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The mechanisms underlying the development of steroid resistance in asthma remain unclear. To establish whether as well as the mechanisms by which the activation of Janus kinases (JAKs) is involved in the development of steroid resistance in asthma, murine steroid-resistant models of the proliferation of group 2 innate lymphoid cells (ILC2s) in vitro and asthmatic airway inflammation in vivo were analysed. ILC2s in the lungs of BALB/c mice were sorted and then incubated with IL-33, thymic stromal lymphopoietin (TSLP), and/or IL-7 with or without dexamethasone (10 nM), the pan-JAK inhibitor, delgocitinib (1-10 000 nM), and/or the Bcl-xL inhibitor, navitoclax (1-100 nM), followed by the detection of viable and apoptotic cells. The anti-apoptotic factor, Bcl-xL was detected in ILC2s by flow cytometry. As a steroid-resistant asthma model, ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at a high dose of 500 μg four times. Dexamethasone (1 mg/kg, i.p.), delgocitinib (3-30 mg/kg, p.o.), or navitoclax (30 mg/kg, p.o.) was administered during the challenges. Cellular infiltration into the lungs was analysed by flow cytometry. Airway remodelling was histologically evaluated. The following results were obtained. (1) Cell proliferation concomitant with a decrease in apoptotic cells was induced when ILC2s were cultured with TSLP and/or IL-7, and was potently inhibited by dexamethasone. In contrast, when the culture with TSLP and IL-7 was performed in the presence of IL-33, the proliferative response exhibited steroid resistance. Steroid-resistant ILC2 proliferation was suppressed by delgocitinib in a concentration-dependent manner. (2) The culture with IL-33, TSLP, and IL-7 induced the overexpression of Bcl-xL, which was clearly inhibited by delgocitinib, but not by dexamethasone. When ILC2s were treated with navitoclax, insensitivity to dexamethasone was significantly cancelled. (3) The development of airway remodelling and the infiltration of ILC2s into the lungs in the asthma model were not suppressed by dexamethasone, but were dose-dependently inhibited by delgocitinib. Combination treatment with dexamethasone and either delgocitinib or navitoclax synergistically suppressed these responses. Therefore, JAKs appear to play significant roles in the induction of steroid resistance by up-regulating Bcl-xL in ILC2s. The inhibition of JAKs and Bcl-xL has potential as pharmacotherapy for steroid-resistant asthma, particularly that mediated by ILC2s., (© 2024 John Wiley & Sons Ltd.)

CCR5 may be involved in the pathogenesis of asthma; however, the underlying mechanisms remain unclear. In comparison with a mild asthma model, subepithelial fibrosis was more severe and CCR5 gene expression in the lungs was significantly higher in our recently developed murine model of steroid-resistant severe asthma. Treatment with the CCR5 antagonist, maraviroc, significantly suppressed the development of subepithelial fibrosis in bronchi, whereas dexamethasone did not. On the other hand, increases in leukocytes related to type 2 inflammation, eosinophils, Th2 cells, and group 2 innate lymphoid cells in the lungs were not affected by the treatment with maraviroc. Increases in neutrophils and total macrophages were also not affected by the CCR5 antagonist. However, increases in transforming growth factor (TGF)-β-producing interstitial macrophages (IMs) were significantly reduced by maraviroc. The present results confirmed increases in CCR5-expressing IMs in the lungs of the severe asthma model. In conclusion, CCR5 on IMs plays significant roles in the development of subepithelial fibrosis in severe asthma through TGF-β production in the lungs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Between 5 and 10% of asthma patients do not respond to glucocorticoid therapy. Experimental animal models are indispensable for investigating the pathogenesis of steroid-resistant asthma; however, the majority of murine asthma models respond well to glucocorticoids. We previously reported that multiple intratracheal administration of ovalbumin (OVA) at a high dose (500 µg/animal) induced steroid-insensitive airway eosinophilia and remodeling with lung fibrosis, whereas a low dose (5 µg/animal) caused steroid-sensitive responses. The aims of the present study were as follows: 1) to clarify whether airway hyperresponsiveness (AHR) in the two models is also insensitive and sensitive to a glucocorticoid, respectively, and 2) to identify steroid-insensitive genes encoding extracellular matrix (ECM) components and pro-fibrotic factors in the lung. In comparisons with non-challenged group, the 5- and 500-µg OVA groups both exhibited AHR to methacholine. Daily intraperitoneal treatment with dexamethasone (1 mg/kg) significantly suppressed the development of AHR in the 5-µg OVA group, but not in the 500-µg OVA group. Among genes encoding ECM components and pro-fibrotic factors, increased gene expressions of fibronectin and collagen types I, III, and IV as ECM components as well as 7 matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, transforming growth factor-β1, and activin A/B as pro-fibrotic factors were insensitive to dexamethasone in the 500-µg OVA group, but were sensitive in the 5-µg OVA group. In conclusion, steroid-insensitive AHR developed in the 500-µg OVA group and steroid-insensitive genes encoding ECM components and pro-fibrotic factors were identified. Drugs targeting these molecules have potential in the treatment of steroid-resistant asthma.

Ceramides are an emerging class of anti-inflammatory lipids, and nanoscale ceramide-delivery systems are potential therapeutic strategies for inflammatory diseases. This study investigated the therapeutic effects of ceramide nanoliposomes (CNL) on type 2 inflammation-based asthma, induced by repeated ovalbumin (OVA) challenges. Asthmatic mice intratracheally treated with ceramide-free liposomes (Ghost) displayed typical airway remodeling including mucosal accumulation and subepithelial fibrosis, whereas, in CNL-treated mice, the degree of airway remodeling was significantly decreased. Compared to the Ghost group, CNL treatment unexpectedly failed to significantly influence formation of type 2 cytokines, including IL-5 and IL-13, known to facilitate pathogenic production of airway mucus predominantly comprising MUC5AC mucin. Interestingly, CNL treatment suppressed OVA-evoked hyperplasia of MUC5AC-generating goblet cells in the airways. This suggests that CNL suppressed goblet cell hyperplasia and airway mucosal accumulation independently of type 2 cytokine formation. Mechanistically, CNL treatment suppressed cell growth and EGF-induced activation of Akt, but not ERK1/2, in a human lung epithelial cell culture system recapitulating airway goblet cell hyperplasia. Taken together, CNL is suggested to have therapeutic effects on airway remodeling in allergic asthma by targeting goblet cell hyperplasia. These findings raise the potential of ceramide-based therapies for airway diseases, such as asthma.

Subgroups of patients with severe asthma showing marked increases in sputum eosinophils and/or neutrophils are insensitive to corticosteroids. Previous reports have shown that exogenous administration of an anti-inflammatory cytokine, interleukin (IL)-10 negatively regulated both eosinophilic and neutrophilic migration into tissues. The objective of this study was to elucidate whether intratracheal IL-10 administration suppresses asthmatic responses in a steroid-insensitive model of mice. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 500 µg/animal four times. Dexamethasone (1 mg/kg, intraperitoneal) or IL-10 (25 ng/mouse, intratracheal) was administered during the multiple challenges. The number of leukocytes, expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IL-10 receptor in the lung, and the development of airway remodeling and hyperresponsiveness were evaluated after the fourth challenge. Consistent with our previous study, dexamethasone hardly suppressed the development of airway remodeling and hyperresponsiveness. Although intratracheal IL-10 administration did not affect the development of airway remodeling, the infiltration of eosinophils and neutrophils, and the development of airway hyperresponsiveness were significantly inhibited. Moreover, IL-10 administration significantly decreased the numbers of ICAM-1 + and VCAM-1 + pulmonary vascular endothelial cells, which express IL-10 receptor 1, even though neither production of eosinophilic nor neutrophilic cytokines in the lung was inhibited. Therefore, IL-10 can suppress eosinophil and neutrophil infiltration by inhibiting the proliferation of ICAM-1 + and VCAM-1 + pulmonary vascular endothelial cells, resulting in inhibition of airway hyperresponsiveness in steroid-insensitive asthmatic mice. IL-10 replacement therapy may be clinically useful for the treatment of steroid-insensitive asthma., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A certain population of asthma patients is resistant to steroid therapy, whereas the mechanisms remain unclear. One of characteristic features of steroid-resistant asthma patients is severe airway eosinophilia based on type-2 inflammation. Aims of this study were: 1) to develop a murine model of steroid-resistant asthma, 2) to elucidate that predominant cellular source of a type-2 cytokine, IL-5 was group 2 innate lymphoid cells (ILC2s), 3) to analyze pathogenic alteration of ILC2s in the severe asthma, and 4) to evaluate therapeutic potential of anti-IL-5 monoclonal antibody (mAb) on the steroid-resistant asthma. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 5 or 500 μg/animal 4 times. Development of airway eosinophilia and remodeling in 5-μg OVA model were significantly suppressed by 1 mg/kg dexamethasone, whereas those in 500-μg OVA model were relatively insensitive to the dose of dexamethasone. ILC2s isolated from the lung of the steroid-insensitive model (500-μg OVA) produced significantly larger amounts of IL-5 in response to IL-33/TSLP than ILC2s from the steroid-sensitive model (5-μg OVA). Interestingly, TSLP receptor expression on ILC2s was up-regulated in the steroid-insensitive model. Treatment with anti-IL-5 mAb in combination with dexamethasone significantly suppressed the airway remodeling of the steroid-insensitive model. In conclusion, multiple intratracheal administration of a high dose of antigen induced steroid-insensitive asthma in sensitized mice. IL-5 was mainly produced from ILC2s, phenotype of which had been pathogenically altered probably through the up-regulation of TSLP receptors. IL-5 blockage could be a useful therapeutic strategy for steroid-resistant asthma., (Copyright © 2021 Elsevier B.V. All rights reserved.)

There is a certain population of intractable asthma patients, who can not be controlled by corticosteroid therapy. It has been suggested that 5-10% of asthma patients have been suffered from steroid resistance. Since it has been difficult to develop a steroid-resistant asthma model, the detailed mechanisms have been unclear. Recently, an intractable asthma model showing steroid insensitivity was developed by the author and colleagues. We found that pathogenic changes in type 2 innate lymphoid cells (ILC2) were induced in the intractable asthma. When ovalbumin (OVA) + Al(OH) 3 -sensitized BALB/c mice were intratracheally challenged with OVA at 5 μg/animal, development of airway remodeling as well as lung eosinophilia and neutrophilia were markedly suppressed by treatment with dexamethasone. In contrast, when increasing the dose of OVA for challenges to 500 μg/animal, those asthmatic responses turned to be steroid insensitive. When Th2 cells and ILC2 in the lung were stimulated in vitro, ILC2 produced larger amounts of type 2 cytokines than Th2 cells. Interestingly, amounts of type 2 cytokines produced by the steroid-insensitive model-derived ILC2 were significantly larger than those by the steroid-sensitive, and that the former ILC2 exhibited higher expression of thymic stromal lymphopoietin (TSLP) receptor and signal transducer and activator of transcription (STAT) 5a gene. Treatment with anti-IL-5 antibody improved the steroid sensitivity. Taken together, ILC2 have been transformed to be pathogenic in the intractable asthma. IL-5 hyper-produced from ILC2 may be involved in the development of steroid resistance. The molecules related to the above mentioned are expected to be targets for development of new therapeutic drugs for intractable asthma.

Asthma patients are classified by phenotype and endotype. Although symptoms in most asthma patients are well controlled by glucocorticoid treatment, certain populations of severe eosinophilic asthma patients in T-helper 2 (Th2)/type 2 asthma and neutrophilic asthma patients in non-Th2/type 2 asthma show insensitivity to inhaled or oral glucocorticoid therapy. In some cases of severe eosinophilic asthma, eosinophils remain in the lungs despite glucocorticoid therapy. It was reported that interleukin (IL)-33-induced activation of type 2 innate lymphoid cells (ILC2) was resistant to glucocorticoid treatment in certain allergic conditions. Regarding neutrophilic airway inflammation in steroid-resistant asthma, IL-17 derived from Th17 cells and IL-8 and tumor necrosis factor-α derived mainly from macrophages were reported to be involved in the pathogenesis. Recently, "NETosis," a specific cell death of neutrophils, has been reported to be involved in asthmatic airway inflammation. When NETosis is induced in asthma, aggravation of inflammation and delay of tissue repair could occur, suggesting that NETosis may be associated with the development of steroid-resistant asthma. This article reviews the pathogenesis of steroid-resistant asthma by focusing mainly on neutrophils.

Introduction: The mechanisms of allergen immunotherapy are not fully elucidated. Here, we sought to develop a murine model to demonstrate the effectiveness of subcutaneous immunotherapy (SCIT) for allergic responses. As excessive antigen dosages may induce immune tolerance in sensitized mice, the effects of SCIT were assessed by varying the antigen dosage. The mechanisms of SCIT were analyzed by focusing on the induction of Foxp3 + Treg cells and IL-10-producing Foxp3 - CD4 + T cells, as well as on the phenotype of the latter cells., Methods: Ovalbumin (OVA) + Al(OH) 3 -sensitized mice received subcutaneous dosages of OVA at 0.01, 0.1 or 1 mg/animal for SCIT, followed by intratracheal challenges with OVA at 5, 50 or 500 μg/animal., Results: The maximum effects of SCIT were observed with 1 mg/animal of OVA for airway inflammation induced by 5 μg/animal of OVA, in which airway eosinophilia and Th2 cytokine production were markedly suppressed. The increase in the OVA-specific IgE level was significantly suppressed by SCIT. The development of bronchial epithelial thickening and mucus accumulation were also suppressed by SCIT. Concomitantly, IL-10-producing Foxp3 - CD4 + T cells were increased in the lungs by SCIT, but Foxp3 + Treg cells were not. Most of the induced IL-10-producing Foxp3 - CD4 + T cells were negative for either IL-5 or LAG-3, but positive for CD49b., Conclusion: We successfully developed an airway allergic model for SCIT. It was suggested that most of IL-10-producing Foxp3 - CD4 + regulatory T cells increased by SCIT in the lungs were CD49b + CD4 + regulatory T cells, but neither Th2 cells nor Tr1 cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Compared with CysLT 1 receptors, the functional role of CysLT 2 receptors in asthma has not been clarified. The purpose of this study was to determine 1) whether CysLT 2 receptors are expressed in the lung of mice and if expression increases in asthmatic mice, and 2) whether CysLT 2 receptors are involved in allergic leukocyte infiltration into the lung and in the development of airway remodeling in asthmatic mice. BALB/c mice were sensitized with ovalbumin (OVA) + Al(OH) 3 , and intratracheally challenged with OVA 4 times. Lung tissue was isolated before and after the 4th OVA challenge for detection of CysLT 2 receptors by immunohistochemistry and flow cytometry. The effect of a CysLT 2 receptor antagonist BayCysLT 2 RA on multiple antigen challenge-induced leukocyte infiltration into the lung and the development of airway remodeling was evaluated. Even in non-challenged mice, CysLT 2 receptors were expressed in bronchial smooth muscle. After multiple challenges, expression was also observed in leukocytes infiltrating into alveolar spaces. CysLT 2 R + leukocytes included alveolar macrophages, conventional dendritic cells, and eosinophils. BayCysLT 2 RA significantly inhibited multiple antigen challenge-induced increases in eosinophils and mononuclear cells in the lung. The development of airway remodeling was tended to be suppressed by CysLT 2 receptor antagonist. In conclusion, CysLT 2 receptors were constitutively expressed in the lung, and expression was strengthened in asthmatic mice. Activation of CysLT 2 receptors was functionally involved in allergic leukocyte infiltration into the lung. The CysLT 2 receptor can be a molecular target for the development of new pharmacotherapies for asthma., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Anti-inflammatory pharmacotherapy for asthma has mainly depended on the inhalation of glucocorticoids, which non-specifically suppress immune responses. If the anti-inflammatory cytokine interleukin (IL)-10 can be induced by a specific antigen, asthmatic airway inflammation could be suppressed when individuals are exposed to the antigen. The purpose of this study was to develop cellular immunotherapeutics for atopic diseases using IL-10-producing CD4 + T cells. Spleen cells isolated from ovalbumin (OVA)-sensitized mice were cultured with the antigen, OVA and growth factors, IL-21, IL-27 and TGF-β for 7 days. After the 7-day culture, the CD4 + T cells were purified using a murine CD4 magnetic beads system. When the induced CD4 + T cells were stimulated by OVA in the presence of antigen-presenting cells, IL-10 was preferentially produced in vitro. When CD4 + T cells were adoptively transferred to OVA-sensitized mice followed by intratracheal OVA challenges, IL-10 was preferentially produced in the serum and bronchoalveolar lavage fluid in vivo. IL-10 production coincided with the inhibition of eosinophilic airway inflammation and epithelial mucus plugging. Most of the IL-10-producing CD4 + T cells were negative for Foxp3 and GATA-3, transcription factors of naturally occurring regulatory T cells and Th2 cells, respectively, but double positive for LAG-3 and CD49b, surface markers of inducible regulatory T cells, Tr1 cells. Collectively, most of the induced IL-10-producing CD4 + T cells could be Tr1 cells, which respond to the antigen to produce IL-10, and effectively suppressed allergic airway inflammation. The induced Tr1 cells may be useful for antigen-specific cellular immunotherapy for atopic diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Elevated allergen-specific IgE levels are a hallmark of allergic asthma, a disease involving chronic airway inflammation characterized by airway hyperresponsiveness (AHR); neutrophilic airway inflammation is found in patients with severe asthma. Furthermore, we have reported that interleukin (IL)-33 and IL-17A contribute to IgE-mediated AHR through neutrophilic inflammation in mice. Meanwhile, semaphorins regulating neuronal and immune function have been focused on in several diseases. Here, we investigated whether semaphorin 7A (SEMA7A) is related to IgE-mediated neutrophilic inflammation in mice. BALB/c mice sensitized with antigen-specific IgE monoclonal antibody were repeatedly challenged by the antigen. When anti-SEMA7A antibody was administered during the fourth to seventh challenges, the infiltration by macrophages, lymphocytes, neutrophils, and eosinophils in the lungs was reduced at the seventh challenge (P<0.05, 0.05, 0.01, and 0.05, respectively). However, the increased production of IL-4, IL-5, IL-13, IL-33, IL-17A, IL-6, and CXCL1 in the lungs was not suppressed. In histological analysis, the epithelial cells, blood vessels, and inflammatory cells in the lungs of IgE-sensitized mice showed SEMA7A expression; plexin C1 for the receptor was expressed in the inflammatory cells. Meanwhile, we examined the effect of anti-SEMA7A antibody on AHR and neutrophilic inflammation enhanced by the collaborative action of IL-33 and IL-17A in normal mice, resulting in the suppression of these responses (P<0.05 and 0.01, respectively). Collectively, we demonstrated that SEMA7A plays a critical role in IgE-mediated neutrophilic airway inflammation. Therefore, SEMA7A may be a potential therapeutic target for severe allergic asthma showing neutrophilic airway inflammation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The expression and functional role of CysLT2 receptors in asthma have not been clarified. In this study, we evaluated CysLT2 receptors expression, and effects of CysLT2-and CysLT1/2-receptor antagonists on antigen-induced bronchoconstriction using isolated lung tissues from both asthma and non-asthma subjects., Methods: CysLT1 and CysLT2 receptors expression in asthma and non-asthma lung tissue preparations was examined in immunohistochemistry experiments, and their functional roles in antigen-induced bronchoconstriction were assessed using ONO-6950, a dual CysLT1/2-receptor antagonist, montelukast, a CysLT1 receptor antagonist, and BayCysLT2RA, a CysLT2 receptor-specific antagonist., Results: CysLT1 receptors were expressed on the bronchial smooth muscle and epithelium, and on alveolar leukocytes in 5 in 5 non-asthma subjects and 2 in 2 asthma subjects. On the other hand, although degrees of CysLT2 receptors expression were variable among the 5 non-asthma subjects, the expression in the asthma lung was detected on bronchial smooth muscle, epithelium and alveolar leukocytes in 2 in 2 asthma subjects. In the non-asthma specimens, antagonism of CysLT2 receptors did not affect antigen-induced bronchial contractions, even after pretreatment with the CysLT1-receptor specific antagonist, montelukast. However, in the bronchus isolated from one of the 2 asthma subjects, antagonism of CysLT2 receptors suppressed contractions, and dual antagonism of CysLT1 and CysLT2 receptors resulted in additive inhibitory effect on anaphylactic contractions., Conclusions: CysLT2 receptors were expressed in lung specimens isolated from asthma subjects. Activation of CysLT2 receptors may contribute to antigen-induced bronchoconstriction in certain asthma population., (Copyright © 2015 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

An orally active dual CysLT1 and CysLT2 antagonist possessing a distinctive structure which consists of triple bond and dicarboxylic acid moieties is described. Gemilukast (ONO-6950) was generated via isomerization of the core indole and the incorporation of a triple bond into a lead compound. Gemilukast exhibited antagonist activities with IC50 values of 1.7 and 25 nM against human CysLT1 and human CysLT2, respectively, and potent efficacy at an oral dose of 0.1 mg/kg given 24 h before LTD4 challenge in a CysLT1-dependent guinea pig asthmatic model. In addition, gemilukast dose-dependently reduced LTC4-induced bronchoconstriction in both CysLT1- and CysLT2-dependent guinea pig asthmatic models, and it reduced antigen-induced constriction of isolated human bronchi. Gemilukast is currently being evaluated in phase II trials for the treatment of asthma.

IgE is known to enhance some antibody responses to specific antigens, but whether this contributes to allergic asthma remains unclear. We have previously found that repeated antigen challenges in mice sensitized with antigen-specific IgE monoclonal antibody (mAb) exacerbated airway inflammation and remodelling accompanied by increased levels of endogenous antigen-specific IgE and IgG1. Here, we investigated whether IgE/antigen-mediated enhancement of endogenous IgE production contributes to the exacerbation of airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA) -specific IgE mAb were challenged with OVA intratracheally seven times; anti-IgE mAb was intraperitoneally administered 1 day before the fourth challenge. Treatment with anti-IgE mAb inhibited the increased level of endogenous OVA-specific IgE in serum, but not OVA-specific IgG1, and a biphasic increase in airway resistance at the fourth challenge. Furthermore, a biphasic increase in airway resistance, airway hyper-responsiveness to methacholine, OVA-specific IgE and IgG1 production, and infiltrations by neutrophils and eosinophils in the lungs at the seventh challenge were suppressed by treatment; airway remodelling, such as goblet cell hyperplasia and sub-epithelial fibrosis, was also reduced. In addition, the production of interleukin-17A, interleukin-33 and CXCL1 in the lungs related to these IgE-mediated responses was decreased by treatment. Collectively, we found that the mechanism leading to the exacerbation of allergic asthma is closely related to IgE/antigen-mediated enhancement of IgE production, suggesting that this may create a vicious circle leading to the chronic status in asthmatic patients having levels of antigen-specific IgE ready to form complexes with antigen., (© 2014 John Wiley & Sons Ltd.)

Neutrophilic airway inflammation is a hallmark of patients with severe asthma. Although we have reported that both IL-33 and IL-17A contributed to IgE-mediated neutrophilic inflammation in mice, the relationship remains unclear. In this article, we examined how IL-17A modifies IL-33-induced neutrophilic inflammation and airway hyperresponsiveness (AHR). IL-33 was intratracheally administered to BALB/c mice on days 0-2; furthermore, on day 7, the effect of the combination of IL-33 and IL-17A was evaluated. Compared with IL-33 or IL-17A alone, the combination exacerbated neutrophilic inflammation and AHR, associated with more increased levels of lung glutamic acid-leucine-arginine(+) CXC chemokines, including CXCL1, CXCL2, and CXCL5, and infiltration by alveolar macrophages expressing CXCR2. Treatment with anti-CXCR2 mAb or depletion of alveolar macrophages repressed neutrophilic inflammation and AHR; in addition, depletion of neutrophils suppressed AHR. These findings prompted us to examine the role of CXCR2 in IgE-sensitized mice; a single treatment with anti-CXCR2 mAb in the seventh Ag challenge inhibited late-phase airway obstruction, AHR, and neutrophilic inflammation. In addition to inhibition, multiple treatments during the fourth to seventh challenge attenuated early-phase airway obstruction, eosinophilic inflammation, and goblet cell hyperplasia associated with the reduction of Th2 cytokine production, including IL-4, IL-5, and IL-13. Collectively, IL-33 cooperated with IL-17A to exacerbate AHR by enhancing neutrophilic inflammation via CXCR2 signaling; furthermore, CXCR2 signaling derived Th2 responses. We thus suggest the underlying mechanisms of IL-33 and IL-17A in allergic asthma and CXCR2 as potential therapeutic targets for the disease.

Interleukin (IL)-33, a member of the IL-1 family of cytokines, is produced when epithelial and endothelial cells are exposed to stimuli. Hematopoietic cells such as macrophages also produce IL-33. IL-33 is considered to function as an 'alarmin', activating various immune cells through its receptor ST2, which leads to the production of various molecules. The IL-33-induced production of pro-inflammatory cytokines is a critical event that aggravates atopic diseases such as asthma, atopic dermatitis, and pollenosis and suggests that IL-33-blocking agents could represent new therapeutic drugs. The anti-IL-33 antibody was effective in allergic models, whereas the anti-ST2 antibody has yielded controversial results because soluble ST2 functions as a decoy receptor for IL-33. IL-33-mediated pulmonary inflammation may be glucocorticoid-resistant especially when other cytokines act synergistically. Anti-tumor necrosis factor (TNF)-α therapy may also be effective against IL-33-mediated diseases. ERK1/2 inhibitors have also been shown to suppress the production of IL-33. On the other hand, activation of β2-receptors enhanced the expression of IL-33 mRNA in dendritic cells by activating protein kinase A (PKA), suggesting that PKA inhibitors may be candidates for IL-33-blocking agents. The effects of IL-33-blocking agents on atopic diseases need to be pharmacologically assessed in experimental and clinical studies.

Background and Purpose: Mast cell hyperplasia has been observed in the lungs of mice with experimental asthma, but few reports have studied basophils. Here, we attempted to discriminate and quantify mast cells and basophils in the lungs in a murine asthma model, determine if both cells were increased by multiple antigen challenges and assess the roles of those cells in asthmatic responses., Experimental Approach: Sensitized Balb/c mice were intratracheally challenged with ovalbumin four times. Mast cells and basophils in enzymatically digested lung tissue were detected by flow cytometry. An anti-FcεRI monoclonal antibody, MAR-1, was i.p. administered during the multiple challenges., Key Results: The numbers of both mast cells (IgE(+) C-kit(+) ) and basophils (IgE(+) C-kit(-) CD49b(+) ) increased in the lungs after three challenges. Treatment with MAR-1 completely abolished the increases; however, a late-phase increase in specific airway resistance (sRaw), and airway eosinophilia and neutrophilia were not affected by the treatment, although the early-phase increase in sRaw was suppressed. MAR-1 reduced antigen-induced airway IL-4 production. Basophils infiltrating the lung clearly produced IL-4 after antigen stimulation in vitro; however, histamine and murine mast cell protease 1 were not increased in the serum after the challenge, indicating that mast cell activation was not evoked., Conclusion and Implications: Both mast cells and basophils infiltrated the lungs by multiple intratracheal antigen challenges in sensitized mice. Neither mast cells nor basophils were involved in late-phase airway obstruction, although early-phase obstruction was mediated by basophils. Targeting basophils in asthma therapy may be useful for an early asthmatic response., (© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

Airway remodeling, pathological changes in the lung structure, is a characteristic feature of chronic asthma. The changes include bronchial epithelial hyperplasia and hypertrophy, excess production of mucus, and fibroblast proliferation in the lung. On the other hand, it has been known that both nitric oxide and superoxide anion are increased in exhaled air of asthmatic patients. These molecules react with each other forming a powerful oxidant, peroxynitrite. In this study, effect of a peroxynitrite scavenger, a metalloporphyrin compound, [tetrakis(4-carboxylatophenyl)porphyrinato]manganese(III) (MnTBAP) on multiple antigen challenge-induced airway remodeling was evaluated in mice. When sensitized BALB/c mice were intratracheally challenged with an antigen, ovalbumin, for 3 times, bronchial epithelial thickening and mucus accumulation in the epithelium were histologically observed. Daily treatment with MnTBAP (3, 10 mg/kg/time/twice a day, intraperitoneally (i.p.)) dose-dependently suppressed both the epithelial thickening and mucus accumulation in the epithelium. On the other hand, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining revealed that the multiple antigen challenges increased the number of apoptotic cells in the bronchial epithelium. The increase in apoptotic cells was also effectively suppressed by the treatment with MnTBAP. Taken together, it was suggested that peroxynitrite could be involved in the formation of epithelial hyperplasia associated with the mucus accumulation through induction of apoptosis of the epithelial cells. Thus, peroxynitrite can be a target molecule for development of new pharmacotherapy for asthma.

Allergen-specific IgE plays an essential role in the pathogenesis of allergic asthma. Although there has been increasing evidence suggesting the involvement of IL-17 in the disease, the relationship between IL-17 and IgE-mediated asthmatic responses has not yet been defined. In this study, we attempted to elucidate the contribution of IL-17 to an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness (AHR). BALB/c mice passively sensitized with an OVA-specific IgE mAb were challenged with OVA intratracheally four times. The fourth challenge caused a late-phase increase in airway resistance associated with elevated levels of IL-17(+)CD4(+) cells in the lungs. Multiple treatments with a C3a receptor antagonist or anti-C3a mAb during the challenges inhibited the increase in IL-17(+)CD4(+) cells. Meanwhile, a single treatment with the antagonist or the mAb at the fourth challenge suppressed the late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid. Because IL-17 production in the lungs was significantly repressed by both treatments, the effect of an anti-IL-17 mAb was examined. The late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid was inhibited. Furthermore, an anti-Gr-1 mAb had a similar effect. Collectively, we found that IgE mediated the increase of IL-17(+)CD4(+) cells in the lungs caused by repeated Ag challenges via C3a. The mechanisms leading to the IgE-mediated late-phase asthmatic response and AHR are closely associated with neutrophilic inflammation through the production of IL-17 induced by C3a.

It has been suggested that interleukin (IL)-10 exerts immunosuppressive effects on allergic inflammation, including asthma, mainly through inhibition of Th2 cell-mediated eosinophilic airway inflammation. In a model of experimental asthma utilizing multiple intratracheal antigen challenges in sensitized mice, IL-10 production as well as eosinophilia and neutrophilia in the lung were induced by the multiple challenges. In this study, we set out to reveal the cellular source of endogenously produced IL-10, and the roles of IL-10 in airway leukocyte inflammation using an anti-IL-10 receptor monoclonal antibody. Balb/c mice were sensitized i.p. with ovalbumin+Al(OH)(3), and then challenged by intratracheal administration of ovalbumin 4 times. Flow cytometric analyses revealed that the cellular source of IL-10 was CD4(+) T cells lacking the transcription factor, forkhead box P3. Treatment with anti-IL-10 receptor monoclonal antibody prior to the 4th challenge significantly augmented airway neutrophilia as well as the production of IL-1β, and CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2, but not airway eosinophilia, Th2 cytokine (IL-4 and IL-5) production, or a late-phase increase in specific airway resistance. Approximately 40% of IL-10 receptor(+) cells expressed the macrophage marker F4/80, whereas only 3-4% of the IL-10 receptor(+) cells were granulocyte differentiation antigen (Gr)-1(high) cells (neutrophils). In conclusion, multiple airway antigen challenges induced the proliferation of IL-10-expressing CD4(+) T cells in regulating airway neutrophilia. Systemic blockade of IL-10 function coincided with increases in IL-1β and CXC chemokines. Thus, IL-1β and CXC chemokines may be targets for development of novel pharmacotherapy for neutrophilic asthma., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Allergen-specific IgE has long been regarded as a major molecular component of allergic asthma. Although IgE plays a central role in the early asthmatic response, its roles in the chronic phase, such as the late asthmatic response, airway hyperresponsiveness (AHR), and airway remodeling (goblet cell hyperplasia and subepithelial fibrosis) have not yet been defined well. In this study, we investigated the hypothesis that chronic responses could be induced by IgE-dependent mechanisms. BALB/c mice passively sensitized with an ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were repeatedly challenged with intratracheal administration of OVA. The first challenge induced early phase airway narrowing without any late response, but the fourth challenge caused not only an early but also a late phase response, AHR, and goblet cell hyperplasia. Macrophages, lymphocytes and neutrophils, but not eosinophils, were significantly increased in the lung 24h after the fourth challenge. Interestingly, levels of OVA-specific IgG1 in serum increased by multiple antigen challenges. A C3a receptor antagonist inhibited the late asthmatic response, AHR, and infiltration by neutrophils. In contrast, no late response, goblet cell hyperplasia, inflammatory cells, or production of IgG1 was observed in severe combined immunodeficient mice. On the other hand, seven challenges in BALB/c mice induced subepithelial fibrosis associated with infiltration by eosinophils. In conclusion, the allergic asthmatic responses induced by passive sensitization with IgE mAb can provide a useful model system to study the pathological roles of IgE in acute and chronic phases of allergic asthma., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The facilitating effects of multiwalled carbon nanotubes (MWCNT) on allergic asthma have not been sufficiently examined, although MWCNT appear to significantly increase the risk of health problems from occupational or environmental exposure. In this study, we examined whether sensitization by the combination of MWCNT with ovalbumin (OVA) promotes allergic asthmatic responses. BALB/c mice administered vehicle, MWCNT, OVA, or MWCNT+OVA through an intranasal route were challenged with OVA intratracheally four times. In the MWCNT+OVA group, the fourth challenge caused not only early- but also late-phase increases in airway resistance, although these responses were not observed in the vehicle, MWCNT, or OVA group; furthermore, the extents of the early and late responses were comparable to those in mice systemically sensitized with OVA+alum. Sensitization with MWCNT and OVA promoted airway inflammation and goblet cell hyperplasia in the lung compared with the vehicle, MWCNT or OVA group. In addition, adjuvant activity for OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a production in serum and increased levels of interleukin-4 (IL-4), IL-5, IL-13, and IL-17 in the lung tissue were observed. In conclusion, these results suggest that exposure to MWCNT and antigen can induce a biphasic increase in airway resistance, airway inflammation, goblet cell hyperplasia, and the production of antigen-specific antibodies. This study highlights the risk of exposure to a combination of MWCNT with antigen.

Aims: Neutrophils have been found increasingly in the lungs of patients with severe asthma; however, it is unclear whether the neutrophils contribute to the induction of the airway obstruction. We determined using a murine model whether neutrophils are involved in the late asthmatic response (LAR), and analyzed mechanisms underlying the antigen-induced airway neutrophilia., Main Methods: BALB/c mice sensitized by ovalbumin (OVA)+Al(OH)(3) were challenged 4 times by intratracheal administration of OVA. Airway mechanics were measured as specific airway resistance., Key Findings: Induction of the LAR after the 4th challenge coincided with airway neutrophilia. In contrast, eosinophil infiltration was established prior to the 4th challenge. A treatment with an anti-Gr-1 monoclonal antibody (mAb) before the 4th challenge selectively suppressed increases in the neutrophil number and myeloperoxidase (MPO) level in bronchoalveolar lavage fluid (BALF), and attenuated the magnitude of LAR by 60-70%. Selective suppression of eosinophilia by anti-IL-5 mAb had little effect on the LAR. The increases in neutrophil number and MPO level were partially inhibited by an anti-CD4 mAb treatment. The CD4(+) cell depletion also significantly inhibited increases in neutrophil chemoattractants, IL-17A, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 in BALF. However, blockade of FcγRII/III failed to suppress the neutrophilia., Significance: These data suggest that neutrophils are key inducers of the LAR, and that the antigen-induced neutrophilia is partially dependent on activated CD4(+) cells that are involved in the production of IL-17A, KC and MIP-2., (Copyright © 2011 Elsevier Inc. All rights reserved.)

It is known that the late asthmatic response (LAR), a characteristic feature of asthma, is closely associated with CD4+ Th2 cell-mediated allergic inflammation. Airway remodeling is also a pathogenesis of asthma, but literature reporting roles of CD4+ cells in the remodeling is controversial. There has been no study that simultaneously assessed the roles of CD4+ cells in both LAR and airway remodeling. Sensitized mice were intratracheally challenged with ovalbumin 4 times. Treatment with an anti-CD4 monoclonal antibody (mAb) before the 1st challenge almost completely abolished increase in CD4+ cells in the tissues after the 4th challenge. The late phase increase in airway resistance after the 4th challenge was also completely inhibited by anti-CD4 mAb. Parameters of airway remodeling, subepithelial fibrosis and epithelial thickening were attenuated by treatment, whereas the inhibition was only 30% - 40%. Bronchial smooth muscle thickening was not affected. Because interleukin (IL)-5 production as well as eosinophilia was effectively suppressed by anti-CD4 mAb, the effect of anti-IL-5 mAb was also examined, resulting in no inhibition of airway remodeling. Collectively, although the LAR was completely dependent on CD4+ cell activation, airway remodeling was only partially dependent on the cell.

In most experimental model of asthma using guinea pigs, the animals are made to inhale an aerosolized antigen which passes through the nasal cavity. In the present study, we attempted to create an animal model of asthma showing a biphasic asthmatic response and airway hyperresponsiveness, in which the allergic responses are restricted to the lung. Guinea pigs were sensitized by the intratracheal instillation of ovalbumin (OVA)+Al(OH)₃ once a day for 7 d, and then intratracheally challenged with OVA 12 d after the last sensitization. The change in specific airway resistance (sRaw) and airway responsiveness to histamine were measured. Pranlukast (100 mg/kg), theophylline (50 mg/kg), and dexamethasone (10 mg/kg) were orally administered 18 and 2 h before the antigen challenge. The challenge caused a marked biphasic elevation of sRaw with peaks at 5 min and 4 h. At 24 h, airway hyperresponsiveness to histamine was observed. Pranlukast, theophylline, and dexamethasone suppressed the late asthmatic response and airway hyperresponsiveness. The early asthmatic response was inhibited by theophylline and dexamethasone. In conclusion, the intratracheal sensitization and challenge caused a biphasic asthmatic response and airway hyperresponsiveness in guinea pigs. This model may be useful for the evaluation of anti-asthma drugs.

Allergic asthma is a chronic inflammatory disorder of the airways characterized by biphasic airway obstruction and airway hyperresponsiveness. In this study, we attempted to elucidate the contribution of the complement C3a to these asthmatic symptoms. BALB/c mice sensitized by i.p. injections of OVA plus alum were challenged with OVA intratracheally four times. The fourth challenge caused a biphasic asthmatic response peaking at 10 min and 3-4 h, as well as airway hyperresponsiveness to methacholine. Histological examination revealed increased expression of C3a receptors in the lung on the fourth challenge. Additionally, the C3 level in serum 4 h after the fourth challenge was significantly reduced compared with that before the challenge. When a C3a receptor antagonist, SB290157, was administered i.p. 30 min before the fourth challenge, the late-phase asthmatic response and airway hyperresponsivness induced by the fourth challenge were significantly inhibited, although the early-phase response was not influenced. In bronchoalveolar lavage fluid, neutrophil infiltration 24 h after the fourth challenge was reduced by the treatment. On the other hand, SB290157 suppressed the increased expression of IL-1beta in the lung in this model, and the intratracheal administration of IL-1beta induced airway obstruction, airway hyperresponsiveness, and neutrophil infiltration in normal mice. These results illustrate that C3a is involved in the development of the late asthmatic response and airway hyperresponsiveness. The mechanism leading to the development of these symptoms may correlate with the recruitment of neutrophils and/or the production of IL-1beta induced by C3a.

To investigate mechanisms underlying the late asthmatic response, we developed a murine model using repetitive intratracheal antigen challenge. BALB/c mice sensitized by i.p. injection with ovalbumin+alum were challenged with ovalbumin intratracheally 4 times. The 1st challenge induced early airway obstruction peaking at 30 min but without a late response; however, the 4th challenge caused not only early but also late airway obstruction at 2-8 h. Eosinophils, and CD4+ and CD8+ T lymphocytes were increased in the airway before the 4th but not before the 1st-3rd challenges. The numbers of IgE+/CD117+ (mast) cells were also increased in the lung before the 4th challenge. Levels of Th2 cytokines were also increased in the airway. Daily administration of dexamethasone during the challenge period suppressed all these inflammatory events. Thus, this experimental late asthmatic response is associated with Th2 cytokine production from inflammatory cells recruited as a consequence of the 1st-3rd challenges.

Background: A toluene-2,4-diisocyanate (TDI)-induced asthma model, in which delayed-type hypersensitivity-like asthmatic airway obstruction is elicited restrictively in the lung, has never been developed., Methods: Guinea pigs were percutaneously sensitized with TDI. For the challenges, once every 2 weeks for a total of 5 times, TDI mists were delivered directly to the lung through an oral cannula, with its tip being positioned in the opening of the trachea. Time-course changes in specific airway resistance (sRaw) were measured by double-flow plethysmography. Basic mechanisms underlying TDI-induced asthma were analyzed., Results: After the 2nd-5th challenges, induction of both an early increase in sRaw that peaked at 10 min and a delayed-type sRaw elevation that peaked at 22 h were observed. Interestingly, in the sensitized/challenged animals, baseline sRaw was elevated by repeated challenge as compared to that seen for non-sensitized animals. Intratracheal administration of a bronchodilator, salbutamol, strongly suppressed the early asthmatic response (EAR) but not the delayed-type asthmatic response (DAR). During DAR, both albumin leakage and fucose secretion into the bronchoalveolar lavage fluid were increased. The cysteinyl leukotriene antagonist pranlukast failed to inhibit either EAR or DAR while the corticosteroid dexamethasone significantly suppressed DAR, without significantly affecting EAR., Conclusions: Effective delivery of TDI to the lung may induce reproducible DAR in sensitized guinea pigs with chronicity that is reflected by an increase in the sRaw baseline. DAR is not mediated by constriction of airway smooth muscles and is probably due to the concurrent presence of mucosal edema and mucus hypersecretion in the airways.

Disodium cromoglycate is an anti-asthmatic drug that has mast cell-stabilizing effects and other anti-inflammatory effects. However, the mechanisms of its anti-inflammatory effects are unclear. In this study, we evaluated effects of disodium cromoglycate on eosinophilia, early and late asthmatic responses, and production of arachidonic acid metabolites in guinea pig lungs. Guinea pigs were alternately sensitized and challenged by exposure to mists of ovalbumin+Al(OH)(3) and ovalbumin, respectively. Disodium cromoglycate (0.5-2 mg/0.1 ml/animal) administered intratracheally before the fifth challenge dose-dependently inhibited asthmatic response, but early asthmatic response was not affected. Disodium cromoglycate at 2 mg/animal potently suppressed increases in cysteinyl leukotrienes (CysLTs) and thromboxane A(2) in the lung during late asthmatic response. Eosinophilia was slightly reduced by disodium cromoglycate. The inhibitory effect of disodium cromoglycate on late asthmatic response is apparently due to inhibition of the release of arachidonic acid metabolites, some of which may be derived from eosinophils that infiltrate the lung.

Background: Allergic airway eosinophilia is suppressed by cysteinyl leukotriene (CysLT) receptor (CysLT1 receptor) antagonists in several species including humans and guinea-pigs, suggesting that CysLTs are directly or indirectly involved in induction of the response., Objective: We examined the effect of CysLT antagonists (pranlukast and MCI-826) on antigen inhalation-induced eosinophilia in peripheral blood and lung, and on IL-5 activity in serum during late increase of airway resistance (late asthmatic response, LAR) in sensitized guinea-pigs., Methods: Guinea-pigs inhaled ovalbumin (OVA) + Al(OH)3 and OVA mists alternately for sensitization and challenge, respectively, once every 2 weeks. At the fifth challenge, the effects of CysLT antagonists and an anti-IL-5 antibody (TRFK-5) on the occurrence of LAR, and blood and lung eosinophilia, which appeared at 5 h after challenge, were examined. The time-course of IL-5 activity in the serum after the challenge was evaluated by measuring in vitro 'eosinophil survival prolongation activity'. The influence of CysLT antagonists on IL-5 activity was assessed., Results: CysLT antagonists and TRFK-5 completely abolished blood and lung eosinophilia. LAR was suppressed by both MCI-826 and TRFK-5 by 40-50%. Sera obtained from sensitized, challenged animals 3 h and 4 h after challenge induced an obvious prolongation of eosinophil survival. The activity of the sera was completely neutralized by prior exposure to TRFK-5, suggesting that it reflected IL-5 activity. Increased IL-5 activity in the serum was inhibited by both pranlukast and MCI-826 by over 90%., Conclusions: CysLTs produced after antigen provocation sequentially induced IL-5 production from some immune component cells via CysLT1 receptor activation. Thus, it is likely that CysLTs indirectly cause antigen-induced eosinophilia through IL-5 production.

Bronchial asthma is characterized by airway nallowing and hypersensitivity, for the occurrence of which (chemical) mediators are almost entirely responsible. Recent clinical as well as experimental reports have strongly suggested that arachidonate metabolites, especially cysteinyl leukotrienes (CysLTs), are involved in the attack of not only allergic asthma but also exercise- or aspirin-induced asthma. Because the receptor of CysLT1 but not CysLT2 largely contributes to the various pulmonary pharmacological actions of CysLTs, CysLT1 antagonists with high potency have rapidly developed recently. Other chemical mediators, tachykinins, endothelins and so on can be also candidates for the cause of the disease.

The contributions of histamine, cysteinyl leukotrienes (CysLTs) and thromboxane A2 (TXA2) to the asthmatic responses and the magnitudes of blood and lung eosinophilia at acute and chronic stages of our asthmatic model were comparatively determined. Guinea pigs were alternately sensitized/challenged by inhalation with ovalbumin+Al(OH)3 and ovalbumin, once every 2 weeks. Effects of mepyramine, pranlukast (a CysLT antagonist) and seratrodast (a TXA2 antagonist) on the early (EAR) and/or the late asthmatic response (LAR) were assessed at the second and fourth antigen challenges. The second challenge caused EAR but not LAR. Although the EAR was decreased at the fourth challenge, a substantial LAR was seen. Both mepyramine and seratrodast inhibited the EAR at the second challenge by approximately 50%. However, at the fourth challenge, these drugs did not inhibit the EAR. The LAR at the fourth challenge was attenuated by pranlukast and seratrodast by 45% and 40%, respectively. Both the blood and lung eosinophilia were modestly and markedly induced 5 h after the second and fourth challenges, respectively. These results strongly suggest that repetition of antigen challenge induces quantitative alterations of chemical mediators participating in the asthmatic responses and a change of the body state under which eosinophils exhibit enhanced migratory activities.

Recently, we reported a reproducible model of asthma in guinea-pigs in vivo, which developed a late asthmatic response (LAR) as well as an early response. In this study, time-related changes in the occurrence of the LAR and leucocyte kinesis were assessed. Furthermore, the state of the activation of eosinophils that migrated into the lower airways was characterized in vitro. Guinea-pigs were alternately sensitized/challenged by inhalation with aerosolized ovalbumin adsorbed on aluminium hydroxide and ovalbumin alone, once every 2 weeks. At defined times before and after the fifth challenge, airway resistance was measured, blood was drawn and bronchoalveolar lavage (BAL) and nasal cavity lavage (NCL) were performed. Superoxide anion (.O2-) production of eosinophils was measured with cytochrome c. Occurrence of LAR and considerable increases in circulating eosinophils coincided with each other 5-7 h after the challenge. After 7 h, eosinophil infiltrations into bronchoalveolar spaces were observed. The capacity of eosinophils from the sensitized animals to produce .O2- was higher than those from the non-sensitized ones, when eosinophils were stimulated by platelet-activating factor. Although an increased number of eosinophils in the NCL fluid was observed, it was much less than that in the BAL fluid. Thus, it has been concluded that eosinophilia in the blood and the lung may participate in the occurrence of the late asthmatic response, which is thought to be preferentially evoked in the lower airways in guinea-pigs in vivo.

To develop a model of chronic experimental asthma in guinea pigs, the animal was forced to inhale the mist of a low dose of ovalbumin (OA) adsorbed on fine Al(OH)3 for sensitization once every 4 weeks. The animal was challenged by inhalation with the mist of OA on day 14 after the respective sensitizations. Either the first or the second antigen challenge markedly induced an early asthmatic response (EAR), whereas there was hardly any late asthmatic response (LAR). At the 3rd challenge, LAR also emerged with some severity. These dual responses were consistently observed until the 10th challenge. On the other hand, repeated inhalation/challenge, once every 2 weeks, with OA alone at the same dose tended to lead to the desensitization of the EAR. In addition, LAR was hardly observed throughout the experiments. In both groups, gamma 1 and IgE levels in the serum were elevated by the repetitive antigen inhalations, yet no obvious relationship between these antibody levels and the intensity of either EAR or LAR was recognized. The present results indicate that the asthmatic model with reproducible EAR and LAR developed in this study appears to be very beneficial for the investigation of bronchial asthma and for the assessment of anti-asthma drugs.