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La bronquiolitis eosinofílica es un trastorno alérgico poco frecuente causado por inflamación eosinofílica en los bronquiolos pulmonares. Se requiere una estrategia de tratamiento eficaz en los casos resistentes a los esteroides. Sin embargo, su fisiopatología sigue sin estar clara debido al escaso número de casos. Presentamos aquí el caso de un hombre de 31 años que experimentó bronquiolitis eosinofílica con ETosis por eosinófilos (EETosis) en los tapones de moco. El paciente fue diagnosticado con asma. Los síntomas respiratorios empeoraron con eosinofilia cuando recibió el tratamiento estándar para el asma, que incluía corticosteroides inhalados, agonistas β2 de acción prolongada, antagonistas muscarínicos de acción prolongada y antagonistas de los receptores de leucotrienos. La tomografía computarizada torácica reveló engrosamiento de la pared bronquial y nódulos centrilobulares en los lóbulos inferiores de ambos pulmones. La broncoscopía mostró obstrucción del bronquio subsegmentario con tapones de moco. El análisis histológico demostró la presencia de abundantes eosinófilos en los tapones mucosos. También se observaron eosinófilos citolíticos junto con formaciones de cristales de Charcot-Leyden y la deposición de proteína básica mayor, lo que indicó la aparición de EETosis. La introducción de benralizumab, un anticuerpo contra el receptor α de la interleucina 5, controló con éxito el estado del paciente y redujo la cantidad de corticosteroides sistémicos administrados. Nuestros resultados confirman que este anticuerpo disminuye notablemente el número de eosinófilos en las vías respiratorias de pacientes con asma grave. Así pues, el benralizumab podría ser un agente terapéutico óptimo para el tratamiento de las enfermedades pulmonares eosinofílicas formadoras de tapones de moco o de EETosis, incluida la bronquiolitis eosinofílica.

Eosinophilic inflammation is primarily characterized by type 2 immune responses against parasitic organisms. In the contemporary human being especially in developed countries, eosinophilic inflammation is strongly associated with allergic/sterile inflammation, and constitutes an undesired immune reaction. This situation is in stark contrast to neutrophilic inflammation, which is indispensable for the host defense against bacterial infections. Among eosinophilic inflammatory disorders, massive accumulation of eosinophils within mucus is observed in certain cases, and is often linked to the distinctive clinical finding of mucus with high viscosity. Eosinophilic mucus is found in a variety of diseases, including chronic allergic keratoconjunctivitis, chronic rhinosinusitis encompassing allergic fungal sinusitis, eosinophilic otitis media, eosinophilic sialodochitis, allergic bronchopulmonary aspergillosis/mycosis, eosinophilic plastic bronchitis, and eosinophilic asthma. In these pathological conditions, chronic inflammation and tissue remodeling coupled with irreversible organ damage due to persistent adhesion of toxic substances and luminal obstruction may impose a significant burden on the body. Eosinophils aggregate in the hyperconcentrated mucus together with cell-derived crystals, macromolecules, and polymers, thereby affecting the biophysical properties of the mucus. This review focuses on the clinically significant challenges of mucus and discusses the consequences of activated eosinophils on the mucosal surface that impact mucus and persistent inflammation.

Eosinophils are short-lived and comprise only a small population of circulating leukocytes; however, they play surprisingly multifunctional roles in homeostasis and various diseases including allergy and infection. Recent research has shed light on active cytolytic eosinophil cell death that releases eosinophil extracellular traps (EETs) and total cellular contents, namely eosinophil extracellular trap cell death (EETosis). The pathological contribution of EETosis was made more cogent by recent findings that a classical pathological finding of eosinophilic inflammation, that of Charcot-Leyden crystals, is closely associated with EETosis. Currently no gold standard methods to identify EETosis exist, but “an active eosinophil lysis that releases cell-free granules and net-like chromatin structure” appears to be a common feature of EETosis. In this review, we describe several approaches that visualize EETs/EETosis in clinical samples and in vitrostudies using isolated human eosinophils. EETs/EETosis can be observed using simple chemical or fluorescence staining, immunostaining, and electron microscopy, although it is noteworthy that visualization of EETs is greatly changed by sample preparation including the extracellular space of EETotic cells and shear flow. Considering the multiple aspects of biological significance, further study into EETs/EETosis is warranted to give a detailed understanding of the roles played in homeostasis and disease pathogenesis.

A predominant protein of human eosinophils is galectin‐10 (Gal‐10), also known as Charcot‐Leyden crystal protein (CLC‐P) because of its remarkable ability to form Charcot‐Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC‐P/Gal‐10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC‐P/Gal‐10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre‐embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC‐P/Gal‐10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC‐P/Gal‐10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250 nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High‐resolution analysis of single cells revealed that CLC‐P/Gal‐10 interacts with the plasma membrane with immunoreactive microdomains of high CLC‐P/Gal‐10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF‐α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC‐P/Gal‐10 as observed by both immunofluorescence and immuno‐EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC‐P/Gal‐10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis). Ascertains the intracellular sites of CLC‐P/Gal‐10 within resting and activated human eosinophils, revealing preformed stores associated with the peripheral cytosol and plasma membrane and not with granules. .

IL-5 is the most potent activator of eosinophils and is produced by Th2 cells and ILC2s. A role for IL-5 in eosinophil extracellular trap cell death, i.e., a proinflammatory cell death, has also been reported. Mepolizumab and benralizumab are humanized mAbs that target IL-5 and the IL-5 receptor α, respectively, and their therapeutic efficacy for severe asthma has been established. Although consistent differences in the efficacies of those drugs have not been proven, benralizumab extensively depleted eosinophils via Ab-dependent cell-mediated cytotoxicity. Blood eosinophil count, but not FeNO or IgE, is the best-established predictive biomarker of the efficacy of anti-IL-5 treatment. Regarding the choice of biologics, the balance between blood eosinophil count and FeNO, indication of comorbidities, longitudinal safety, and interval of injection should be considered. Mepolizumab was also effective in maintaining the remission of refractory eosinophilic granulomatous polyangiitis. Moreover, mepolizumab decreased the proportion of patients who required surgery and lowered the nasal polyp score in patients with chronic rhinosinusitis with nasal polyps; a further extensive trial is currently under way. In a phase II benralizumab study performed in Japan, no significant effect on nasal polyp score at week 12 was observed, suggesting a requirement for longer treatment. In this review, the role of IL-5 in eosinophil biology and the current status of anti-IL-5 therapy are discussed. The longitudinal safety of anti-IL-5 therapy has been increasingly established, and this strategy will be continuously indicated for eosinophilic diseases as a specific treatment for eosinophilic inflammation.

Protein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.

Protein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.

Allergic fungal rhinosinusitis (AFRS) and allergic bronchopulmonary mycosis (ABPM) are inflammatory disorders of the respiratory tract resulting from type 1 and 3 hypersensitivity reactions against fungi. The hallmark features of both diseases are eosinophil infiltration into the airway mucosa caused by localized type 2 inflammation and concomitant viscid secretions in the airways. Eosinophilic mucin-induced compression of adjacent anatomic structures leads to bone erosion and central bronchiectasis in the upper and lower respiratory tracts, respectively. Although these diseases share common features in their pathogenesis, they also exhibit notable differences. Epidemiologic findings are diverse, with AFRS typically presenting at a younger age, exhibiting less complicated bronchial asthma, and displaying lower total immunoglobulin E levels in laboratory findings compared with ABPM. Furthermore, despite their similar pathogenesis, the rarity of sinio-bronchial allergic mycosis in both AFRS and ABPM underscores the distinctions between these two diseases. This review aims to clarify the similarities and differences in the pathogenesis of AFRS and ABPM to determine what can be learned about AFRS from ABPM, where more is known.

Intracellular trafficking of CD63 is connected to granule‐derived secretory pathways in human eosinophils. Eosinophil activation leads to secretion of presynthesized, granule‐stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane‐4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil‐specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63+large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule‐derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.

The chemokine receptor, CC-chemokine receptor 3 (CCR3), and its major ligands, eotaxin, RANTES, and MCP-4, are involved in eosinophil chemotaxis. It is thought that CCR3 plays an important role in the recruitment and activation of eosinophils in nasal polyposis. We examined nasal polyp extract-induced eosinophil chemotaxis and the effect of a CCR3 antagonist using EZ-TAXIScan, a novel real-time chemotaxis assay device.

This study investigated a new method of flow cytometry (FCM) for analysis of red blood cell (RBC) osmotic fragility. Venous peripheral blood collected in a sampling tube coated with EDTA 2Na was analyzed using FCM to determine RBC osmotic fragility. RBCs were represented as a double-peaked forward scatter (FSC) histogram in FCM. RBCs showed ballooning and spherical shape change in hypotonic solutions before hemolysis. The ballooning of RBCs was expressed as a disappearance of the minor peak and by narrowing and a shift to the right of the FSC histogram. The process of hemolysis was expressed as shrinking of the RBC cytogram in the right upper quadrant on the scatter plot of side scatter versus FSC and by emergence of a cell debris cytogram in the left lower quadrant. The ratio of intact RBCs in hypotonic solution was available as an indicator of osmotic fragility. Acidic solution made erythrocytes less tolerant to hypotonic solution by inducing spherical shape change. In conclusion, FCM can be used to assess RBC osmotic fragility.

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Prostaglandin D2 (PGD2) regulates various immunological responses via two distinct PGD2 receptors, prostaglandin D receptor (DP), and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Recent studies have demonstrated that PGD2 induces the migration of eosinophils through CRTH2. Although human eosinophils express both DP and CRTH2, it is unclear whether the function of DP is involved in eosinophil migration.

Asthma is a chronic airway inflammatory disease caused by immune cells such as T lymphocytes and eosinophils. Recently, highly sensitive C-reactive protein (hs-CRP) assays have become available for detecting small changes in CRP levels within the reference range, allowing for the evaluation of clinical inflammation.

Eosinophils are considered to be the major inflammatory cells in asthma. Since regulated on activation, normal T expressed and secreted (RANTES) is a potent chemoattractant for various important inflammatory cells such as eosinophils as well as memory T cells potentially recruiting these cells to an inflamed focus, RANTES has been considered to play a key role in various allergic disorders such as asthma.

Adhesion molecules participate in an important part of inflammatory process in relation to the accumulation of inflammatory cells, such as eosinophils. The expression of adhesion molecules differs depending upon the cells and tissue. In the present study, to elucidate these differences, a comparative study was performed on the prolongation of eosinophil survival via adhesion molecules.

Recently, a second prostaglandin D2(PGD2) receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), was identified. Because PGD2 was reported to have chemotactic activity on eosinophils, CRTH2 expressed on eosinophils attracted interest as a receptor associated with eosinophil migration to, and accumulation at, inflammatory sites. To elucidate the mechanism regulating the expression of CRTH2 on eosinophils, the effects of PGD2, interleukin (IL)-4, IL-5 and interferon (IFN)-γ on CRTH2 expression were investigated.

Background Eosinophilic cholecystitis (EC) is a rare condition that presents in a manner comparable to acute cholecystitis. The diagnosis is based on classical symptoms of cholecystitis with excessive eosinophilic infiltration within the gallbladder. EC has been reported alone or in combination with manifestations, such as eosinophilic gastrointestinal tract inflammation. However, association with airway inflammation in patients with EC is rare. Case Presentation:We report the case of a 65-year-old man who had refractory eosinophilic chronic rhinosinusitis with bronchial asthma. A second endoscopic sinus surgery (ESS) was performed as treatment for recurrent nasal polyps. EC occurred while inhaled corticosteroids were reduced after ESS. Pathologic examination of the excised gallbladder demonstrated submucosal infiltration with a number of eosinophils. Furthermore, immunohistostaining revealed many galectin-10-positive cells in both the gallbladder mucosa and the paranasal sinus mucosa. Galectin-10 is a major constituent of human eosinophils, also known as the Charcot–Leyden crystal protein, which has been linked with eosinophilic inflammation. Interestingly, nasal polyps were reduced without any additional treatments 1 month after the cholecystectomy.Conclusions We experienced a rare case wherein EC onset occurred in a patient with refractory eosinophilic airway inflammation during inhaled corticosteroid tapering. Galectin-10 might help diagnose rare cases of eosinophilic inflammation in multiple organs.

Nakao: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Matsuda:GlaxoSmithKline K.K.: Honoraria; Novartis Pharma K. K.: Honoraria; Chugai Pharmaceutical Co, Ltd.: Honoraria; Kyowa Hakko Kirin Co, Ltd.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Celgene Corporation: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Sanofi K.K.: Honoraria; Beckman Coulter K.K.: Honoraria. Mitani:Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Bristol-Myesr Squibb: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Chugai: Research Funding; Astellas: Research Funding; Sumitomo Dainippon: Research Funding; Novartis: Research Funding; Toyama Chemical: Research Funding.

Background.Idiopathic pure red cell aplasia (PRCA) and secondary PRCA not responding to the treatment of the underlying diseases are generally thought be immune-mediated and treated by immunosuppressive therapy. We previously conducted the PRCA2004/2006 study and reported that poor response to induction therapy and relapse of anemia were associated with death. Principal causes of death were infections and organ failure. Based on the literatures, idiopathic PRCA may represents the prodrome to myelodysplastic syndromes. Theoretically, there are two potential mechanisms of unresponsiveness to immunosuppression; the clonal hematopoiesis by the stem/progenitor cells that have undergone somatic mutations during disease progression of PRCA and the clonal changes of auto-aggressive lymphocytes reacting against erythroid progenitors.

Background.Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by anti-platelet autoantibodies. The underlying mechanism is thought to involve the production of IgG autoantibodies specific for platelet membrane antigens such as glycoprotein (GP) IIb/IIIa and GPIb/IX, although the anti-platelet autoantibody testing is less sensitive. The ASH and the IWG guidelines for the management of ITP do not recommend routine testing of anti-platelet autoantibodies for the diagnosis of ITP, and thus diagnostic biomarkers for ITP remain to be developed. Although the principal pathophysiology of ITP is believed to be an IgG-mediated autoimmune disease, there are few evidence for the B-cell receptor (BCR) repertoires which are associated or related to this disorder.