Koichiro Asano

Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids.

Eotaxin is a potent and specific eosinophil chemoattractant that is mobilized in the respiratory epithelium after allergic stimulation. Pulmonary levels of eotaxin mRNA are known to increase after allergen exposure in sensitized animals. In this study we demonstrate that TNF alpha and IL-1beta induce the accumulation of eotaxin mRNA in the pulmonary epithelial cell lines A549 and BEAS 2B in a dose-dependent manner. Cytokine-induced A549 cell mRNA accumulation was maximal at 4 h and was significantly enhanced when the cells were costimulated with IFNgamma. TNFalpha- and IL-1beta-induced increases in eotaxin mRNA were diminished in a dose-dependent manner by the glucocorticoid dexamethasone and were augmented by the protein synthesis inhibitor cycloheximide. Cytokine-induced increases in eotaxin mRNA expression correlated with increased eotaxin protein production and secretion, and dexamethasone inhibition of cytokine-induced eotaxin mRNA augmentation was associated with diminished eotaxin protein secretion. These findings, together with the known kinetics of TNF alpha and IL-1beta mobilization in asthmatic airways and the potent eosinophil chemotactic effects of eotaxin, define a mechanism linking inflammatory cytokine mobilization to eosinophil recruitment that may be relevant to the pathogenesis of asthma.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

The Anti-Inflammatory and Proresolving Mediator Resolvin E1 Protects Mice from Bacterial Pneumonia and Acute Lung Injury

Whereas pneumonia is the most common cause of death and disability worldwide, most cases of pneumonia spontaneously resolve. Mechanisms that promote pneumonia resolution remain to be determined. Resolvin E1 (RvE1) is an endogenous mediator that displays proresolving actions in sterile inflammation. In this study, we developed a new model of aspiration pneumonia to evaluate the effect of RvE1 on acute lung injury caused by acid aspiration and subsequent bacterial challenge. Mice received hydrochloric acid into the left lung followed by the enteric pathogen Escherichia coli. I.v. administration of RvE1 (∼0.005 mg/kg) prior to acid injury selectively decreased lung neutrophil accumulation by 55% and enhanced clearance of E. coli. RvE1 significantly decreased lung tissue levels of several proinflammatory chemokines and cytokines, including IL-1β, IL-6, HMGB-1, MIP-1α, MIP-1β, keratinocyte-derived chemokine, and MCP-1, in a manner independent of the anti-inflammatory mediators IL-10 and lipoxin A4. In addition, animals treated with RvE1 had a marked improvement in survival. These findings in experimental aspiration pneumonia have uncovered protective roles for RvE1 in pathogen-mediated inflammation that are both anti-inflammatory for neutrophils and protective for host defense, suggesting that RvE1 represents the first candidate for a novel therapeutic strategy for acute lung injury and pneumonia that harnesses natural resolution mechanisms.

Leukotriene C4 synthase gene A(-444)C polymorphism and clinical response to a CYS-LT1 antagonist, pranlukast, in Japanese patients with moderate asthma

CysLT(1) antagonists are effective for a subset of patients with asthma; however, there has been no good way to predict a given patients response. We examined the interaction between the clinical response to a cysLT(1) antagonist, pranlukast, and DNA sequence variant A(-444)C in leukotriene C(4) synthase (LTC(4) S) gene in Japanese patients with moderate asthma. The frequency of LTC(4) S C(-444) allele was 21.6% in the Japanese general population (n = 171) and 19.4% in the asthmatic subjects ( n= 349). A 4-week prospective, open trial of pranlukast (225 mg twice daily) was performed in 50 patients with moderate asthma who had been well controlled with inhaled corticosteroid (beclomethasone 400-800 microg/day or fluticasone 200-400 microg/day). The C(-444) allele carriers (n = 16) responded better to pranlukast compared to the A(-444) allele homozygotes ( n= 31) [14.3 5.3% vs. 3.1 2.4% improvement of forced expiratory volume in one second (FEV(1) ), 0.01], while LTC(4) S genotype-stratified response to inhaled beta-agonist salbutamol (200 microg) was not observed (17.5 2.1% vs. 18.7 2.2% improvement of FEV(1) ). Univariate analysis demonstrated that the better response to pranlukast (more than 10% improvement of FEV(1) ) was correlated with LTC(4) S genotype (P < 0.01) and pretreatment airway reversibility to salbutamol (P < 0.01), but not with sex, age, atopic status, urinary leukotriene E(4) excretion rate, or daily dose of inhaled corticosteroid. Furthermore, multivariate regression analysis suggested that LTC(4) S genotype and the bronchodilatory effect of salbutamol were independent variables to predict the clinical response to pranlukast (P < 0.05). We conclude that LTC(4) S genotype is predictive of the clinical response to a cysLT(1) antagonist, pranlukast, in Japanese patients with moderate asthma.

Interferon and IL-27 antagonize the function of group 2 innate lymphoid cells and type 2 innate immune responses

Group 2 innate lymphoid cells (ILC2 cells) are type 2 cytokine–producing cells of the innate immune system with important roles in helminth infection and allergic inflammation. Here we found that tissue-resident ILC2 cells proliferated in situ without migrating during inflammatory responses. Both type I and type II interferons and interleukin 27 (IL-27) suppressed ILC2 function in a manner dependent on the transcription factor STAT1. ILC2-mediated lung inflammation was enhanced in the absence of the interferon-γ (IFN-γ) receptor on ILC2 cells in vivo. IFN-γ effectively suppressed the function of tissue-resident ILC2 cells but not that of inflammatory ILC2 cells, and IL-27 suppressed tissue-resident ILC2 cells but not tissue-resident TH2 cells during lung inflammation induced by Alternaria alternata. Our results demonstrate that suppression mediated by interferon and IL-27 is a negative feedback mechanism for ILC2 function in vivo.

Thymic stromal lymphopoietin induces corticosteroid resistance in natural helper cells during airway inflammation

Type-2 innate immune responses that occur in airways and are accompanied by goblet-cell hyperplasia and mucus production are largely driven by interleukin-33 (IL-33) and natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2s) and the major target of IL-33. Here we report that the corticosteroid resistance observed as a result of airway inflammation triggered by sensitization and exposure to allergen is induced via the IL-33/NH-cell axis. Thymic stromal lymphopoietin (TSLP) synthesized during airway inflammation plays a pivotal role in the induction of NH-cell corticosteroid resistance in vitro and in vivo, by controlling phosphorylation of STAT5 and expression of Bcl-xL in NH cells. Blockade of TSLP with a neutralizing antibody or blocking the TSLP/STAT5 signalling pathway with low molecular-weight STAT5 inhibitors such as pimozide restores corticosteroid sensitivity. Thus, the TSLP-STAT5 pathway could be a new therapeutic target in severe, corticosteroid-resistant asthma.

New bronchoscopic microsample probe to measure the biochemical constituents in epithelial lining fluid of patients with acute respiratory distress syndrome

ObjectiveA noninvasive bronchoscopic microsampling (BMS) probe was developed to sample biochemical constituents of the epithelial lining fluid in small airways. DesignObservational, controlled study. SettingIntensive care unit of academic medical center. Patients and Procedure BMS was applied in a control group of seven patients who had hemoptysis or small solitary peripheral nodules but no hypoxemia or other signs of acute inflammation and in four patients with acute respiratory distress syndrome (ARDS), to test whether BMS can ascertain the presence of acute pulmonary inflammation without complications. Measurements and Results Complications, including a significant decrease in arterial oxygen saturation, were observed neither during nor after BMS. In the ARDS group, albumin, lactate dehydrogenase, interleukin-6, basic fibroblast growth factor, and neutrophil elastase concentrations in epithelial lining fluid were significantly higher (p < .0001, p = .012, p < .0001, p < .0001, and p < .0001, respectively) than in the control group. Serial BMS was safely performed in one patient with ARDS, allowing us to observe a correlation between changes in the concentration of inflammation-related biochemical markers and clinical course of the disease. ConclusionsThese results suggest that BMS is safe and useful to monitor pulmonary biochemical events in ARDS.

Diurnal variation of urinary leukotriene E4 and histamine excretion rates in normal subjects and patients with mild-to-moderate asthma☆☆☆★

BACKGROUND Leukotriene E4 (LTE4) and histamine excreted into the urine reflect the in vivo synthesis and release of cysteinyl leukotrienes and histamine, respectively. We examined the diurnal variation of the excretion rate of these mediators over 4 consecutive days in normal subjects (n = 5) and patients with stable mild-to-moderate asthma (n = 8). METHODS Sixteen consecutive 6-hour urine samples were collected over 4 days. Urinary LTE4 concentrations were determined by reverse-phase high-pressure liquid chromatography, followed by ELISA. Urinary histamine concentrations were measured by ELISA. The excretion rates of these compounds were normalized relative to urinary creatinine content. RESULTS The mean urinary LTE4 excretion rate was 83.8 +/- 38.2 pg/mg creatinine (mean +/- SD) in normal subjects; in patients with asthma, the urinary LTE4 excretion rate (110.0 +/- 59.2 pg/mg creatinine) was significantly higher than that in normal subjects (p < 0.05). The urinary histamine excretion rate was not different between normal subjects (24.0 +/- 12.5 ng/mg creatinine) and patients with asthma (31.5 +/- 25.8 ng/mg creatinine). A robust and systematic within-day variation (p < 0.01), but no day-to-day variation, was observed in histamine excretion rate. Although the magnitude of variation in LTE4 excretion within a day was significantly greater in patients with asthma than in normal subjects (p < 0.05), we could not identify any specific diurnal variation pattern in either the normal or the asthma group. No significant correlation was observed between urinary LTE4 and histamine excretion rate within any subject. CONCLUSIONS Patients with asthma excrete LTE4 in the urine at a greater rate than normal subjects. Although no systematic variation in urinary LTE4 excretion rates over the course of a day was observed in either normal subjects or patients with stable asthma, the presence of a systematic diurnal variation of urinary histamine excretion exists in both groups.

Glucocorticoid protects rodent hearts from ischemia/reperfusion injury by activating lipocalin-type prostaglandin D synthase–derived PGD2 biosynthesis

Lipocalin-type prostaglandin D synthase (L-PGDS), which was originally identified as an enzyme responsible for PGD2 biosynthesis in the brain, is highly expressed in the myocardium, including in cardiomyocytes. However, the factors that control expression of the gene encoding L-PGDS and the pathophysiologic role of L-PGDS in cardiomyocytes are poorly understood. In the present study, we demonstrate that glucocorticoids, which act as repressors of prostaglandin biosynthesis in most cell types, upregulated the expression of L-PGDS together with cytosolic calcium-dependent phospholipase A2 and COX2 via the glucocorticoid receptor (GR) in rat cardiomyocytes. Accordingly, PGD2 was the most prominently induced prostaglandin in vivo in mouse hearts and in vitro in cultured rat cardiomyocytes after exposure to GR-selective agonists. In isolated Langendorff-perfused mouse hearts, dexamethasone alleviated ischemia/reperfusion injury. This cardioprotective effect was completely abrogated by either pharmacologic inhibition of COX2 or disruption of the gene encoding L-PGDS. In in vivo ischemia/reperfusion experiments, dexamethasone reduced infarct size in wild-type mice. This cardioprotective effect of dexamethasone was markedly reduced in L-PGDS-deficient mice. In cultured rat cardiomyocytes, PGD2 protected against cell death induced by anoxia/reoxygenation via the D-type prostanoid receptor and the ERK1/2-mediated pathway. Taken together, these results suggest what we believe to be a novel interaction between glucocorticoid-GR signaling and the cardiomyocyte survival pathway mediated by the arachidonic acid cascade.

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.