Koichi Sayama

Cyclooxygenase-2/Prostaglandin D2/CRTH2 Pathway Mediates Double-Stranded RNA-Induced Enhancement of Allergic Airway Inflammation

Respiratory RNA viruses responsible for the common cold often worsen airway inflammation and bronchial responsiveness, two characteristic features of human asthma. We studied the effects of dsRNA, a nucleotide synthesized during viral replication, on airway inflammation and bronchial hyperresponsiveness in murine models of asthma. Intratracheal instillation of poly I:C, a synthetic dsRNA, increased the airway eosinophilia and enhanced bronchial hyperresponsiveness to methacholine in OVA-sensitized, exposed rats. These changes were associated with induction of cyclooxygenase-2 (COX-2) expression and COX-2-dependent PGD2 synthesis in the lungs, particularly in alveolar macrophages. The direct intratracheal instillation of PGD2 enhanced the eosinophilic inflammation in OVA-exposed animals, whereas pretreatment with a dual antagonist against the PGD2 receptor-(CRTH2) and the thromboxane A2 receptor, but not with a thromboxane A2 receptor-specific antagonist, nearly completely eliminated the dsRNA-induced worsening of airway inflammation and bronchial hyperresponsiveness. CRTH2-deficient mice had the same degree of allergen-induced airway eosinophilia as wild-type mice, but they did not exhibit a dsRNA-induced increase in eosinophil accumulation. Our data demonstrate that COX-2-dependent production of PGD2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenetic factors responsible for the dsRNA-induced enhancement of airway inflammation and responsiveness.

Dysregulated synthesis of protectin D1 in eosinophils from patients with severe asthma

BACKGROUND Protectin D1 (PD1) is an anti-inflammatory and proresolving lipid mediator biosynthesized from the omega-3 fatty acid docosahexaenoic acid (DHA). Exogenous PD1 conferred protection against eosinophilic inflammation in animals with experimental asthma, although its endogenous cellular source and functions in human airways are of interest. OBJECTIVE We sought to investigate the synthesizing capacity of PD1 in eosinophils from healthy subjects and patients with severe asthma and its direct effects on eosinophil functions. METHODS Human eosinophil-derived metabolites of arachidonic acid and DHA were analyzed with liquid chromatography-tandem mass spectrometry-based lipidomic analysis. The biological activities of PD1 on the function of human eosinophils, including chemotaxis, adhesion molecule expressions, degranulation, superoxide anion generation, or survival, were examined. RESULTS We identified PD1 as one of the main anti-inflammatory and proresolving molecules synthesized in human eosinophils. PD1, in nanomolar concentrations, suppressed the chemotaxis induced by CCL11/eotaxin-1 or 5-oxo-eicosatetraenoic acid and modulated the expression of the adhesion molecules CD11b and L-selectin, although it had no effects on the degranulation, superoxide anion generation, or survival of the eosinophils. Compared with the cells harvested from healthy subjects, we observed a prominent decrease in the biosynthesis of PD1 by eosinophils from patients with severe asthma, even in presence of DHA. CONCLUSION These observations are a first indication that activated human eosinophils represent a major source of PD1, which can act as a self-resolving machinery in eosinophilic inflammation, whereas the production of PD1 is impaired in patients with severe asthma.

Acute exacerbation of subclinical idiopathic pulmonary fibrosis triggered by hypofractionated stereotactic body radiotherapy in a patient with primary lung cancer and slightly focal honeycombing

Hypofractionated stereotactic body radiotherapy (SBRT) for pulmonary lesions provides a high local control rate, allows completely painless ambulatory treatment, and is not associated with adverse reactions in most cases. Here we report a 70-year-old lung cancer patient with slight focal pulmonary honeycombing in whom subclinical idiopathic pulmonary fibrosis was exacerbated by SBRT. This experience has important implications for the development of selection criteria prior to SBRT for pulmonary lesions. For SBRT candidates with lung tumors, attention must be paid to the presence of co-morbid interstitial pneumonia even if findings are minimal. Such patients must be informed of potential risks, and careful decision-making must take place when SBRT is being considered.

TLR3-Mediated Synthesis and Release of Eotaxin-1/CCL11 from Human Bronchial Smooth Muscle Cells Stimulated with Double-Stranded RNA

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.

Analysis of comorbid factors that increase the COPD assessment test scores

BackgroundThe chronic obstructive pulmonary disease (COPD) Assessment Test (CAT) is a concise health status measure for COPD. COPD patients have a variety of comorbidities, but little is known about their impact on quality of life. This study was designed to investigate comorbid factors that may contribute to high CAT scores.MethodsAn observational study at Keio University and affiliated hospitals enrolled 336 COPD patients and 67 non-COPD subjects. Health status was assessed by the CAT, the St. Georges Respiratory Questionnaire (SGRQ), and all components of the Medical Outcomes Study Short-Form 36-Item (SF-36) version 2, which is a generic measure of health. Comorbidities were identified based on patients’ reports, physicians’ records, and questionnaires, including the Frequency Scale for the Symptoms of Gastro-esophageal reflux disease (GERD) and the Hospital Anxiety and Depression Scale. Dual X-ray absorptiometry measurements of bone mineral density were performed.ResultsThe CAT showed moderate-good correlations with the SGRQ and all components of the SF-36. The presence of GERD, depression, arrhythmia, and anxiety was significantly associated with a high CAT score in the COPD patients.ConclusionsSymptomatic COPD patients have a high prevalence of comorbidities. A high CAT score should alert the clinician to a higher likelihood of certain comorbidities such as GERD and depression, because these diseases may co-exist unrecognized.Trial registrationClinical trial registered with UMIN (UMIN000003470).

Predictors of Osteoporosis and Vertebral Fractures in Patients Presenting with Moderate-to-Severe Chronic Obstructive Lung Disease

Abstract Bone mineral density (BMD) alone does not reliably predict osteoporotic fractures. The Fracture Risk Assessment Tool (FRAX) was developed to estimate the risk of fracture in the general population. This study was designed to identify predictors of osteoporosis and vertebral fractures in patients presenting with chronic obstructive pulmonary disease (COPD). We studied 85 patients (mean age = 75 years; 92% men) with moderate to very severe COPD. Osteoporosis and vertebral fractures were diagnosed with dual energy X-ray absorptiometric scan and vertebral X-rays, respectively. Patient characteristics, including age, gender, body mass index (BMI), and results of pulmonary function tests, chest computed tomography scan, blood and urinary biomarkers of bone turnover were recorded, and a FRAX score was calculated by a computer-based algorithm. Osteoporosis, defined as a T score < –2.5, found in 20 patients (24%), was associated with female gender, BMI, dyspnea scale, long-term oxygen therapy (LTOT), vital capacity (VC), emphysema score on computed tomography, measurements of serum and urinary biomarkers of bone turnover. Vertebral fractures, diagnosed in 29 patients (35%), were strongly correlated with age, LTOT, VC, and forced expiratory volume in 1 sec, treatment with oral corticosteroid or warfarin, and weakly associated with the presence of osteoporosis. There was no correlation between FRAX score and prevalence of vertebral fractures, suggesting that neither BMD alone nor FRAX score would predict the presence of vertebral fractures in COPD patients. A disease-specific algorithm to predict osteoporotic fractures is needed to improve the management of patients suffering from COPD.

Acute Exacerbation of Usual Interstitial Pneumonia After Resection of Lung Cancer

BACKGROUND Acute exacerbation of usual interstitial pneumonia is one of the most life-threatening complications after resection of lung cancer. It is important to detect patients at high risk for acute exacerbation. The purpose of this study was to investigate the incidence of postoperative acute exacerbation and the importance of the finding of honeycombing in predicting the risk of it. METHODS From 992 consecutive pulmonary resections for lung cancer, the background lung was retrospectively assessed histologically. Preoperative chest computed tomographic images of those patients with histologically proven usual interstitial pneumonia were retrospectively reassessed and classified into three groups: typical honeycombing, possible honeycombing, and no honeycombing. Records of patients who experienced postoperative acute exacerbation were extracted from the clinical database, and the correlation between computed tomographic findings and incidence of acute exacerbation was analyzed. RESULTS Of the 992 lung cancer patients, 12 (1.2%) experienced postoperative acute exacerbation. Usual interstitial pneumonia was histologically confirmed in 249 patients, and 4.8% of them experienced postoperative acute exacerbation. No patient without usual interstitial pneumonia experienced it. Preoperative computed tomographic images were available in 205 of the 249 patients. Postoperative acute exacerbation developed in 13.6% of patients (6 of 44 patients) in the typical honeycombing group, 6.4% of patients (5 of 78 patients) in the possible honeycombing group, and 0% of patients (0 of 83 patients) in the no-honeycombing group. Compared with the no-honeycombing group, the incidence of acute exacerbation in the typical and possible honeycombing groups was significantly higher (p=0.001, p=0.025, respectively). CONCLUSIONS Physicians should consider the finding of possible honeycombing, as well as typical honeycombing, a potential risk factor for acute exacerbation after pulmonary resection for lung cancer.

Induction of Mucin and MUC5AC Expression by the Protease Activity of Aspergillus fumigatus in Airway Epithelial Cells

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α–converting enzyme (TACE), critical for the cleavage of membrane-bound pro–TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

Strain-Specific Phenotypes of Airway Inflammation and Bronchial Hyperresponsiveness Induced by Epicutaneous Allergen Sensitization in BALB/c and C57BL/6 Mice

Background: Allergen sensitization through a disrupted skin barrier appears to play a prominent role in the development of atopic diseases, including allergic asthma. The role of the genetic background in immunological and physiological phenotypes induced by epicutaneous sensitization is undetermined. Methods: BALB/c and C57BL/6 mice were sensitized either epicutaneously by patch application of ovalbumin (OVA) or systemically by intraperitoneal injection of OVA with alum before exposure to aerosolized OVA. The concentrations of OVA-specific immunoglobulin in serum and cytokines in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The severity of airway inflammation was evaluated by cell counts in BALF, and bronchial responsiveness to methacholine was measured by the flexiVent system. Results: The production of OVA-specific IgG1 and IgE was greater in the epicutaneously sensitized BALB/c than C57BL/6 mice. In contrast, both eosinophilic airway inflammation and bronchial responsiveness to methacholine were more prominent in the C57BL/6 than in the BALB/c mice. The concentrations of interleukin-4 increased significantly in the BALF from C57BL/6 mice only. No between-strain differences were observed after intraperitoneal sensitization. Conclusions: The C57BL/6 mouse is a more appropriate model than the BALB/c mouse to study the relationship between skin barrier dysfunction and the pathogenesis of allergic asthma.

Association between β-adrenoceptor gene polymorphisms and relative response to β2-agonists and anticholinergic drugs in Japanese asthmatic patients

Background and objective:  Whether β2‐adrenoceptor gene (ADRB2) polymorphisms are associated with airway responsiveness to β2‐agonist medications remains controversial, partly due to factors that may confound pharmacogenetic associations, including age, cigarette smoking and airway remodelling. To overcome these problems, we performed an analysis using parameters that reflected the specific bronchodilator response to β2‐agonists.