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The assessment and management of patients with asthma is challenging because of the complexity of the underlying inflammatory mechanisms and heterogeneity of their clinical presentation. Optimizing disease management requires therapy individualization that should rely on reliable biomarkers to unravel the phenotypes and endotypes of asthma. The secretory activity and turnover of eosinophils, as assessed by measuring eosinophil-derived proteins, may provide an accurate and complementary tool that mirrors the eosinophil activation status. Emerging evidence suggests that eosinophil-derived neurotoxin has considerable potential as a precision medicine biomarker. In this review, we explore the suitability of eosinophil-derived neurotoxin as a biomarker in asthma management, with particular emphasis on its clinical significance in the management of both pediatric and adult populations., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Airway remodeling is responsible for the progressive decline of lung function in bronchial asthma. Matrix metalloproteinase-2 and fibroblast-to-myofibroblast transition are involved in tissue remodeling. Here we evaluated whether eosinophils play a role in fibroblasts-to-myofibroblasts transition and in the expression of matrix metalloproteinase-2. We co-cultured human eosinophils with human fetal lung fibroblast-1 cells, assessed the expression of remodeling-associated molecules by immunoassays and polymerase-chain reaction, and eosinophils-mediated migration of human fetal lung fibroblast-1 cells using a Boyden chamber. To clarify the participation of matrix metalloproteinase-2 in airway remodeling we administered bone marrow-derived eosinophils by intra-tracheal route to transgenic mice overexpressing the human matrix metalloproteinase-2. The expression of α-smooth muscle actin significantly increased in human fetal lung fibroblast-1 cells co-cultured with human eosinophils compared to controls. There was enhanced expression of matrix metalloproteinase-2 during fibroblast-to-myofibroblast transition. An inhibitor of matrix metalloproteinases blocked eosinophils-associated fibroblast-to-myofibroblast transition and increased migration of fibroblasts. The human matrix metalloproteinase-2 transgenic mice receiving adoptive transfer of mouse eosinophils exhibited increased inflammation and advanced airway remodeling compared to wild type mice. This study demonstrated that eosinophils induce fibroblast-to-myofibroblast transition, secretion of matrix metalloproteinase-2, accelerated migration of fibroblasts, and promote matrix metalloproteinase-2-related airway remodeling. These findings provide a novel mechanistic pathway for eosinophil-associated airway remodeling in bronchial asthma.

Background: Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis., Methods: Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system., Results: Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation., Conclusions: Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling., (Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Epithelial-mesenchymal transition is currently recognized as an important mechanism for the increased number of myofibroblasts in cancer and fibrotic diseases. We have already reported that epithelial-mesenchymal transition is involved in airway remodeling induced by eosinophils. Procaterol is a selective and full β 2 adrenergic agonist that is used as a rescue of asthmatic attack inhaler form and orally as a controller. In this study, we evaluated whether procaterol can suppress epithelial-mesenchymal transition of airway epithelial cells induced by eosinophils., Methods: Epithelial-mesenchymal transition was assessed using a co-culture system of human bronchial epithelial cells and primary human eosinophils or an eosinophilic leukemia cell line., Results: Procaterol significantly inhibited co-culture associated morphological changes of bronchial epithelial cells, decreased the expression of vimentin, and increased the expression of E-cadherin compared to control. Butoxamine, a specific β 2 -adrenergic antagonist, significantly blocked changes induced by procaterol. In addition, procaterol inhibited the expression of adhesion molecules induced during the interaction between eosinophils and bronchial epithelial cells, suggesting the involvement of adhesion molecules in the process of epithelial-mesenchymal transition. Forskolin, a cyclic adenosine monophosphate-promoting agent, exhibits similar inhibitory activity of procaterol., Conclusions: Overall, these observations support the beneficial effect of procaterol on airway remodeling frequently associated with chronic obstructive pulmonary diseases.

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

Background: It has been suggested that there is a complex interaction between microbiota and various human diseases. Some bacteria have been reported to be involved in the inception and progression of asthma, and others in the protection against asthma. We know very little about the mechanisms by which bacteria do harm or good with regard to asthma. This study investigated whether bacteria exert differential effects on the functions of eosinophils, major effector cells in airway inflammation in asthma., Methods: Eosinophils were purified from healthy adult volunteers by Percoll density gradient centrifugation and negative immunomagnetic bead selection using anti-CD16 microbeads. Three kinds of heat-killed bacteria that have been implicated in asthma, namely Staphylococcus aureus (SA), Haemophilus influenzae (HI) and a Prevotella sp. (PS), were tested for their effects on the secretion of eosinophil-derived neurotoxin (EDN), the generation of superoxides and the production of cytokines/chemokines., Results: SA, but not HI or PS, induced significant EDN release in a dose-dependent manner. Superoxide generation was significantly enhanced by each of the bacterial species, but most strongly by SA, which induced significantly greater TNF-α production by eosinophils than either HI or PS. Conversely, interleukin 10, an anti-inflammatory cytokine, was more strongly induced by HI and PS than by SA., Conclusions: Bacteria exert differential effects on eosinophils. Based on these results, SA may be involved in the exacerbation of, and HI and PS in the inhibition of, eosinophilic inflammation in asthma., (Copyright © 2013 S. Karger AG, Basel.)

Colonization by SA is associated with exacerbation of AD. Eosinophilic inflammation is a cardinal pathological feature of AD, but little is known about possible direct interaction between SA and eosinophils. PAFR appears to be involved in phagocytosis of Gram-positive bacteria by leukocytes. The objective of this study was to investigate whether SA directly induces eosinophil effector functions via PAFR in the context of AD pathogenesis. Peripheral blood eosinophils were cultured with heat-killed SA, and EDN release, superoxide generation, and adhesion to fibronectin-coated plates were measured. Cytokines, released in the supernatants, were quantified by multiplex bead immunoassays. FISH-labeled SA was incubated with eosinophils and visualized by confocal laser-scanning microscopy. PAFR-blocking peptide and PAFR antagonists were tested for inhibitory effects on SA-induced reactions. SA induced EDN release and superoxide generation by eosinophils in a dose-dependent manner. IL-5 significantly enhanced SA-induced EDN release. IL-5 and IL-17A significantly enhanced SA-induced superoxide generation. SA enhanced eosinophil adhesion to fibronectin, which was blocked by anti-CD49d, and induced eosinophil secretion of various cytokines/chemokines (IL-2R, IL-9, TNFR, IL-1 β, IL-17A, IP-10, TNF-α, PDGF-bb, VEGF, and FGF-basic). After incubation of eosinophils with SA, FISH-labeled SA was visualized in the eosinophils' cytoplasm, indicating phagocytosis. A PAFR-blocking peptide and two PAFR antagonists completely inhibited those reactions. In conclusion, SA directly induced eosinophil activation via PAFR. Blockade of PAFR may be a novel, therapeutic approach for AD colonized by SA.

Background: Viral respiratory tract infections play an important role in the inception and exacerbation of asthma. Eosinophils, major effector cells in asthma, often accumulate in the airways during viral infections and are possibly activated by respiratory RNA viruses through Toll-like receptor (TLR) 7. We investigated the effect of a β(2)-agonist, i.e. procaterol, and a corticosteroid, i.e. budesonide, that are commonly used for viral-induced asthma, on TLR7 ligand-induced activation of eosinophils in vitro., Methods: Purified peripheral blood eosinophils were incubated with procaterol and/or budesonide and stimulated with a TLR7 ligand, i.e. R-837. Expression of CD11b was analyzed by flow cytometry. Superoxide generation was measured via the cytochrome C reduction method. IL-8 in the supernatants was assayed by ELISA., Results: Although procaterol or budesonide alone did not inhibit R-837-induced CD11b expression, combinations of the 2 drugs significantly inhibited CD11b. Likewise, the combinations significantly inhibited O(2)(-) generation at low concentrations. Budesonide significantly inhibited R-837-induced IL-8 production in a concentration-dependent manner, and procaterol potentiated inhibition by budesonide although single-agent procaterol had no effect., Conclusion: A combination of procaterol and budesonide inhibits the TLR7-mediated effector function of eosinophils, indicating their possible anti-inflammatory effect for virus-induced asthma., (Copyright © 2011 S. Karger AG, Basel.)

Th2 cytokine IL-5 and CC chemokine eotaxin are thought to be key regulators of eosinophils in bronchial asthma. However, their involvement in children with stable asthma (SA) has not been determined. We investigated the roles of IL-5 and eotaxin in eosinophil degranulation in children with SA. Induced sputum was obtained from 30 SA, 21 allergic rhinitis (AR), and 22 non-atopic healthy control (HC) children. We measured sputum levels of IL-5, eotaxin, and eosinophil indices [percentage eosinophils, eosinophil-derived neurotoxin (EDN), and eosinophil-cationic protein (ECP)]. We also examined correlations of IL-5 and eotaxin with eosinophil indices. Sputum percentage eosinophils and EDN and ECP levels were significantly higher in the SA group than in the HC group, while only the sputum EDN and ECP levels were significantly higher in the AR group than in the HC group. Unexpectedly, sputum levels of IL-5 were not significantly different among the three groups; however, the levels of eotaxin were higher in the SA group when compared to the HC group. No significant correlations were found between IL-5 and percentage eosinophils, EDN, or ECP levels; in contrast, eotaxin levels correlated significantly with percentage eosinophils (R(s) = 0.638; p = 0.0001), EDN (R(s) = 0.522; p = 0.003), and ECP levels (R(s) = 0.630 and p = 0.0002). The elevated levels and good correlations of eotaxin with sputum eosinophil indices, and no elevation or correlation of IL-5 with these indices, suggest that CC chemokine eotaxin may play a more important role in eosinophil degranulation in children with SA.

The purpose of our study was to investigate whether tumor necrosis factor (TNF)-alpha correlates with eosinophilic inflammation that occurs during a lower respiratory tract infection with the respiratory syncytial virus (RSV) in children. Sixty children with RSV bronchiolitis (RSV group) and 20 healthy children with no respiratory symptoms (Control group) were enrolled. We measured the nasal lavage fluid (NLF) Th2 cytokine (IL-5), proinflammatory cytokine (TNF-alpha, IL-8), eosinophil-active cytokine [granulocyte-macrophage colony stimulating factor (GM-CSF), IFN-gamma], and eosinophil-active chemokine (eotaxin, regulated on activation normal T cell excreted and secreted) levels for both groups. We also measured serum eosinophil-degranulation product (eosinophil-derived neurotoxin; EDN, eosinophil cationic protein; ECP) levels from RSV group. TNF-alpha, IL-8, GM-CSF, IFN-gamma, and eotaxin levels were significantly higher in the RSV group compared with the Control group. TNF-alpha correlated with GM-CSF (r = 0.87, p < 0.0001), IFN-gamma (r = 0.92, p < 0.0001), eotaxin (r = 0.64, p < 0.0001), and IL-8 (r = 0.84, p < 0.0001). TNF-alpha may have an important role in eosinophilic inflammation of airways in children with RSV bronchiolitis.

Background: Recent studies suggest that probiotics alleviate pathophysiological processes of allergic diseases and inflammatory bowel diseases, whereas 'non-probiotic' microflora has negative effects. However, the underlying mechanisms are not well known, especially in relation to eosinophils, the major effector cells of these inflammatory diseases., Objective: To investigate the effects of probiotic Bifidobacterium bifidum (BB) on human eosinophil functions compared with pathogenic Clostridium difficile (CD)., Methods: Peripheral human eosinophils were cultured with heat-killed BB or CD. FISH-labeled CD and BB were incubated with eosinophils visualized by confocal laser scanning microscopy. Superoxide generation and degranulation of eosinophils were measured with the cytochrome c reduction method and the eosinophil-derived neurotoxin (EDN) release assay, respectively., Results: Confocal microscopy revealed that Cy3-labeled CD and BB were apparently ingested by eosinophils. Both bacteria induced minimal superoxide generation. However, CD elicited significantly higher EDN release than BB. GM-CSF significantly enhanced EDN release by CD but not by BB. Bacterial-induced EDN release was calcium dependent., Conclusion: The beneficial effect of probiotic BB might be explained, at least in part, by its ability to decrease EDN release from eosinophils compared with 'pathogenic' CD., ((c) 2010 S. Karger AG, Basel.)

Background: Eosinophils play a pivotal role in allergic inflammation. Recent evidence suggests that they not only function as terminal effector cells but have potential to interact with allergen and initiate immune responses. We investigated cytokine production from eosinophils through direct interaction with a major allergen, house dust mite (HDM) ., Methods: Purified eosinophils from HDM-sensitized or non-sensitized donors were cultured with HDM extract or lipopolysaccharide (LPS) for 18 or 40 h. A panel of cytokine gene expression in eosinophils was examined by means of real-time RT-PCR. Released cytokines in the culture supernatants were assessed with a specific ELISA. In some experiments, HDM was pretreated with protease inhibitors, then added to the culture. Cytokines tested for gene expression were interleukin (IL)-2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 16,17, 18, TGF-beta1 and GM-CSF,., Results: LPS induced small enhancement of GM-CSF gene expression at 18 h. At 40 h, HDM induced about 60-fold enhancement of IL-9 gene expression. IL-9 protein was also detected in the culture supernatants at 60 h. Those reactions were observed regardless of HDM sensitization status of the donors. HDM-induced IL-9 expression was completely inhibited with a serine protease inhibitor, AEBSF, not with a cysteine protease inhibitor, E-64., Conclusions: Accumulated eosinophils in the airways in asthma may directly react with HDM and produce IL-9 to further promote Th2-type immune responses. Protease-activated receptor 2, a ligand for serine proteases, which contained in HDM, may be involved in the reaction.

Background: Recent evidence suggests that both neutrophilic and eosinophilic inflammation persist in the airways of patients with severe asthma. Mechanisms for interaction between neutrophils and eosinophils are still to be understood. Since eosinophils express protease-activated receptor 2, neutrophil-derived serine proteases may activate eosinophils., Objective: We investigated the effect of neutrophil serine proteases on eosinophil effector functions., Methods: Peripheral blood eosinophils were stimulated with elastase, cathepsin G and proteinase 3. Superoxide generation was quantitated with the cytochrome C reduction method. A panel of cytokines and chemokines in the culture supernatants were measured with a multiplex beads array system. Effects of an elastase inhibitor, sivelestat, and a serine protease inhibitor, PMSF, on the protease-induced reactions were also tested., Results: Neutrophil proteases significantly induced superoxide production from eosinophils. Elastase was the most potent among them. Sivelestat and PMSF inhibited the reaction. The proteases induced production of IL-6, IL-8, TNF-alpha and GRO-alpha, that have a possible connection with neutrophilic inflammation., Conclusion: Neutrophil proteases activate eosinophils to produce superoxide, proinflammatory cytokines and neutrophilotactic chemokines and may further aggravate airway inflammation in patients with severe asthma., (Copyright 2008 S. Karger AG, Basel.)

Eosinophils may play an important role in the pathogenesis of airway remodeling in asthma through the production of various fibrogenic cytokines such as transforming growth factor-beta1 (TGF-beta1). Cysteinyl leukotrienes are also suggested to be involved in remodeling with their potential to induce proliferation of airway smooth muscle cells. Since massive eosinophil infiltration and the release of cysteinyl leukotrienes in airway secretions are often seen in asthma, we hypothesized that cysteinyl leukotrienes may be involved in airway remodeling through induction of TGF-beta1 from eosinophils. Peripheral blood eosinophils were cultured with leukotriene D(4) (LTD(4)) and/or interleukin-5 (IL-5) or granulocyte colony-stimulating factor (GM-CSF) for 16 h and gene expression of TGF-beta1 was quantified with real-time PCR. A combination of LTD(4) and IL-5 or LTD(4) and GM-CSF synergistically induced TGF-beta1 expression in eosinophils although stimulation with single factor, LTD(4), IL-5 or GM-CSF did not induce the gene expression. LTD(4) also induced significant gene expression in eosinophils cultured in an intercellular adhesion molecule-1-coated plate. The results suggested that CysLTs stimulate eosinophils to induce TGF-beta1 production in allergic inflammation where IL-5 and GM-CSF are abundant and may be involved in the pathogenesis of airway remodeling., (Copyright 2005 S. Karger AG, Basel.)

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine that is constitutively expressed by the liver parenchymal cells and present in the normal plasma at high concentrations. Previous studies have shown that CCL16 is a low-affinity ligand for CCR1, CCR2, CCR5, and CCR8 and attracts monocytes and T cells. Recently, a novel histamine receptor termed type 4 (H4) has been identified and shown to be selectively expressed by eosinophils and mast cells. In this study, we demonstrated that CCL16 induced pertussis toxin-sensitive calcium mobilization and chemotaxis in murine L1.2 cells expressing H4 but not those expressing histamine receptor type 1 (H1) or type 2 (H2). CCL16 bound to H4 with a K(d) of 17 nM. By RT-PCR, human and mouse eosinophils express H4 but not H3. Accordingly, CCL16 induced efficient migratory responses in human and mouse eosinophils. Furthermore, the responses of human and mouse eosinophils to CCL16 were effectively suppressed by thioperamide, an antagonist for H3 and H4. Intravenous injection of CCL16 into mice induced a rapid mobilization of eosinophils from bone marrow to peripheral blood, which was also suppressed by thioperamide. Collectively, CCL16 is a novel functional ligand for H4 and may have a role in trafficking of eosinophils.

Background: The cysteinyl leukotrienes (CysLTs) mediate their biological actions through two receptors: CysLT(1) receptor and CysLT(2) receptor., Objective: This study was undertaken to examine the direct effects of CysLTs on eosinophils, such as chemotaxis and degranulation, focusing on CysLT(1)., Methods: Eosinophils were isolated from venous blood from normal volunteers who had no history of allergy (purity >99%). They were subjected to reverse transcription-PCR analysis and flow-cytometric analysis for CysLT(1). Binding assays were performed with [(3)H]LTD(4). Purified eosinophils loaded with Fura-2 acetoxymethyl ester were stimulated with CysLTs, and Ca(2+) influx was measured. Eosinophil migration in response to CysLTs was measured using a 96-well multiwell Boyden chamber. Eosinophils were treated with LTD(4) at 10(-6) M for 60 min followed by incubation for 4 h at 37 degrees C in the presence or absence of IL-5 and eosinophil-derived neurotoxin (EDN) release was evaluated., Results: The expression of the mRNA and protein of CysLT(1) on eosinophils and [(3)H]LTD(4)-specific binding to eosinophils were observed. Neither Th1 cytokine (IFN-gamma) nor Th2 cytokines (IL-4 or IL-5) affected CysLT(1) expression in eosinophils. CysLTs induced an increase in intracellular free Ca(2+) in eosinophils via CysLT(1), as suggested by the efficient inhibition by a CysLT(1) antagonist, pranlukast, in addition to the rank order of potency being LTD(4), LTC(4) and LTE(4). LTD(4) stimulated eosinophils to migrate at 10(-6) M via CysLT(1). LTE(4) also induced significant eosinophil migration at 10(-6) M. LTD(4) enhanced EDN release induced by IL-5 via CysLT(1)., Conclusion: CysLTs induce migration and enhance degranulation in eosinophils via CysLT(1). Accordingly, interaction of CysLTs and CysLT(1) on eosinophils has the potential to play a prominent role in the pathophysiology of asthma., (Copyright 2002 S. Karger AG, Basel)

Inhibiting the release of toxic granule proteins from eosinophils is a possible means of treating allergic inflammation. This study was performed to examine whether procaterol and theophylline, commonly used bronchodilators in asthma, inhibit eosinophil degranulation induced by interleukin (IL)-5. Purified eosinophils from patients with asthma were incubated with IL-5 for 24 hr in the presence of theophylline, procaterol, combinations of theophylline and procaterol, or dexamethasone. Levels of eosinophil-derived neurotoxin (EDN) in the supernatants were measured with radioimmunoassay. Theophylline inhibited IL-5-induced release of EDN in a concentration-dependent manner. Procaterol inhibited degranulation only at high concentrations. However, procaterol at 10(-9) M and 10(-8) M, which are physiologic concentrations, together with theophylline at 10(-5) M, which is a concentration commonly found in the serum of patients receiving low-dose theophylline, inhibited degranulation by 43.8%. This finding indicates that theophylline and procaterol have synergistic effects. The inhibition was comparable to that with dexamethasone at 10(-9) M. Our results suggest that a combination of low-dose theophylline and procaterol exhibits antiinflammatory effects in asthma by inhibiting eosinophil-effector functions.

Background: Benralizumab is an anti‐interleukin‐5 receptor α monoclonal antibody approved as an add‐on maintenance treatment for patients with uncontrolled severe asthma. Prior Phase 3 studies have evaluated benralizumab in patients aged ≥12 years with severe uncontrolled asthma. The TATE study evaluated the pharmacokinetics (PK), pharmacodynamics (PD), and safety of benralizumab treatment in children. Methods: TATE was an open‐label, Phase 3 study of benralizumab in children aged 6–11 years from the United States and Japan (plus participants aged 12–14 years from Japan) with severe eosinophilic asthma. Participants received benralizumab 10/30 mg according to weight (<35/≥35 kg). Primary endpoints included maximum serum concentration (Cmax), clearance, half‐life (t1/2), and blood eosinophil count. Clearance and t1/2 were derived from a population PK (popPK) analysis. Safety and tolerability were also assessed. Results: Twenty‐eight children aged 6–11 years were included, with an additional two participants from Japan aged 12–14 years also included in the popPK analysis. Mean Cmax was 1901.2 and 3118.7 ng/mL in the 10 mg/<35 kg and 30 mg/≥35 kg groups, respectively. Clearance was 0.257, and mean t1/2 was 14.5 days. Near‐complete depletion of blood eosinophils was shown across dose/weight groups. Exploratory efficacy analyses found numerical improvements in mean FEV1, mean ACQ‐IA, patient/clinician global impression of change, and exacerbation rates. Adverse events occurred in 22/28 (78.6%) of participants; none led to discontinuation/death. Conclusion: PK, PD, and safety data support long‐term benralizumab in children with severe eosinophilic asthma, and were similar to findings in adolescents and adults. Trial Registration: ClinicalTrials.gov‐ID: NCT04305405. [ABSTRACT FROM AUTHOR]

Background: Allergen-specific immunotherapy is currently the only disease-modifying treatment for allergic asthma, and it has been shown to improve control of asthma while reducing both drug use and asthma exacerbations. However, its effects on lung function—especially its long-term effects—remain controversial. We aimed to identify factors associated with a possible beneficial effect of allergen-specific immunotherapy on lung function in asthma by retrospectively evaluating the long-term changes in lung function in children with asthma who received house dust mite subcutaneous immunotherapy (HDM-SCIT). Methods: We enrolled children with asthma who had undergone HDM-SCIT for more than 1 year. Clinical information and lung function measurements were retrieved from the electronic chart system. To characterize the trajectory of lung function change, we performed linear regression analysis to evaluate the maximal expiratory flow at 50% of the forced vital capacity during two periods: before and during HDM-SCIT. Slopes from a least-squares regression line for the two periods, i.e., S1 before HDM-SCIT and S2 during HDM-SCIT, were compared. The subjects were then classified into two groups: an improving group (Group I) defined as S2 − S1 > 0, and a declining group (Group D) defined as S2 − S1 < 0. The clinical factors at the start of HDM-SCIT were compared between the two groups. Results: A total of 16 patients were analyzed. Eight patients were classified into each of Group I and Group D. The mean ages were 10.5 and 11.8 years, and the mean treatment periods were 4.1 and 3.9 years. Group I had a significantly lower blood eosinophil count and a significantly higher HDM-specific IgE level than Group D. Logistic regression showed a strong relationship between those two markers and the lung function trajectory. Conclusion: Control of the blood eosinophil count in highly HDM-sensitized patients may increase the beneficial effect of HDM-SCIT on lung function. [ABSTRACT FROM AUTHOR]

Background: Epidemiological studies suggest that vitamin D may be protective against the inception and exacerbation of allergic diseases. However, the direct effect of vitamin D on eosinophils, the major effector cells in allergic inflammation, is not known. It has been reported that C-X-C chemokine receptor type 4 (CXCR4) in eosinophils is induced in non-Th2 cytokine milieu or in response to glucocorticoids, recruiting the cell to noninflammatory sites. Objectives: To test whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3 or calcitriol], the active metabolite of vitamin D, acts directly on eosinophils to induce upregulation of CXCR4. Methods: Peripheral blood eosinophils from normal volunteers were isolated by CD16 immunomagnetic beads. Vitamin D receptor (VDR) expression was detected by RT-PCR. Eosinophils were cultured with 1,25-(OH)2D3 and the survival and expression of CXCR4 on eosinophils were measured by flowcytometry. Eosinophil migration by CXCL-12/SDF-1 in the presence of 1,25-(OH)2D3 was also analyzed. Results: Eosinophils expressed VDR. 1,25-(OH)2D3 prolonged eosinophil survival and upregulated eosinophil surface expression of CXCR4 in a concentration-dependent manner. Interleukin (IL)-5 significantly reduced CXCR4 expression and migration induced by the ligand CXCL-12/SDF-1. 1,25-(OH)2D3 reversed the negative effects of IL-5 on the CXCR4-CXCL12 pathway. Conclusion: 1,25-(OH)2D3 regulates CXCR4 expression in eosinophils. The mechanism may be involved in eosinophil recruitment to noninflammatory sites where the ligand of CXCR4 is constitutively expressed. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Study Objective: This study was designed to investigate the possible role of IFN-γ in eosinophil degranulation that occurs during respiratory syncytial virus (RSV) bronchiolitis. Methods: Sixty-seven infants, 2-24 months old and hospitalized with their first episode of acute RSV bronchiolitis, were selected for this study. Eosinophil-active cytokine and chemokine profiles in nasal lavage supernatants taken within the first 48 h of admission were determined by a multiplex bead array system (Luminex). Comparisons were made with control (Control group) subjects ( n = 20). Results: Nasal IFN-γ levels were significantly higher ( P < 0.0001) in RSV bronchiolitis (median = 4.4 pg/ml) infants compared to controls (0.0 pg/ml). IFN-γ levels correlated significantly with the levels of nasal eotaxin ( r = 0.566, P < 0.0001), RANTES ( r = 0.627, P < 0.0001), GM-CSF ( r = 0.849, P < 0.0001), and EDN ( r = 0.693, P < 0.001). Nasal interleukin (IL)-4, IL-5, and IL-13 were below sensitivity levels in most RSV bronchiolitis and control subjects. Conclusion: These results suggest that IFN-γ may play an important role in eosinophilic inflammation in RSV bronchiolitis. [ABSTRACT FROM AUTHOR]

Choi J, Callaway Z, Kim HB, Fujisawa T, Kim CK. The role of TNF-α in eosinophilic inflammation associated with RSV bronchiolitis. Pediatr Allergy Immunol 2010: 21: 474–479. © 2009 John Wiley & Sons A/S The purpose of our study was to investigate whether tumor necrosis factor (TNF)-α correlates with eosinophilic inflammation that occurs during a lower respiratory tract infection with the respiratory syncytial virus (RSV) in children. Sixty children with RSV bronchiolitis (RSV group) and 20 healthy children with no respiratory symptoms (Control group) were enrolled. We measured the nasal lavage fluid (NLF) Th2 cytokine (IL-5), proinflammatory cytokine (TNF-α, IL-8), eosinophil-active cytokine [granulocyte-macrophage colony stimulating factor (GM-CSF), IFN-γ], and eosinophil-active chemokine (eotaxin, regulated on activation normal T cell excreted and secreted) levels for both groups. We also measured serum eosinophil-degranulation product (eosinophil-derived neurotoxin; EDN, eosinophil cationic protein; ECP) levels from RSV group. TNF-α, IL-8, GM-CSF, IFN-γ, and eotaxin levels were significantly higher in the RSV group compared with the Control group. TNF-α correlated with GM-CSF ( r = 0.87, p < 0.0001), IFN-γ ( r = 0.92, p < 0.0001), eotaxin ( r = 0.64, p < 0.0001), and IL-8 ( r = 0.84, p < 0.0001). TNF-α may have an important role in eosinophilic inflammation of airways in children with RSV bronchiolitis. [ABSTRACT FROM AUTHOR]

Eosinophils may play an important role in the pathogenesis of airway remodeling in asthma through the production of various fibrogenic cytokines such as transforming growth factor-β1 (TGF-β1). Cysteinyl leukotrienes are also suggested to be involved in remodeling with their potential to induce proliferation of airway smooth muscle cells. Since massive eosinophil infiltration and the release of cysteinyl leukotrienes in airway secretions are often seen in asthma, we hypothesized that cysteinyl leukotrienes may be involved in airway remodeling through induction of TGF-β1 from eosinophils. Peripheral blood eosinophils were cultured with leukotriene D4 (LTD4) and/or interleukin-5 (IL-5) or granulocyte colony-stimulating factor (GM-CSF) for 16 h and gene expression of TGF-β1 was quantified with real-time PCR. A combination of LTD4 and IL-5 or LTD4 and GM-CSF synergistically induced TGF-β1 expression in eosinophils although stimulation with single factor, LTD4, IL-5 or GM-CSF did not induce the gene expression. LTD4 also induced significant gene expression in eosinophils cultured in an intercellular adhesion molecule-1-coated plate. The results suggested that CysLTs stimulate eosinophils to induce TGF-β1 production in allergic inflammation where IL-5 and GM-CSF are abundant and may be involved in the pathogenesis of airway remodeling. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The cysteinyl leukotrienes (CysLTs) mediate their biological actions through two receptors: CysLT[sub 1] receptor and CysLT[sub 2] receptor. Objective: This study was undertaken to examine the direct effects of CysLTs on eosinophils, such as chemotaxis and degranulation, focusing on CysLT[sub 1] . Methods: Eosinophils were isolated from venous blood from normal volunteers who had no history of allergy (purity >99%). They were subjected to reverse transcription-PCR analysis and flow-cytometric analysis for CysLT[sub 1] . Binding assays were performed with [[sup 3] H]LTD[sub 4] . Purified eosinophils loaded with Fura-2 acetoxymethyl ester were stimulated with CysLTs, and Ca[sup 2+] influx was measured. Eosinophil migration in response to CysLTs was measured using a 96-well multiwell Boyden chamber. Eosinophils were treated with LTD[sub 4] at 10[sup –6] M for 60 min followed by incubation for 4 h at 37°C in the presence or absence of IL-5 and eosinophil-derived neurotoxin (EDN) release was evaluated. Results: The expression of the mRNA and protein of CysLT[sub 1] on eosinophils and [[sup 3] H]LTD[sub 4] -specific binding to eosinophils were observed. Neither Th1 cytokine (IFN-γ) nor Th2 cytokines (IL-4 or IL-5) affected CysLT[sub 1] expression in eosinophils. CysLTs induced an increase in intracellular free Ca[sup 2+] in eosinophils via CysLT[sub 1] , as suggested by the efficient inhibition by a CysLT[sub 1] antagonist, pranlukast, in addition to the rank order of potency being LTD[sub 4] , LTC[sub 4] and LTE[sub 4] . LTD[sub 4] stimulated eosinophils to migrate at 10[sup –6] M via CysLT[sub 1] . LTE[sub 4] also induced significant eosinophil migration at 10[sup –6] M. LTD[sub 4] enhanced EDN release induced by IL-5 via CysLT[sub 1] . Conclusion: CysLTs induce migration and enhance degranulation in eosinophils via CysLT[sub 1] . Accordingly, interaction of CysLTs and CysLT[sub 1] on eosinophils has the potential to play a prominent role in the pathophysiology of asthma.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Recent studies have demonstrated that eosinophils from allergic patients express low levels of FcεRI on their surface, but the regulatory mechanisms of eosinophil surface FcεRI expression are not fully understood. We investigated whether IL-4 and IgE, which are reported to regulate surface FcεRI expression on human mast cells, are able to affect surface FcεRI expression in normal human eosinophils. Methods: Eosinophils purified from peripheral blood were cultured with IL-5 and with or without IL-4 and/or IgE, and surface FcεRI expression was analyzed by flow cytometry using an anti-FcεRI mAb, CRA-1. Results: Apparent FcεRI expression (∼1% of mast cell FcεRI levels) was observed in eosinophils cultured with both IL-4 and IgE. A combination of IL-4 (≥1 ng/ml) and IgE (≥ 0.5 μg/ml) was necessary for the maximal induction of surface FcεRI expression. In the presence of IL-4 and IgE, eosinophils cultured for 2 days demonstrated low but statistically significant levels of surface FcεRI, which reached a plateau after 7 days of culture. However, cross-linkage of surface FcεRI molecules by CRA-1 or anti-IgE did not induce any eosinophil activation. Conclusions: IL-4 and IgE can affect the levels of surface FcεRI on normal human eosinophils. FcεRI expression on eosinophils may be regulated by a mechanism similar to that in mast cells.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Measurement of eosinophil cationic protein (ECP) in serum has been utilized as a marker for allergic inflammation. The serum level of ECP represents the level found in vivo plus additional proteins released in vitro from peripheral blood eosinophils during the coagulation period. The mechanisms of release, however, are unclear. We investigated a possible involvement of adhesion molecules in the ECP release. Materials and Methods: Venous blood was drawn in the presence of EDTA from allergic donors. The blood was incubated with neutralizing monoclonal antibodies to CD18, CD11a, CD11b, CD29, CD49d, CD54, α[sub 4] β[sub 7] , or isotype-matched control antibodies, respectively, at 4°C for 30 min. Calcium gluconate (calcium) was then added to induce coagulation. The blood was further incubated for 90 min and centrifuged to obtain the serum. ECP in the serum was measured with RIA. In some experiments, purified eosinophils were incubated with plasma and calcium, then ECP in the supernatants was assayed. Results: ECP in the samples with calcium was significantly higher than in those without calcium. Purified eosinophils released ECP upon plasma coagulation. Anti-CD18, CD49d, and α[sub 4] β[sub 7] antibodies significantly suppressed ECP levels in the serum. Conclusions: These results suggest that ECP release in the serum is calcium and plasma coagulation-dependent and that cell adhesion through α[sub L] β[sub 2] , α[sub M] β[sub 2] , α[sub 4] β[sub 1] and α[sub 4] β[sub 7] integrins is at least in part responsible for ECP release. [ABSTRACT FROM AUTHOR]

Background: Matrix metalloproteinases (MMPs) are major contributors to tumor invasion, remodeling of connective tissue and infiltration of inflammatory cells and may be important mediators in developing allergic inflammation. Overexpression of MMP-9 mRNA by eosinophils in the asthmatic airways has been reported. To clarify the relative significance of MMP as an inflammatory mediator from eosinophils, we determined the content of MMP-9 in the peripheral blood eosinophils and compared it with the other leukocyte fractions. Methods: Peripheral blood eosinophils, neutrophils, and mononuclear cells were purified from normal and allergic donors with Percoll gradient centrifugation and CD16 negative selection. Cell lysate and culture supernatants stimulated with IL-5, PAF, and PMA were tested for MMP-9 with gelatin zymography and ELISA. Results: The amount of MMP-9 in highly purified eosinophils, neutrophils, and mononuclear cells was 2.5 ± 0.9, 4,073 ± 581, and 7.6 ± 1.4 ng/5 × 10[sup 6] cells, respectively. There was no difference in MMP-9 content of eosinophils between normal donors and patients with asthma. Culture of peripheral blood eosinophils with IL-5 for 4 days did not induce MMP-9 production. The stimulation of eosinophils with PMA and other secretogogues caused only small amounts of MMP-9 secretion as compared with neutrophils. Conclusions: These findings suggest that circulating eosinophils normally have only small amounts of MMP-9 and that eosinophils may need complex activation signals to produce significant amounts of MMP as seen in tissues of allergic inflammation. [ABSTRACT FROM AUTHOR]

Presents an overview of several studies on allergic inflammation. Role of eosinophils in the pathogenesis of asthma; Reduction of eosinophil in the lung and peripheral blood by a humanized anti-interleukin-5 monoclonal antibody (SB-240563); Topics presented and discussed at the 14th Annual Workshop on Eosinophils in Allergy and Related Diseases 2001.

Highlights: [•] Inhibition of EMT by montelukast was evaluated. [•] EMT was assessed using a co-culture system of epithelial cells and eosinophils. [•] Montelukast inhibited EMT induced in the co-culture system. [•] Montelukast decreased TGF-β1 production and Smad3 phosphorylation. [•] Eosinophils significantly enhanced CysLTs production but montelukast inhibited it. [Copyright &y& Elsevier]

IL-3, IL-5, and GM-CSF exert various overlapping functions in basophils. We investigated the receptor expression profiles and concentration-dependent effects of IL-3, IL-5, and GM-CSF on several basophil functions in comparison with their effects on eosinophils. The order of the receptor expression levels was IL-3Rα>IL-5Rα>GM-CSFRα in basophils and IL-5Rα&ges;GM-CSFRα>IL-3Rα in eosinophils. Compared with eosinophils, basophils expressed a much higher level of IL-3Rα and similar levels of IL-5Rα and GM-CSFRα. The order of potency was IL-3>IL-5=GM-CSF for degranulation, survival, and CD11b expression in basophils, and IL-5=GM-CSF&ges;IL-3 for survival and CD11b expression in eosinophils. However, IL-3 induced CD69 expression preferentially in basophils. Our results indicate that IL-3 is the most potent activator of human basophils, and that the rank order of potency of hemopoietic growth factors virtually corresponded to their receptor expression levels in both cell types. [Copyright &y& Elsevier]