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Introduction: The muscarinic M3 receptor antagonist, tiotropium, has a bronchodilatory effect on asthma patients. Additionally, tiotropium inhibits allergic airway inflammation and remodeling in a murine asthma model. However, the underlying mechanisms of this M3 receptor antagonist remain unclear. Therefore, we investigated the effect of muscarinic M3 receptor blockage on M2 macrophage development during allergic airway inflammation., Methods: BALB/c mice were sensitized and challenged with ovalbumin to develop a murine model of allergic airway inflammation mimicking human atopic asthma. During the challenge phase, mice were treated with or without tiotropium. Lung cells were isolated 24 h after the last treatment and gated using CD68-positive cells. Relm-α and Arginase-1 (Arg1) (M2 macrophage markers) expression was determined by flow cytometry. Mouse bone marrow mononuclear cell-derived macrophages (mBMMacs) and human peripheral blood mononuclear cells (PBMCs)-derived macrophages were stimulated with IL-4 and treated with a muscarinic M3 receptor antagonist in vitro., Results: The total cells, eosinophils, and IL-5 and IL-13 levels in BAL fluids were markedly decreased in the asthma group treated with tiotropium compared to that in the untreated asthma group. The Relm-α and Arg1 expression in macrophages was reduced considerably in the asthma group treated with tiotropium compared to that in the untreated asthma group, suggesting that the development of M2 macrophages was inhibited by muscarinic M3 receptor blockage. Additionally, muscarinic M3 receptor blockage in vitro significantly inhibited M2 macrophage development in both mBMMacs- and PBMCs-derived macrophages., Conclusions: Muscarinic M3 receptor blockage inhibits M2 macrophage development and prevents allergic airway inflammation. Moreover, muscarinic M3 receptors might be involved in the differentiation of immature macrophages into M2 macrophages., (© 2024 S. Karger AG, Basel.)

Introduction: Inhalation of fungal allergens induces airway epithelial damage following airway inflammation and excessive mucus secretion, which can lead to severe asthma with fungal sensitization (SAFS). Comprehensive gene expression analysis in Alternaria-exposed mouse airways, a model of SAFS, has not been conducted., Methods: BALB/c mice received intranasal administration of Alternaria extract or phosphate-buffered saline twice a week for 6 weeks. Lung sections and bronchoalveolar lavage fluid were obtained to assess airway inflammation. RNA-Seq in the central airway was performed, and gene ontology (GO) analysis and gene set enrichment analysis (GSEA) were conducted for pathway analyses. An in vitro experiment using human airway epithelial cell 16HBE14o- was performed to validate the RNA-Seq findings., Results: Eosinophilic airway inflammation with mucus overproduction and airway remodeling was observed in mice exposed to Alternaria. RNA-Seq analysis revealed 403 upregulated and 108 downregulated genes in airways of Alternaria-exposed mice. In GO analysis, the functions of immunoglobulin (Ig) receptor binding, Ig production, inflammatory response, and T-cell activation were upregulated, while those of keratinization and defense response to other organisms were downregulated. GSEA revealed positive enrichment in T-cell receptor complex, immunological synapse, antigen binding, mast cell activation, and Ig receptor binding, and negative enrichment in keratinization and cornification in Alternaria-exposed mice relative to control. Alternaria exposure to 16HBE14o- cells validated the downregulation of epithelial keratinization-related genes, including SPRR1A, SPRR1B, and KRT6B., Conclusion: RNA-Seq analysis showed that Alternaria exposure induced inflammatory response and impaired defense mechanisms in mice airway epithelium, which might be therapeutic targets for SAFS., (© 2021 S. Karger AG, Basel.)

Introduction: Bronchoconstriction was recently shown to cause airway remodeling and induce allergic airway inflammation in asthma. However, the mechanisms how mechanical stress via bronchoconstriction could induce airway inflammation and remodeling remain unclear., Objective: We investigated the effect of bronchoconstriction induced by methacholine inhalation in a murine model of asthma., Methods: BALB/c female mice were sensitized and challenged with ovalbumin (OVA), followed by treatment with methacholine by a nebulizer twice a day for 7 days. Twenty-four hours after the last methacholine treatment, the bronchoalveolar lavage fluid (BALF) and lung tissues were collected. The BALF was analyzed for total and differential cell counts and cytokine levels. The lung tissues were analyzed for goblet cell metaplasia, thickness of the smooth muscle, and lung fibrosis. The expression of cytokines in the lung was also examined., Results: OVA sensitization and challenge induced infiltration of total cells, macrophages, and eosinophils in the BALF along with goblet cell metaplasia and increased airway smooth muscle hypertrophy. Seven days after the last OVA challenge, untreated mice achieved reduction in airway inflammation, while methacholine maintained the number of BALF total cells, macrophages, and eosinophils. The percentage of goblet cells and the thickness of airway smooth muscle were also maintained by methacholine. Moreover, the treatment of methacholine induced the expression of transforming growth factor (TGF)-β in the lung. This result indicates that the production of TGF-β is involved in induction of airway remodeling caused by bronchoconstriction with methacholine., Conclusions: Repeated bronchoconstriction caused by methacholine inhalation elicited allergic airway inflammation and airway remodeling., (© 2020 S. Karger AG, Basel.)

Background: Chronic obstructive pulmonary disease (COPD) and asthma have similar clinical features and are both exacerbated by airway infection., Objective: To determine whether garenoxacin mesylate hydrate (GRNX) added to the standard care for bacterial infection-induced acute exacerbation of asthma or COPD in adults has clinical benefits., Method: This single-arm clinical trial was conducted from January 2015 to March 2016. Adults with a history of asthma or COPD for more than 12 months were recruited within 48 h of presentation with fever and acute deterioration of asthma or COPD requiring additional intervention. Participants were administered 400 mg GRNX daily for 7 days without additional systemic corticosteroids or other antibiotics. The primary outcome was efficacy of GRNX based on clinical symptoms and blood test results after 7 days of treatment. Secondary outcomes were: (1) comparison of the blood test results, radiograph findings, and bacterial culture surveillance before and after treatment; (2) effectiveness of GRNX after 3 days of administration; (3) analyzation of patient symptoms based on patient diary; and (4) continued effectiveness of GRNX on 14th day after the treatment (visit 3)., Results: The study included 44 febrile patients (34 asthma and 10 COPD). Frequently isolated bacteria included Moraxella catarrhalis (n = 6) and Klebsiella pneumoniae (n = 4). On visit 2, 40 patients responded, and no severe adverse events were observed. All secondary outcomes showed favorable results., Conclusion: GRNX effectively treated asthma and COPD patients with acute bacterial infection without severe adverse events. Further research with a larger study population is needed., (© 2019 S. Karger AG, Basel.)

Background: Interleukin (IL)-33, a new member of the IL-1 cytokine family, is involved in T helper (Th)2-type responses in a wide range of diseases and is mediated by expression of the ST2 receptor in many immune cells. As the effects of IL-33 on dendritic cells (DCs) remain controversial, we investigated the ability of IL-33 to modulate the functions of these cells., Methods: DCs were derived from mouse bone marrow, and the expression of the IL-33 receptor ST2 was examined by fluorescence-activated cell sorting and RT-PCR. The responses of the DCs to IL-33 were examined by RT-PCR and ELISA, and activation of mitogen-activated protein kinases (MAPKs) was determined by Western blotting., Results: ST2 ligand mRNA and protein were detectable in DCs. IL-33 induced the production of thymus and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 and the activation of extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 MAPK., Conclusions: DCs respond directly to IL-33 through ST2. The interaction between IL-33 and DCs may represent a new pathway to initiate Th2-type immune responses. IL-33 and ST2 may play important roles in allergic inflammation., (Copyright © 2013 S. Karger AG, Basel.)

Background: Viral infection can exacerbate asthma by inducing the accumulation of inflammatory cells in the airway. We have previously reported that double-stranded RNA (dsRNA), a viral product and ligand of the Toll-like receptor-3 (TLR3), activates the transcription factors NF-κB and IRF-3 and upregulates the expression of inflammatory chemokines in airway epithelial cells. Here, we examined the effects of the glucocorticoid fluticasone propionate (FP) on the expression of the inflammatory chemokines CCL5, CXCL8 and CXCL10., Methods: The airway epithelial cell line BEAS-2B was used for this study. Expression of CCL5, CXCL8 and CXCL10 mRNA and protein was quantified by real-time PCR and ELISA assay, respectively. To examine the association of FP with the physiology of chemokine production, we included several methods. Nuclear translocation of transcription factors was determined by performing Western blot analysis. Histone deacetylase (HDAC) activity in nuclear extracts was measured using a colorimetric assay. Stability of the chemokine mRNAs was examined in cells incubated with actinomycin D. The activities of the CCL5 promoter and the transcription factors NF-κB and IRF-3 were assessed using luciferase reporter assays., Results: Treatment of BEAS-2B cells with FP significantly and dose-dependently (10(-9) to 10(-6)M) inhibited dsRNA-induced expression of CCL5, CXCL8 and CXCL10 protein and mRNA, but did not affect mRNA stability. FP also significantly inhibited dsRNA-stimulated CCL5 promoter activity. However, FP had no effect on the activity of HDAC or the nuclear translocation of NF-κB and IRF-3., Conclusions: FP inhibits the dsRNA-stimulated expression of inflammatory chemokines in airway epithelial cells. FP may act by inhibiting chemokine transcription through an as yet unidentified mechanism., (Copyright © 2013 S. Karger AG, Basel.)

Eosinophilic gastroenteritis (EGE) is characterized by eosinophilic infiltration of the digestive organs, most commonly of the stomach and the duodenum. Symptoms of EGE are nonspecific and include nausea, vomiting, abdominal pain, dyspepsia, malabsorption, ascites and weight loss. The various symptoms of EGE depend on its location and the depth of gastrointestinal eosinophil infiltration. We report a case presenting with acute pancreatitis caused by a milk allergy. The patient's symptoms rapidly improved after treatment with corticosteroids, and he remained symptom-free for more than 20 months by the elimination of cow's milk from his diet. Serum titers of pancreatic enzymes and total bilirubin simultaneously recovered and blood eosinophil counts normalized. The causative allergens of EGE are too various to detect; however, allergologic exams revealed that a cow's milk allergy had provoked EGE in our case. Adult-onset cow's milk allergies are rare; when seen, however, they may present severe complications such as anaphylaxis, gastroenteritis and pancreatitis. When unaccountable gastrointestinal symptoms are observed, EGE caused by food allergies should be included in the differential diagnosis., (Copyright © 2012 S. Karger AG, Basel.)

Background: Respiratory syncytial virus (RSV) infection in infants with Th2 predisposition is thought to increase the risk of allergic sensitization, recurrent wheezing, and bronchial asthma during childhood. We attempted to clarify the molecular mechanisms by which Th1/Th2 predisposition in the host alters RSV infection and facilitates airway inflammation., Methods: A549 human airway epithelial cells were inoculated with live or UV-treated RSV after pretreatment with either a combination of tumor necrosis factor (TNF)-α and interferon-γ (Th1-primed) or a combination of TNF-α and interleukin-4 (Th2-primed) for 48 h. The gene and protein expression profiles of RSV-infected A549 cells were examined., Results: GeneChip analysis indicated that, at 96 h after inoculation with RSV, the expression of 62 genes was specifically enhanced (more than 2-fold by normalized data) in Th2-primed cells compared to that in unprimed or Th1-primed cells. An increase in mRNA and protein levels of monocyte chemoattractant protein (MCP)-1/CCL2 among those 62 genes was confirmed by real-time PCR and cytometric bead assay, respectively. RSV replication was markedly diminished in Th1-primed airway epithelial cells but not in Th2-primed cells, which was presumably caused at least in part by the early induction of antiviral genes., Conclusions: These results suggest that Th1/Th2 predisposition in the host prior to RSV infection critically regulates inflammatory reactions in the airways through alteration of gene expression, and that MCP-1/CCL2 plays an important role in the pathogenesis of severe RSV infection and the subsequent development of asthma in Th2-predisposed hosts., (Copyright © 2010 S. Karger AG, Basel.)

Background: Interleukin (IL)-33, a new member of the IL-1 cytokine family, has been recognized as a key cytokine that enhances T helper 2-balanced immune regulation through its receptor ST2; however, the function and relationship of the IL-33 and ST2 pathways in bronchial asthma are still unclear. We investigated the cellular origin and regulation of IL-33 and ST2 in allergic bronchial asthma in vivo and in vitro., Methods: BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Mice were exposed to aerosolized 1% OVA for 30 min a day for 7 days. These mice were then challenged with aerosolized 1% OVA 2 days after the last day of exposure. After the OVA challenge, the mice were sacrificed and their lung tissues were obtained. Mouse lung fibroblasts were cultured and treated with IL-33 or IL-13., Results: The levels of IL-33 mRNA and IL-33 protein in lung tissue increased after the OVA challenge. Most IL-33-expressing cells were CD11c+ cells and epithelial cells, and many ST2-expressing cells were stained lung fibroblasts and inflammatory cells. IL-33 induced eotaxin/CCL11 production in lung fibroblasts. IL-33 and IL-13 synergistically induced eotaxin expression., Conclusions: IL-33 may contribute to the induction and maintenance of eosinophilic inflammation in the airways by acting on lung fibroblasts. IL-33 and ST2 may play important roles in allergic bronchial asthma., (Copyright © 2011 S. Karger AG, Basel.)

Background: CCL5/RANTES contributes to prolonged eosinophilic inflammation and asthma exacerbation after a viral infection. We studied the mechanism of CCL5 expression using viral product double-stranded RNA (dsRNA), a ligand of Toll-like receptor 3 (TLR3), and inflammatory cytokines in airway epithelial cells., Methods: The airway epithelial cell line BEAS-2B was used in our in vitro study, and the levels of CCL5 mRNA and CCL5 protein expression were determined using real-time PCR and ELISA. The activity of the CCL5 promoter region and nuclear factor (NF)-kappaB was assessed by dual luciferase assay using specific luciferase reporter plasmids. We used actinomycin D to assess the stability of mRNA. Phosphorylation of signal transducer and activator of transcription 1 (STAT-1) was analyzed by Western blot., Results: Synthetic dsRNA up-regulated the expression of CCL5 mRNA and CCL5 protein. Adding TNF-alpha or IFN-gamma to dsRNA further increased the expression of CCL5. The combination of TNF-alpha and dsRNA cooperatively activated the CCL5 promoter region and the NF-kappaB-specific reporter. IFN-gamma did not activate these reporters. However, it increased the stability of CCL5 mRNA induced by dsRNA. IFN-gamma phosphorylated STAT-1, but dsRNA did not. The effects of IFN-gamma were not evident in the cells transfected with short interfering RNA for STAT-1., Conclusions: Cross-talk between TLR3 signaling and inflammatory cytokines regulates the expression of CCL5 in airway epithelial cells. In this mechanism, TNF-alpha may activate NF-kappaB, in cooperation with TLR3 signaling. IFN-gamma may stabilize CCL5 mRNA up-regulated by TLR3. This mechanism may depend on STAT-1., ((c) 2010 S. Karger AG, Basel.)

Background: Osteopontin (OPN) contributes to the development of T helper 1 (Th1)-mediated immunity and Th1-associated diseases. However, the role of OPN in bronchial asthma is unclear. Corticosteroids reduce airway inflammation, as reflected by the low eosinophil and T-cell counts, and the low level of cytokine expression. We investigated OPN production and the inhibitory effects of corticosteroids on OPN production in a murine model of allergic asthma., Methods: BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Some mice received daily injections of dexamethasone (DEX) or phosphate-buffered saline for 1 week. All OVA-challenged mice were exposed to aerosolized 1% OVA for 30 min an hour after these injections. After the OVA challenge, the mice were killed, and bronchoalveolar lavage (BAL) fluid and lung tissue were examined., Results: The levels of OPN protein in BAL fluid and OPN mRNA in lung tissue increased after OVA challenge. Most OPN-expressing cells were CD11c+ cells and some were T cells. DEX decreased the levels of OPN protein in the BAL fluid, and those of OPN mRNA and OPN protein in lung tissue., Conclusions: OPN may play an important role in allergic bronchial asthma. Corticosteroids inhibit OPN production in mice with allergic asthma. The beneficial effect of corticosteroids in bronchial asthma is partly due to their inhibitory effects on OPN production., (Copyright 2009 S. Karger AG, Basel.)

Background: Oxidants including reactive oxygen species have been indicated to play an important role in the pathogenesis of asthma., Objective: We investigated oxidative status in patients with acute exacerbations of asthma and evaluated the therapeutic response using the D-ROM test which is simple to use and quick., Methods: We measured reactive oxygen metabolite (ROM) levels in the serum of 42 outpatients with acute exacerbations of asthma, 11 outpatients with stable asthma and 40 healthy subjects using the D-ROM test. Seven inpatients admitted due to acute exacerbations of asthma were also enrolled to evaluate the effects of treatment. Serum eosinophil cationic protein and plasma polymorphonuclear elastase were also measured by EIA or ELISA to evaluate the correlation between inflammation and oxidative status., Results: Serum ROM levels were significantly higher in patients with acute exacerbation of asthma than in patients with stable asthma or healthy subjects. Levels of serum eosinophil cationic protein and plasma polymorphonuclear elastase were increased in acute exacerbation and moderately correlated to ROM levels. Levels of ROM were significantly decreased after treatment with systemic steroids and bronchodilators., Conclusion: These findings suggest that acute exacerbation of asthma is associated with increased oxidative stress. Serum ROM levels would partly reflect the inflammation with eosinophils and neutrophils and may be useful as biomarkers of asthma., (Copyright 2008 S. Karger AG, Basel.)

Background: Airway smooth muscle (ASM) cells may contribute to the pathogenesis of asthma including airway inflammation and remodeling. We focused our study on the regulation of chemokine expression by cytokines and analyzed the mechanisms of eotaxin/CCL-11 expression in ASM cells., Methods: Human ASM cells were cultured in vitro and treated with IL-4, interferon-gamma (IFNgamma), and tumor necrosis factor-alpha (TNFalpha). Secretion of chemokines into the culture medium was analyzed by ELISA. Expression of eotaxin mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Binding of transcription factor signal transducer activator of transcription (STAT) 6 to the eotaxin promoter-derived DNA was analyzed by pull-down Western blot. To assess transcriptional regulation of eotaxin, cells were transfected with eotaxin promoter-luciferase reporter plasmids, and activity was determined by dual luciferase assay., Results: The Th2 cytokine IL-4 preferentially stimulated the expression of the CC chemokine receptor (CCR) 3-ligand chemokines eotaxin, eotaxin-3, and MCP-4. The Th1 cytokine IFNgamma stimulated the expression of chemokines IP-10 and RANTES. IL-4 stimulated nuclear translocation of signal transducer activator of transcription 6 (STAT6) and its binding to the eotaxin promoter region. IL-4 activated the eotaxin promoter and its activity was inhibited by mutation of the binding site for STAT6 in the promoter., Conclusions: The Th2 cytokine IL-4 preferentially stimulated the expression of CCR3 ligand chemokines including eotaxin in ASM cells. The transcription factor STAT6 may play a pivotal role in the activation of eotaxin transcription in response to IL-4.

Background: We hypothesized that synthetic double-stranded (ds)RNA may mimic viral infection and reported that dsRNA stimulates expression of inflammatory chemokines through a receptor of dsRNA Toll-like receptor (TLR) 3 in airway epithelial cells. In this study, we focused our study on the role of other receptors for dsRNA, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and double-stranded RNA-dependent protein kinase (PKR)., Methods: Airway epithelial cell BEAS-2B was cultured in vitro. Expression of target RNA and protein were analyzed by PCR and ELISA. To analyze the role of receptors for dsRNA, knockdown of theses genes was performed with short interfering RNA (siRNA)., Results: We first investigated the effects of chloroquine, an inhibitor of lysosomal acidification, on the expression of chemokines. Preincubation with 100 microM chloroquine significantly inhibited the expression of mRNA for RANTES, IP-10, and IL-8, stimulated by poly I:C, indicating that poly I:C may react with a receptor expressed inside the cells. RIG-I, MDA-5, and PKR are supposed to be expressed inside the airway epithelial cells. However, the expression of chemokines stimulated with poly I:C was not significantly inhibited for these putative receptors in the cells which were transfected with siRNA., Conclusions: Synthetic dsRNA poly I:C stimulates the expression of inflammatory chemokines in airway epithelial cells, but the putative receptors for dsRNA such as RIG-I, MDA-5, or PKR may not play pivotal roles in this process. TLR3 may play a major role as reported previously.

Background: Interleukin (IL)-17F is a recently discovered cytokine and is derived from a panel of limited cell types, such as activated CD4+ T cells, basophils, and mast cells. IL-17F is known to induce several cytokines and chemokines. However, its involvement in airway inflammation has not been well understood. To this end, the expression of IL-17F and the inhibitory effects of glucocorticoids on its expression in a mouse model of asthma were examined., Methods: Five-week-old BALB/c male mice were sensitized by intraperitoneal injection (i.p.) of ovalbumin (OVA) with alum, and challenged by daily inhalation of aerosolized 1% OVA. 24 h after last challenge (OVA/OVA), the expression of IL-17F was examined in lung tissues by immunohistochemistry and reverse-transcription polymerase chain reaction. Control mice were sensitized and challenged with saline (Sham/Sham). In addition, a group OF OVA-sensitized mice received i.p. injection of water-soluble dexamethasone (DEX) in saline 1 h before ova challenge (OVA/DEX)., Results: In sham-challenged mice, IL-17F was not expressed in the lungs, while, in contrast, IL-17F was predominantly expressed in bronchial epithelial cells in addition to the infiltrating inflammatory cells in OVA/OVA mice. Further, the expression of IL-17 F was significantly attenuated by the treatment of mice with DEX., Conclusion: These results suggest that bronchial epithelium-derived IL-17F may represent a new pharmacological target for glucocorticoids and may play a role in allergic asthma.

Background: The physiological roles of eosinophils that accumulate in the uterus during pregnancy and of uterus-dwelling mast cells remain unknown. In the present study, we investigated the degranulation of eosinophils and mast cells within the normal course of pregnancy and after delivery by measuring the urinary concentrations of eosinophil-derived neurotoxin (EDN) and N-methylhistamine., Methods: Spot urine samples from 65 pregnant women and 15 nonpregnant, age-matched women were examined. Urinary EDN and N-methylhistamine concentrations were measured by radioimmunoassay and standardized with urinary creatinine concentration., Results: A significant increase in the urinary EDN concentration was observed until the second trimester in the normal pregnancies. The elevated urinary EDN levels decreased after the onset of labor in the third trimester and normalized within 1 month after normal vaginal delivery. In women who underwent a cesarean section, the urinary EDN concentration was significantly higher for up to 1 month after delivery, compared to that in women who underwent a vaginal delivery. In contrast, the urinary N-methylhistamine concentration did not change until the second trimester and was significantly decreased during the third trimester. No significant correlation between the peripheral blood eosinophil count and the urinary EDN concentration was observed in these subjects., Conclusions: Eosinophils appear to play a role in the progression of pregnancy and recovery after a cesarean section through the degranulation of eosinophils. In addition, mast cell degranulation does not appear to be related to the contraction of uterine smooth muscle during labor., (Copyright 2003 S. Karger AG, Basel)