Kita, H

Abstract Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in ly- sates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (X ± SEM) in ng/106 eosinophils (and in nM/106 eosinophils) were: MBP, 8,982 ± 611 (641.6); EDN, 3,283 ± 116 (178.4); ECP, 5,269 ± 283 (250.9); and EPO, 12,174 ± 859 (171.5). Basophils from a normal person contained (in ng/106 cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/106 cells) MBP, 3 ± 0.5; EDN, 72 ± 9; and ECP, 50 ± 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

Abstract Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112 ± 4 ng/106 cells, and ECP, 163 ± 2 ng/106 cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.

Abstract Group 2 innate lymphoid cells (ILC2s) reside in multiple organs in the body, where they play roles in immunity, tissue homeostasis, and metabolic regulation. However, little is known about the regulatory mechanisms of ILC2s in different organs. Here, we identified ILC2s in the mouse uterus and found that they express cell surface molecules, including the IL-33 receptor, ST2, that are roughly comparable to those expressed by lung ILC2s. Both in vivo and in vitro treatment with IL-33 induced type 2 cytokine production in uterine ILC2s, suggesting that they respond to IL-33 in a manner similar to ILC2s in other organs. Importantly, uterine ILC2s were nearly absent in ovariectomized mice and were increased in wild-type mice by estrogen administration, whereas lung ILC2s were unaffected by both ovariectomy and estrogen administration. Likewise, a marked reduction in uterine ILC2s was observed in mice deficient in estrogen receptor α or estrogen receptor β. Furthermore, uterine ILC2s highly expressed estrogen receptor α, and in vitro culture of isolated uterine ILC2s with 17β-estradiol modified expression of a number of genes. Finally, an increased prevalence in neonatal mortality was observed in litters from dams lacking the IL-33 receptor, ST2. Taken together, our findings indicate that unlike lung IL2Cs, uterine ILC2s are regulated by female sex hormones, which may specialize them for specific physiological functions.

Summary Interleukin ( IL )‐33 is a key cytokine involved in type 2 immunity and allergic airway diseases. Abundantly expressed in lung epithelial cells, IL ‐33 plays critical roles in both innate and adaptive immune responses in mucosal organs. In innate immunity, IL ‐33 and group 2 innate lymphoid cells ( ILC 2s) provide an essential axis for rapid immune responses and tissue homeostasis. In adaptive immunity, IL ‐33 interacts with dendritic cells, Th2 cells, follicular T cells, and regulatory T cells, where IL ‐33 influences the development of chronic airway inflammation and tissue remodeling. The clinical findings that both the IL ‐33 and ILC 2 levels are elevated in patients with allergic airway diseases suggest that IL ‐33 plays an important role in the pathogenesis of these diseases. IL ‐33 and ILC 2 may also serve as biomarkers for disease classification and to monitor the progression of diseases. In this article, we reviewed the current knowledge of the biology of IL ‐33 and discussed the roles of the IL ‐33 in regulating airway immune responses and allergic airway diseases.

Aeroallergens produced by Alternaria alternata can elicit life-threatening exacerbations of asthma in patients sensitized to this fungus. In this study, the effect of Alternaria on ion transport mechanisms underlying mucociliary clearance and airway epithelial barrier function was investigated in human airway epithelial cells. Apical exposure to Alternaria induced an increase in anion secretion that was inhibited by blockers of CFTR and Ca2+-activated Cl− channels. Stimulation of anion secretion was dependent on Ca2+ uptake from the apical solution. Alternaria exposure also produced an increase in reactive oxygen species (ROS) that was blocked by pretreatment with the oxidant scavenger glutathione (GSH). GSH and the NADPH oxidase inhibitor/complex 1 electron transport inhibitor diphenylene iodonium chloride (DPI) blocked ATP release and the increase in intracellular [Ca2+] evoked by Alternaria. Alternaria also decreased transepithelial resistance, and a portion of this effect was dependent on the increase in ROS. However, the Alternaria-induced increase in unidirectional dextran (molecular mass = 4,000 Da) flux across the epithelium could not be accounted for by increased oxidative stress. These results support the conclusion that oxidative stress induced by Alternaria was responsible for regulating Ca2+-dependent anion secretion and tight junction electrical resistance that would be expected to affect mucociliary clearance.

Objective: The study aimed to evaluate symptoms described by patients with chronic rhinosinusitis with polypoid changes/nasal polyps and their correlation with computed tomography (CT), nasal endoscopy, and intranasal biomarkers. Study Design: Prospective multicenter study symptom data from postsurgical adult chronic rhinosinusitis study participants with recurrent disease refractory to medical therapy were analyzed in comparison with objective data. Methods: Using logistic regression analysis, participant-rated 16-question surveys from 258 participants were assessed for correlation with nasal endoscopy scores, CT percentage of sinus occlusion, and intranasal biomarkers of fungal antigens (Alternaria and Aspergillus), eosinophilic inflammation (eosinophil-derived neurotoxin [EDN] and major basic protein [MBP]), and inflammatory cytokines (interleukins 5 and 13). Results: Study participant assessments revealed increased CT occlusion in participants presenting with greater inability to smell ( P < .019). Mucosal inflammation identified on nasal endoscopy was positively correlated with congestion ( P < .028), runny nose ( P < .002), and ear pain ( P < .007). Elevated EDN was positively correlated in patients with bothersome congestion ( P < .031) and runny nose ( P < .011). Sneezing was positively correlated with multiple markers: Alternaria ( P < .024), interleukin-13 ( P < .027), MBP ( P < .034), and interleukin-5 ( P < .019). Conclusion: Nasal endoscopy, not CT imaging, has the strongest correlation with the 2 cardinal symptoms of congestion and runny nose in CRS patients; these correlate with biomarkers of eosinophilic inflammation.

Abstract Rationale Difficulty of asthma ascertainment and its associated methodologic heterogeneity have created significant barriers to asthma care and research. Objectives We evaluated the validity of an existing natural language processing (NLP) algorithm for asthma criteria to enable an automated chart review using electronic medical records (EMRs). Methods The study was designed as a retrospective birth cohort study using a random sample of 500 subjects from the 1997–2007 Mayo Birth Cohort who were born at Mayo Clinic and enrolled in primary pediatric care at Mayo Clinic Rochester. Performance of NLP-based asthma ascertainment using predetermined asthma criteria was assessed by determining both criterion validity (chart review of EMRs by abstractor as a gold standard) and construct validity (association with known risk factors for asthma, such as allergic rhinitis). Measurements and Main Results After excluding three subjects whose respiratory symptoms could be attributed to other conditions (e.g., tracheomalacia), among the remaining eligible 497 subjects, 51% were male, 77% white persons, and the median age at last follow-up date was 11.5 years. The asthma prevalence was 31% in the study cohort. Sensitivity, specificity, positive predictive value, and negative predictive value for NLP algorithm in predicting asthma status were 97%, 95%, 90%, and 98%, respectively. The risk factors for asthma (e.g., allergic rhinitis) that were identified either by NLP or the abstractor were the same. Conclusions Asthma ascertainment through NLP should be considered in the era of EMRs because it can enable large-scale clinical studies in a more time-efficient manner and improve the recognition and care of childhood asthma in practice.

AbstractBackgroundInterleukin (IL)‐33 is implicated in the pathophysiology of asthma and allergic diseases. However, our knowledge is limited regarding how IL‐33 release is controlled. The transcription factor nuclear factor‐erythroid‐2‐related factor 2 (Nrf2) plays a key role in antioxidant response regulation.ObjectiveThe goal of this project was to investigate the role of cellular oxidative stress in controlling IL‐33 release in airway epithelium.MethodsComplementary approaches were used that included human bronchial epithelial cells and mouse models of airway type‐2 immunity that were exposed to fungus Alternaria extract. The clinically available Nrf2 activator 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid methyl ester (CDDO‐Me) was used to evaluate the role of Nrf2‐induced antioxidant molecules.ResultsHuman bronchial epithelial cells produced reactive oxygen species (ROS) when they were exposed to Alternaria extract. ROS scavengers, such as glutathione (GSH) and N‐acetyl cysteine, prevented extracellular secretion of ATP and increases in intracellular calcium concentrations that precede IL‐33 release. Administration of CDDO‐Me to mice enhanced expression of a number of antioxidant molecules in the lungs and elevated lung levels of endogenous GSH. Importantly, CDDO‐Me treatment reduced allergen‐induced ATP secretion and IL‐33 release by airway epithelial cells in vitro and protected mice from IL‐33 release and asthma‐like pathological changes in the lungs.ConclusionsThe balance between oxidative stress and antioxidant responses plays a key role in controlling IL‐33 release in airway epithelium. The therapeutic potential of Nrf2 activators needs to be considered for asthma and allergic airway diseases.

Background Endotyping chronic rhinosinusitis (CRS) through simplified cytokine assays may help direct individualized therapy such as corticosteroids, antibiotics, or biologics. We performed an unsupervised network analysis to endotype CRS and control subjects using a commercially available cytokine‐chemokine immunoassay. Methods A 41‐plex cytokine‐chemokine array along with major basic protein (MBP) assay was performed on sinonasal surgical tissue of 32 adults. Subjects were defined as non‐CRS controls (n = 6), CRS with nasal polyps (CRSwNP; n = 13), and CRS without nasal polyps (CRSsNP; n = 13). Unsupervised network modeling was performed to reveal association cytokine‐chemokine (“analyte”) clusters and “subject” groups. Results Network mapping and unsupervised clustering revealed 3 analyte clusters and 3 subject groups. Analyte cluster‐1 was composed of T helper 1 (Th1)/Th17 type markers, analyte cluster‐2 Th2 markers, and analyte cluster‐3 chemokines (CC) and growth factors (GF). Subject group‐1 was devoid of CRSwNP, had fewer asthmatics, and was associated most strongly with analyte cluster‐3 (CC/GF) ( p < 0.001). Subject group‐2 was characterized with the most asthmatics (86%) and CRSwNP (100%) patients, and was associated with analyte cluster‐2 (Th2; p < 0.001). Subject group‐3 was associated with both analyte cluster‐1 (Th1/Th17) and analyte cluster‐3 (CC/GF) ( p < 0.001), and had the highest proportion of CRSsNP patients (62.5%). Tissue levels of MBP, eosinophilia, and computed tomography (CT) scores were significantly higher in subject group‐2 vs other groups ( p ≤ 0.05). Conclusion An unbiased network‐mapping approach using a commercially available immunoassay kit reveals 3 distinct tissue cytokine‐chemokine signatures that endotype CRS patients and controls. These signatures are prominent even in a limited number of patients, and may help formulate individualized therapy and optimize outcomes.

Abstract Rationale Cystatin A and SPINK5 are endogenous protease inhibitors (EPIs) that may play key roles in epithelial barrier function. Objectives To investigate the roles of EPIs in the pathogenesis of chronic rhinosinusitis (CRS). Methods We examined the expression of cystatin A and SPINK5 in the nasal epithelial cells of patients with CRS. Additionally, the in vitro effects of recombinant EPIs on the secretion of the epithelial-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin in airway epithelial cells, and the in vivo effects of recombinant EPIs in the nasal epithelium of mice exposed to multiple airborne allergens (MAA) were examined. Measurements and Main Results Compared with control subjects and patients with noneosinophilic CRS, patients with eosinophilic CRS showed significantly lower protein and mRNA expression of cystatin A and SPINK5 in the nasal epithelium. Allergen-induced production of IL-25, IL-33, and thymic stromal lymphopoietin in normal human bronchial epithelial cells was inhibited by treatment with recombinant cystatin A or SPINK5. Conversely, the production of these cytokines was increased when cystatin A or SPINK5 were knocked down with small interfering RNA. Chronic MAA exposure induced goblet cell metaplasia and epithelial disruption in mouse nasal epithelium and decreased the tissue expression and nasal lavage levels of cystatin A and SPINK5. Intranasal instillations of recombinant EPIs attenuated this MAA-induced pathology. Conclusions Cystatin A and SPINK5 play an important role in protecting the airway epithelium from exogenous proteases. The preservation of EPIs may have a therapeutic benefit in intractable airway inflammation, such as eosinophilic CRS.