Kazutetsu Aoshiba

Effect of suplatast tosilate, a Th2 cytokine inhibitor, on steroid-dependent asthma: a double-blind randomised study

BACKGROUND Th2 cytokines play an important part in the pathogenesis of asthma. Our aim was to study the effect of suplatast tosilate, a selective Th2 cytokine inhibitor, on asthma control and asthma exacerbations during reduction of inhaled corticosteroid dose in patients with steroid-dependent asthma. METHODS 85 patients with moderate to severe asthma taking high doses (> or = 1500 microg per day) of inhaled beclometasone dipropionate, were assigned suplatast tosilate (100 mg three times daily) or placebo for 8 weeks in a double-blind, randomised, parallel-group, multicentre trial. During the first 4 weeks, other medications remained unchanged (add-on phase); during the next 4 weeks, the doses of beclometasone were halved (steroid-reduction phase). Main outcome measures were pulmonary function, asthma symptoms, and use of beta2-agonists. FINDINGS Data were available from 77 patients. During the add-on phase, suplatast tosilate treatment, compared with placebo, was associated with higher forced expiratory volume in 1 s (mean difference between groups for changes from baseline at week 4, 0.20 L [95% CI 0.16-0.24], p=0.043), morning peak expiratory flow (18.6 L/min [14.1-23.1], p=0.037), and less diurnal variation in peak expiratory flow rate, asthma symptom scores (7.1 [6.6-7.6], p=0.029), and serum concentrations of eosinophil cationic protein and IgE. In the steroid-reduction phase, pulmonary function, asthma symptoms, and use of beta2-agonist deteriorated significantly more in the placebo group than in the suplatast group. INTERPRETATION Treatment with a Th2 cytokine inhibitor in steroid-dependent asthma improves pulmonary function and symptom control, and allows a decrease in dose of inhaled corticosteroid without significant side-effects. Some improvements in pharmacokinetics are, however, needed.

Erythromycin shortens neutrophil survival by accelerating apoptosis.

Erythromycin is reported to have an anti-inflammatory action, which may account for its clinical effectiveness in the treatment of chronic inflammatory diseases such as diffuse panbronchiolitis. To evaluate the anti-inflammatory action of erythromycin, we examined the survival of isolated neutrophils with and without erythromycin. Erythromycin shortened neutrophil survival in a dose-dependent fashion, with a maximum effect at 10 micrograms/ml [corrected] and above. Survival at 24 h was 63.4% in medium with 10 micrograms of erythromycin per ml compared with 82.7% in control medium (P < 0.01). This shortening of survival was brought about by acceleration of apoptosis, as evidenced by transmission electron microscopy. In a manner similar to that of erythromycin, other macrolide antibiotics, i.e., clarithromycin, roxithromycin, and midecamycin, also shortened neutrophil survival, but neither the beta-lactams ampicillin and cefazolin nor the aminoglycoside gentamicin affected their survival. Erythromycin increased intracellular levels of cyclic AMP (cAMP) to 150% of control levels in neutrophils. Forskolin, rolipram, and dibutyryl-cAMP, which are known to increase intracellular cAMP levels, also shortened neutrophil survival. H-89, an inhibitor of cAMP-dependent protein kinase A, partially blocked the survival-shortening effect of erythromycin. Our findings suggest that erythromycin shortens neutrophil survival at least in part through elevation of intracellular cAMP levels.

Senescence Hypothesis for the Pathogenetic Mechanism of Chronic Obstructive Pulmonary Disease

We report herein that pulmonary emphysematous lesions appear to be a dynamic phenomenon that involves not only the gradual loss of alveolar structure but also apoptosis, cellular proliferation, and cellular senescence. Cellular proliferation compensates for increased alveolar cell apoptosis in patients with chronic obstructive pulmonary disease (COPD). However, smoking, age, and the increased cell cycle turnover that compensates for apoptosis accelerate alveolar cell senescence, thereby halting cellular proliferation and tipping the balance toward apoptosis, which, in turn, promotes the formation of emphysematous lesions. As a result, alveolar cells disappear and the emphysematous lesions progress. At the same time, cellular senescence is believed to induce inflammation. More specifically, senescent alveolar cells induce inflammation by producing various inflammatory cytokines in tissue. Lymphocytes and Clara cells may also age more rapidly in the lungs of patients with COPD. Lymphocyte senescence may induce an autoimmune reaction and increase susceptibility to infection, and Clara cell senescence may impair airway regeneration as well as sustain airway inflammation. Thus, cellular senescence may be involved in arrested tissue repair, chronic inflammation, and increased susceptibility to infection, which are the typical features of COPD.

Alveolar cell senescence exacerbates pulmonary inflammation in patients with chronic obstructive pulmonary disease.

Background: Alveolar cell senescence is accelerated in patients with chronic obstructive pulmonary disease (COPD). Objectives: We tested the hypothesis that alveolar cell senescence contributes to the chronic inflammation that affects the lungs of COPD patients. Methods: We exposed alveolar type II-like epithelial (A549) cells to a G-quadruplex-interacting telomerase inhibitor in vitro to induce cellular senescence and analyzed the production of proinflammatory cytokines and the activation of NF-ĸB. Human dermal microvascular endothelial cells (HDMECs) were serially passaged to induce replicative senescence. We also immunostained human lung tissue sections obtained from COPD patients, asymptomatic smokers and asymptomatic nonsmokers and examined correlations between type II cell senescence and inflammation. Results: Senescent A549 cells and HDMECs, whether stimulated with lipopolysaccharide or not, produced greater amounts of IL-6, IL-8 and TNF-α, which paralleled NF-ĸB activation, than did presenescent cells. There were positive correlations between the percentages of senescent type II cells that expressed p16INK4a and the percentages of type II cells that expressed phosphorylated NF-ĸB. The lung tissue of the COPD patients contained higher percentages of proinflammatory senescent type II cells that co-expressed p16INK4a and phosphorylated NF-ĸB than the tissue from asymptomatic smokers and asymptomatic nonsmokers. Higher percentages of p16INK4a-positive senescent type II cells than of p16INK4a-negative presenescent type II cells were positive for phosphorylated NF-ĸB. Conclusions: Senescence of alveolar epithelial cells is associated with functional alterations of the cells to a proinflammatory phenotype and may contribute to the pathogenesis of COPD.

Nicotine prolongs neutrophil survival by suppressing apoptosis

Neutrophil accumulation in the lung is implicated in the pathogenesis of pulmonary emphysema and chronic bronchitis associated with cigarette smoking. To determine whether nicotine contributes to this accumulation through the prolongation of neutrophil survival, we examined the survival rates of isolated neutrophils cultured with or without nicotine. We found that nicotine prolonged neutrophil survival in a dose-dependent fashion, with a maximum effect at 10(-6) mol/L. The survival rate at 72 hours was 35.6% +/- 1.2% in medium with 10(-6) mol/L nicotine, compared with 15.5% +/- 0.5% in control medium (mean +/- SEM; p < 0.01), as determined by trypan blue dye exclusion. This prolongation was brought about by suppression of apoptosis, as evidenced by both transmission electron and fluorescence microscopy, and was associated with the preservation of neutrophil functions such as chemotaxis and O2- generation. The prolongation of survival caused by nicotine was abrogated by the addition of Pro-Lys-Arg-NH2, a competitive inhibitor of the specific binding of nicotine to noncholinergic receptors on neutrophils. However, the prolongation of survival caused by nicotine was not suppressed in the presence of K-252b, an inhibitor of protein kinase C. These findings suggest that nicotine prolongs neutrophil survival through noncholinergic nicotine receptors and new protein synthesis, without activation of protein kinase C.

Platinum nanoparticle antioxidants inhibit pulmonary inflammation in mice exposed to cigarette smoke

Recent evidence implicates increased oxidative stress as an important mechanism of the pulmonary inflammation that occurs in cigarette smokers. Since cigarette smoke (CS) contains and generates a large amount of reactive oxygen species (ROS) that elicit pulmonary inflammation, antioxidants may become effective therapeutic agents for CS-related inflammatory lung diseases, such as chronic obstructive pulmonary disease. Platinum nanoparticles stabilized with polyacrylate to form a stable colloid solution (PAA-Pt) are a new class of antioxidants that has been shown to efficiently quench ROS. In the present study we investigated the therapeutic effects of PAA-Pt on pulmonary inflammation in smoking mice. PAA-Pt or saline was administered intranasally to DBA/2 mice, which were then exposed to CS or control air daily for 3 days. Mice were sacrificed 4h after their final exposure to CS or control air. CS exposure caused depletion of antioxidant capacity, NFkappaB activation, and neutrophilic inflammation in the lungs of mice, and intranasal administration of PAA-Pt prior to CS exposure was found to inhibit these changes. Intranasal administration of PAA-Pt alone did not elicit pulmonary inflammation or toxicity. In in vitro experiments, treatment of alveolar-type-II-like A549 cells with PAA-Pt inhibited cell death after exposure to a CS extract. These results suggest that platinum nanoparticles act as antioxidants that inhibit pulmonary inflammation induced by acute cigarette smoking.

Therapeutic effects of erythropoietin in murine models of endotoxin shock

Objective:Erythropoietin has recently emerged as a cytoprotective cytokine, which possesses the ability to protect many tissues, including the brain, heart, and kidneys, against ischemia or traumatic injury. We investigated the therapeutic effects of erythropoietin in a murine model of endotoxin shock. Design:Prospective, randomized study. Setting:University-based research laboratory. Subjects:Male BALB/c mice. Interventions:Mice intraperitoneally received either lipopolysaccharide (LPS) from Escherichia coli or vehicle. Erythropoietin was administered at a dose of 1000 IU/kg subcutaneously at different time points after LPS administration. We also investigated the effect of erythropoietin on the development of septic shock caused by cecal perforation. Measurements and Main Results:Treatment of mice with erythropoietin, within 2 hours after LPS administration, improved the mortality rate. Treatment of cecal perforated mice with erythropoietin extended survival by 12 hours, but all animals died by 72 hours in both groups. Erythropoietin attenuated apoptosis in the lungs, liver, small intestine, thymus, and spleen, as assessed by terminal deoxynucleotidyl transferase-mediated nucleotide nick-end labeling staining, active caspase-3 immunostaining and immunoblotting, and measurements of caspase-3/7 activity. Erythropoietin also reduced inducible nitric oxide synthase expression, nitric oxide production, peroxynitrite formation, and tissue hypoxia. In contrast, erythropoietin did not affect the degree of LPS-induced inflammation, as assessed by measurements of blood levels of interleukin-1&bgr;, interleukin-6, tumor necrosis factor-&agr;, growth-related oncogene/keratinocyte-derived cytokine, and high mobility group box 1, the phosphorylation levels of nuclear factor &kgr;B, and the number of neutrophils infiltrating the lungs and the liver. Conclusions:The results of the study demonstrate that administration of a large dose of erythropoietin after induction of experimental endotoxemia improved survival and that the beneficial effects of erythropoietin were associated with inhibition of apoptosis, nitric oxide production, and tissue hypoxia, without alterations in inflammatory responses.

Bleomycin induces cellular senescence in alveolar epithelial cells

Cellular senescence is a state of irreversible growth arrest. In this paper the authors examined whether bleomycin, an agent that causes pulmonary fibrosis, induces the senescence of alveolar epithelial cells. Type II-like alveolar epithelial (A549) cells or rat primary type II cells were exposed to bleomycin and then evaluated for markers of cellular senescence. Bleomycin was also administered intratracheally in C57BL/6 mice. The authors found that exposure to bleomycin induced cellular senescence in A549 cells and rat primary type II cells. The senescence was characterised by a dose- and time-dependent increase in senescence-associated β‐galactosidase activity, senescence-associated changes in cell morphology, an increase in cell size and lysosomal mass, the overexpression of p21 protein, and irreversible growth arrest. The intratracheal injection of bleomycin in mice induced an increase in senescence-associated β‐galactosidase activity in type II epithelial cells, reaching a maximum at day 7. These results suggest that bleomycin induces a phenotype that is indistinguishable from that of senescence in alveolar epithelial cells. The induction of epithelial senescence by bleomycin may contribute to the pathway of impaired re-epithelialisation leading to pulmonary fibrosis.

Differences in airway remodeling between asthma and chronic obstructive pulmonary disease

The functional consequence of asthma and chronic obstructive pulmonary disease (COPD) is airflow limitation, which is mostly reversible in asthma and not fully reversible in COPD. In both diseases, inflammatory conditions are associated with cellular and structural changes, referred to as remodeling, and these structural changes may lead to thickening of the airway wall, thereby promoting airway narrowing and airflow limitation. However, the pattern of infilatrated cells and the pattern of structural changes occur differently in the two diseases. In asthma, CD4+, T lymphocytes, eosinophils, and mast cells are the predominant cells involved, whereas COPD, CD8+, T lymphocytes, and macrophages are predominantly involved. In severe cases of asthma and COPD, neutrophil infiltration becomes evident. Regarding structural changes, epithelial injury and early thickening of reticular basement membrane are highly characteristic of the airway wall of asthmatics. Increases in airway smooth muscle mass occur in large airways of severe asthmatics and in small airways of patients with COPD. Thickening of the airway wall, golblet cell hyperplasia, mucous gland hypertrophy, and the luminal obstruction caused by inflammatory exudates and mucous are features of both asthma and COPD. Squamous epithelial metaplasia and airway wall fibrosis are commonly observed characteristics of COPD. Destruction and fibrosis of the alveolar wall occur in COPD but not in asthma. The remodeling processes accompanied by chronic inflammatory infiltrates interact in a complex fashion and contribute to the development of airflow limitation in both asthma and COPD.

DNA damage as a molecular link in the pathogenesis of COPD in smokers

In this study, we investigated whether DNA double-strand breaks (DSBs) contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). We immunofluorescence-stained lung tissue samples obtained from COPD patients, asymptomatic smokers and nonsmokers for markers of DSBs. The numbers of DSB foci (phosphorylated histone 2AX (&ggr;H2AX), phosphorylated ATM (ataxia telangiectasia mutated) substrate and phosphorylated p53-binding protein-1 foci) per cell in alveolar type I and II cells and endothelial cells were higher in the COPD patients than in the asymptomatic smokers and nonsmokers. The lung tissue in which type II cells contained higher numbers of &ggr;H2AX foci per cell had higher percentages of type II cells that expressed p16INK4a (p16), phosphorylated nuclear factor (NF)-&kgr;B and interleukin (IL)-6, and of alveolar wall cells that expressed active caspase-3. The type II cells that contained higher numbers of &ggr;H2AX foci per cell had higher rates of expression of p16, phosphorylated NF-&kgr;B, and IL-6. Half of the alveolar wall cells that expressed active-caspase-3 contained &ggr;H2AX foci. Type II cells that stained positive for 8-hydroxy-2-deoxyguanosine contained a higher number of &ggr;H2AX foci per cell than the type II cells that stained negative. In conclusion, DSBs, at least in part caused by oxidative stress, appear to contribute to the pathogenesis of COPD by inducing apoptosis, cell senescence and pro-inflammatory responses.