level. In activated eosinophils, EoSVs and specific granules with ultrastructural signs of degranulation were polarized toward tumor cells. Ultrastructural changes in paraptosis-like cell death, such as mitochondrial swelling, dilation of the nuclear envelope, cytoplasmic vacuoles, and nuclear chromatin condensation, but without margination of the chromatin, were observed in these tumor cells. Our data support the notion that eosinophils may exert an antitumoral role in gastric cancer. Finally, the case reported provides, for the first time, ultrastructural evidence of classical and compound exocytosis of eosinophils in the tumor stroma of human adenocarcinoma.

Label-free imaging and evaluation of characteristic properties of asthma-derived eosinophils using optical diffraction tomography.

Optical diffraction tomography (ODT), an emerging imaging technique that does not require fluorescent staining, can measure the three-dimensional distribution of the refractive index (RI) of organelles. In this study, we used ODT to characterize the pathological characteristics of human eosinophils derived from asthma patients presenting with eosinophilia. In addition to morphological information about organelles appearing in eosinophils, including the cytoplasm, nucleus, and vacuole, we succeeded in imaging specific granules and quantifying the RI values of the granules. Interestingly, ODT analysis showed that the RI (i.e., molecular density) of granules was significantly different between eosinophils from asthma patients and healthy individuals without eosinophilia, and that vacuoles were frequently found in the cells of asthma patients. Our results suggest that the physicochemical properties of eosinophils derived from patients with asthma can be quantitatively distinguished from those of healthy individuals. The method will provide insight into efficient evaluation of the characteristics of eosinophils at the organelle level for various diseases with eosinophilia.

Human pluripotent stem cell-derived eosinophils reveal potent cytotoxicity against solid tumors.

Eosinophils are attractive innate immune cells to use to potentiate T cell antitumor efficacy because they are capable of infiltrating tumors at early stages and modulating the tumor microenvironment. However, the limited number of functional eosinophils caused by the scarcity and short life of primary eosinophils in peripheral blood has greatly impeded the development of eosinophil-based immunotherapy. In this study, we established an efficient chemically defined protocol to generate a large quantity of functional eosinophils from human pluripotent stem cells (hPSCs) with nearly 100% purity expressing eosinophil peroxidase. These hPSC-derived eosinophils transcriptionally resembled their primary counterpart. Moreover, hPSC-derived eosinophils showed competent tumor killing capacity in established solid tumors. Furthermore, the combination of hPSC-derived eosinophils with CAR-T cells exhibited potential synergistic effects, inhibiting tumor growth and enhancing mouse survival. Our study opens up new avenues for the development of eosinophil-based immunotherapies to treat cancer.

Eosinophilic dermatoses.

Eosinophilic dermatoses are a heterogeneous group of diseases, characterized by an eosinophil-rich infiltrate and/or degranulation of eosinophils. Blood eosinophilia may be an associated feature. Typical, albeit not specific histological findings include 'flame figures', which are caused by the accumulation of cationic proteins released by eosinophils and subsequent collagen denaturation. "Classic" eosinophilic dermatoses include eosinophilic cellulitis (Wells syndrome), granuloma faciale, eosinophilic fasciitis (Shulman syndrome) and eosinophilic folliculitis (Ofuji disease). In addition, there is a multitude of skin diseases that present with varying degrees of eosinophilic infiltration. These include atopic dermatitis, bullous pemphigoid, urticaria, allergic contact dermatitis, prurigo nodularis, arthropod bite reaction, parasitic infections, and drug hypersensitivity. Even though these disorders share a common characteristic (tissue eosinophilia), they differ greatly in their clinical presentation.

Eosinophils capture viruses, a capacity that is defective in asthma.

BACKGROUND: Activated eosinophils cause major pathology in stable and exacerbating asthma; however, they can also display protective properties like an extracellular antiviral activity. Initial murine studies led us to further explore a potential intracellular antiviral activity by eosinophils. METHODS: To follow eosinophil-virus interaction, respiratory syncytial virus (RSV) and influenza virus were labeled with a fluorescent lipophilic dye (DiD). Interactions with eosinophils were visualized by confocal microscopy, electron microscopy, and flow cytometry. Eosinophil activation was assessed by both flow cytometry and ELISA. In a separate study, eosinophils were depleted in asthma patients using anti-IL-5 (mepolizumab), followed by a challenge with rhinovirus-16 (RV16). RESULTS: DiD-RSV and DiD-influenza rapidly adhered to human eosinophils and were internalized and inactivated (95% in ≤ 2 hours) as reflected by a reduced replication in epithelial cells. The capacity of eosinophils to capture virus was reduced up to 75% with increasing severity of asthma. Eosinophils were activated by virus in vitro and in vivo. In vivo this correlated with virus-induced loss of asthma control. CONCLUSIONS: This previously unrecognized and in asthma attenuated antiviral property provides a new perspective to eosinophils in asthma. This is indicative of an imbalance between protective and cytotoxic properties by eosinophils that may underlie asthma exacerbations.

Impact of eosinophil-peroxidase (EPX) deficiency on eosinophil structure and function in mouse airways.

Eosinophil peroxidase (EPX) is a major constituent of the large cytoplasmic granules of both human and mouse eosinophilic leukocytes. Human EPX deficiency is a rare, autosomal-recessive disorder limited to the eosinophil lineage. Our intent was to explore the impact of EPX gene deletion on eosinophil content, structure, and function. In response to repetitive intranasal challenge with a filtrate of the allergen, Alternaria alternata, we found significantly fewer eosinophils peripherally and in the respiratory tracts of EPX-/- mice compared to wild-type controls; furthermore, both the major population (Gr1-/lo ) and the smaller population of Gr1hi eosinophils from EPX-/- mice displayed lower median fluorescence intensities (MFIs) for Siglec F. Quantitative evaluation of transmission electron micrographs of lung eosinophils confirmed the relative reduction in granule outer matrix volume in cells from the EPX-/- mice, a finding analogous to that observed in human EPX deficiency. Despite the reduced size of the granule matrix, the cytokine content of eosinophils isolated from allergen-challenged EPX-/- and wild-type mice were largely comparable to one another, although the EPX-/- eosinophils contained reduced concentrations of IL-3. Other distinguishing features of lung eosinophils from allergen-challenged EPX-/- mice included a reduced fraction of surface TLR4+ cells and reduced MFI for NOD1. Interestingly, the EPX gene deletion had no impact on eosinophil-mediated clearance of gram-negative Haemophilus influenzae from the airways. As such, although no clinical findings have been associated with human EPX deficiency, our findings suggest that further evaluation for alterations in eosinophil structure and function may be warranted.

Activation of Eosinophils and Mast Cells in Functional Dyspepsia: an Ultrastructural Evaluation.

We recently identified mucosal mast cell and eosinophil hyperplasia in association with a duodenal impaired barrier function in functional dyspepsia (FD). We aimed to further describe the implication of these immune cells by assessing their activation state at the ultrastructural level and by evaluating the association between impaired epithelial integrity and immune activation. Duodenal biopsies were obtained from 24 FD patients and 37 healthy controls. The ultrastructure of mast cells and eosinophils was analyzed by transmission electron microscopy. Transepithelial electrical resistance and paracellular permeability were measured to evaluate epithelial barrier function. The type of degranulation in eosinophils and mast cells was piecemeal. Eosinophils displayed higher degree of degranulation in FD patients than in controls (p < 0.0001). Quantification revealed a decreased granular density in eosinophils of FD patients (p < 0.0001). The degree of degranulation in mast cells was similar in both groups. However, a more heterogeneous profile was found in the FD group (p < 0.0001). No association between epithelial integrity and the number and activation state of mucosal eosinophils and mast cells was found. We demonstrated ultrastructural changes in degranulation state of eosinophils and mast cells, suggesting that eosinophil and mast cell activation play a role in the pathophysiology of FD.

Characterization of Cotton Rat (Sigmodon hispidus) Eosinophils, Including Their Response to Respiratory Syncytial Virus Infection.

Eosinophils have been postulated to play a protective role against infection with respiratory syncytial virus (RSV), increase the severity of allergic asthma during respiratory viral infection, and drive vaccine-enhanced disease. To address these questions in the cotton rat model of RSV infection, we characterized cotton rat eosinophils by electron microscopy as well as by bronchoalveolar lavage and histology of lung sections. Using these methods, we demonstrated that eosinophils comprise approximately half of all cells in the bronchoalveolar lavage fluids from cotton rats. The function of these cells was comparable to that of eosinophils of other species. Ex vivo, eosinophils stimulated with calcium ionophores secreted eosinophil peroxidase. In vivo, treatment with house dust mite antigen increased eosinophil numbers in lung. Infection with Staphylococcus aureus lead to a marked increase in neutrophils without an increase in eosinophils, and eosinophil numbers were not influenced by infection with influenza virus or measles virus. Similarly, infection with RSV did not result in an increase in eosinophils. Lastly, RSV infection did not increase eosinophil recruitment into the lung after challenge with house dust mite antigen, but it did increase eosinophil recruitment into the lungs of cotton rats previously immunized with formalin-inactivated RSV vaccine, thus contributing to vaccine-enhanced disease.

Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Schistosoma mansoni Infection.

Eosinophils have been long associated with helminthic infections, although their functions in these diseases remain unclear. During schistosomiasis caused by the trematode Schistosoma mansoni, eosinophils are specifically recruited and migrate to sites of granulomatous responses where they degranulate. However, little is known about the mechanisms of eosinophil secretion during this disease. Here, we investigated the degranulation patterns, including the cellular mechanisms of major basic protein-1 (MBP-1) release, from inflammatory eosinophils in a mouse model of S. mansoni infection (acute phase). Fragments of the liver, a major target organ of this disease, were processed for histologic analyses (whole slide imaging), conventional transmission electron microscopy (TEM), and immunonanogold EM using a pre-embedding approach for precise localization of major basic protein 1 (MBP-1), a typical cationic protein stored pre-synthesized in eosinophil secretory (specific) granules. A well-characterized granulomatous inflammatory response with a high number of infiltrating eosinophils surrounding S. mansoni eggs was observed in the livers of infected mice. Moreover, significant elevations in the levels of plasma Th2 cytokines (IL-4, IL-13, and IL-10) and serum enzymes (alanine aminotransferase and aspartate aminotransferase) reflecting altered liver function were detected in response to the infection. TEM quantitative analyses revealed that while 19.1% of eosinophils were intact, most of them showed distinct degranulation processes: cytolysis (13.0%), classical and/or compound exocytosis identified by granule fusions (1.5%), and mainly piecemeal degranulation (PMD) (66.4%), which is mediated by vesicular trafficking. Immunonanogold EM showed a consistent labeling for MBP-1 associated with secretory granules. Most MBP-1-positive granules had PMD features (79.0 ± 4.8%). MBP-1 was also present extracellularly and on vesicles distributed in the cytoplasm and attached to/surrounding the surface of emptying granules. Our data demonstrated that liver-infiltrating mouse eosinophils are able to degranulate through different secretory processes during acute experimental S. mansoni infections with PMD being the predominant mechanism of eosinophil secretion. This means that a selective secretion of MBP-1 is occurring. Moreover, our study demonstrates, for the first time, a vesicular trafficking of MBP-1 within mouse eosinophils elicited by a helminth infection. Vesicle-mediated secretion of MBP-1 may be relevant for the rapid release of small concentrations of MBP-1 under cell activation.

Functions of tissue-resident eosinophils.

Eosinophils are a prominent cell type in particular host responses such as the response to helminth infection and allergic disease. Their effector functions have been attributed to their capacity to release cationic proteins stored in cytoplasmic granules by degranulation. However, eosinophils are now being recognized for more varied functions in previously underappreciated diverse tissue sites, based on the ability of eosinophils to release cytokines (often preformed) that mediate a broad range of activities into the local environment. In this Review, we consider evolving insights into the tissue distribution of eosinophils and their functional immunobiology, which enable eosinophils to secrete in a selective manner cytokines and other mediators that have diverse, 'non-effector' functions in health and disease.

The changing aetiology of eosinophilia in migrants and returning travellers in the Hospital for Tropical Diseases, London 2002-2015: An observational study.

INTRODUCTION: Determining the cause of eosinophilia in patients returning from the tropics continues to present a diagnostic challenge. The history, symptoms and degree of eosinophilia are often poor predictors of eventual diagnosis, but helminths are an important cause. The current British Infection Association recommendations use travel history to guide investigation of eosinophilia. However the global burden of helminth disease and travel patterns have changed over the last 3 decades and guidelines based on previous epidemiology need to be reviewed in the light of current data. METHODS: Consecutive patients presenting with, or referred for, investigation of eosinophilia were identified prospectively. Case notes, laboratory results and electronic records were reviewed for demographic and clinical data. Patients with an eosinophil count ≥0.50 × 109/L were included, and grouped based on lifetime history of travel to: West Africa, elsewhere in Africa, and the rest of the world. Results were compared to published data from 1997 to 2002 collected at the same centre. RESULTS: Of 410 patients who met the inclusion criteria, 407 had a documented travel history. Average yearly referrals for eosinophilia fell from 58 per year between 1997 and 2002, to 33 per year (2002-2015). The proportion of eosinophilia cases diagnosed with a parasitic cause fell from 64% to 50%, and yields for all parasitological investigations fell, the largest reduction in stool microscopy (20% yield to 9%) and day bloods for microfilariae (14% yield to 3%). Strongyloides stercoralis was the commonest diagnosis overall in our cohort, accounting for 50% of the total parasites diagnosed, and was present in 38% of patients from West Africa, 19% from rest of Africa, and 34% from rest of world; a relative increase compared to previous data. Schistosomiasis is slightly less common in those who had travelled to West Africa than the rest of Africa, and overall point prevalence has fallen from 33% (1997-2002) to 17% (2002-2015). Travellers were significantly less likely than patients who had immigrated to the UK to be diagnosed with any parasite (OR 0.54 95% CI 0.378-0.778 p = 0.0009). DISCUSSION: A parasitic cause will still be found in half of people returning from the tropics with an eosinophilia, but we observed a fall in the overall prevalence of parasitic diagnoses when compared with the earlier data. This may, in part, be explained by the impact of control programmes on the prevalence of parasites globally, especially filarial disease. S. stercoralis now represents the majority of parasites diagnosed in our cohort from all continents. We identified significantly higher rates of strongyloidiasis in immigrants than returning travellers. Despite the falling yields of stool microscopy and filarial serology the current guidelines based on travel history remain relevant with adequate yield.

Evidence of eosinophil extracellular trap cell death in COPD: does it represent the trigger that switches on the disease?

In spite of the numerous studies on chronic obstructive pulmonary disease (COPD), the cellular and molecular basis of the disease's development remain unclear. Neutrophils and eosinophils are known to be key players in COPD. Recently, neutrophil extracellular trap cell death (NETosis), a mechanism due to decondensation and extrusion of chromatin to form extracellular traps, has been demonstrated in COPD. However, there is limited knowledge about eosinophil extracellular trap cell death (EETosis) and its role in the pathogenesis of COPD. The aim of this study was to evaluate EETosis in stable COPD. Induced sputum obtained from healthy smokers and low exacerbation risk COPD A or B group patients or high exacerbation risk COPD C or D group patients were included. Samples were examined using electron microscopy and immunofluorescence. Healthy smokers (n=10) and COPD A (n=19) group exhibited neutrophilic or paucigranulocytic phenotypes, with NETosis being absent in these patients. In contrast, COPD B (n=29), with eosinophilic or mixed phenotypes, showed EETosis and incipient NETosis. COPD C (n=18) and COPD D groups (n=13) were differentiated from low exacerbation rate-COPD group by the abundant cellular debris, with COPD C group having an eosinophilic pattern and numerous cells undergoing EETosis. A hallmark of this group was the abundant released membranes that often appeared phagocytosed by neutrophils, which coincidentally exhibited early NETosis changes. The COPD D group included patients with a neutrophilic or mixed pattern, with abundant neutrophil extracellular trap-derived material. This study is the first to demonstrate EETosis at different stages of stable COPD. The results suggest a role for eosinophils in COPD pathophysiology, especially at the beginning and during the persistence of the disease, regardless of whether the patient quit smoking, with EETosis debris probably triggering uncontrolled NETosis. The main target of these findings should be young smokers with the potential to develop COPD.

Special dyeing, histochemistry, immunohistochemistry and ultrastructure: A study of mast cells/eosinophilic granules cells (MCs/EGC) from Centropomus parallelus intestine.

Intestine mast cells/eosinophilic granule cells (MCs/EGC) of the marine species Centropomus parallelus (fat snook) were first studied using light and electron microscopy techniques. Mast cells are cells from the connective tissue found in almost all organs and tissues of vertebrates. In fish, they appear in greater numbers in parts of their bodies that are exposed to their environment, such as skin, gills and intestine. The granules in fat snook's mast cell contain a variety of substances, such as histamine, heparin, chondroitin sulfate, serotonin, proteases and cytokines. The present study of intestine MCs/EGC was carried out in 20 specimens of fat snook. Samples of tissue were fixed in Bouin solution and in buffered formalin. Ferric hematoxylin - Congo red, pH6 acridine orange, pH2.5 and pH0,5 Alcian Blue (AB), toluidine blue, PAS, AB + PAS and immunohistochemistry protocols were used. In the mucosa and submucosa layers, MCs/EGCs granules with basic contents were evidenced by Congo red staining, and with acid contents granules were identified through pH 2.5 and 0,5 AB, and acridine orange. Basic and acid contents were simultaneously evidenced using ferric hematoxylin - Congo red stain. Metachromasia was observed in both mucosal and submucosal mast cells. Neutral glycoproteins were evidenced by using PAS protocol, glycosaminoglycan through AB and both simultaneously through AB + PAS. In immunohistochemistry assays, MCs/EGC were positive for tryptase, chymase and serotonin. As in mammals, the study of samples fixed in modified Karnovsky for transmission electron microscopy evidenced that most of the MCs granules were spherical and showed varying electron density, as described in previous reports on other teleost fish species. The metachromasia observed and the identification of tryptase, chymase and serotonin suggest a great similarity between fat snook's MCs/EGC and those described in the mucosa of mammals.

Genetic background modulates outcome of therapeutic amyloid peptides in treatment of neuroinflammation.

Amyloid hexapeptide molecules are effective in the treatment of the murine model of neuroinflammation, known as experimental autoimmune encephalomyelitis (EAE). Efficacy however differs between two inbred mouse strains, C57BL/6J (B6) and C57BL/10SnJ (B10). Amyloid hexapeptide treatments improved the clinical outcomes of B6, but not B10 mice, indicating that genetic background influences therapeutic efficacy. Moreover, although previous studies indicated that prion protein deficiency results in more severe EAE in B6 mice, we observed no such effect in B10 mice. In addition, we found that amyloid hexapeptide treatments of B10 and B6 mice elicited differential IL4 responses. Thus, the modulatory potential of prion protein and related treatments with other amyloid hexapeptides in EAE depends on mouse strain.

The effect of hepatocyte growth factor on secretory functions in human eosinophils.

Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is now recognized as a humoral mediator in inflammatory and immune responses. Previous studies indicated that HGF negatively regulated allergic airway inflammation. In view of eosinophils playing a role in the pathogenesis of asthma, especially in airway remodeling as a rich source of pro-fibrogenic mediators, the effects of HGF on the different types of eosinophil secretory functions were examined in this study. We found that HGF significantly inhibited IL-5-induced secretion of TGF-ß and VEGF from human eosinophils. The inhibitory effect is not associated with TGF-ß transcription; rather, it is associated with ultrastructural granule emptying and loss of intracellular TGF-ß contents, indicating HGF inhibits the process of piecemeal degranulation. The effect of HGF on extracellular trap cell death (ETosis) that mediates cytolytic degranulation was also investigated; however, immobilized IgG- or phorbol myristate acetate-induced ETosis was only minimally attenuated by HGF. These results reveal the effect of HGF on the distinct pathways of eosinophil secretory functions and also provide novel insights into the role of HGF in the pathogenesis of allergic inflammation.

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy.

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles - EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses.

Morphology and staining behavior of neutrophilic and eosinophilic granulocytes of the common marmoset (Callithrix jacchus).

Common marmosets (Callithrix jacchus) are frequently used as translational animal models for human diseases. However, a comparative study of cytological and histochemical detection methods as well as morphometric and ultrastructural characterization of neutrophils and eosinophils in this species is lacking. Blood samples of house dust mite sensitized and allergen challenged as well as lipopolysaccharide (LPS) challenged marmosets were analyzed with different cytological and histological staining methods. Furthermore, cell size and number of nuclear segments were compared between neutrophils and eosinophils. Electron microscopy was performed to characterize the ultrastructure of granulocytes. Of all applied cytological stains, three allowed differentiation of eosinophils and neutrophils and, thus, reliable quantification in blood smears: May-Grünwald-Giemsa stain, Congo Red and Naphthol AS-D Chloroacetate-Esterase. For histology, Hematoxylin-Eosin (H&E) could not demonstrate clear differences, whereas Sirius Red, Congo Red, and Naphthol AS-D Chloroacetate Esterase showed capable results for identification of eosinophils or neutrophils in lung tissue. Morphometry revealed that marmoset neutrophils have more nuclear segments and are slightly larger than eosinophils. Ultrastructurally, eosinophils presented with large homogeneous electron-dense granules without crystalloid cores, while neutrophils were characterized by heterogeneous granules of different size and density. Additionally, sombrero-like vesicles were detected in tissue eosinophils of atopic marmosets, indicative for hypersensitivity-related piecemeal degranulation. In conclusion, we provide a detailed overview of marmoset eosinophils and neutrophils, important for phenotypic characterization of marmoset models for human airway diseases.