Takahiro Nakajima

Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice

Besides lowering cholesterol, statins exert multiple effects, such as anti-inflammatory activity and improvement of endothelial cell function. We examined whether simvastatin (SS) protects against the development of elastase-induced pulmonary emphysema in mice by using mean linear intercepts of alveoli (Lm) as a morphometric parameter of emphysema. After injection of intratracheal elastase on day 0, C57BL/6 mice were treated daily with SS (SS+ group) or PBS (SS- group) for 2 wk. A 21% decrease in Lm on day 7 was observed in the SS+ group vs. the SS- group. Anti-inflammatory effects of SS were observed as a decrease in percentage of neutrophils up to day 3, and in hydroxyproline concentration on day 3, in bronchoalveolar lavage fluid (BALF). SS also increased the number of proliferating cell nuclear antigen (PCNA)-positive alveolar epithelial cells between days 3 and 14. To confirm the role of statins in promoting proliferation of alveolar cells, mice were treated with SS (SS+) vs. PBS (SS-) for 12 days, starting 3 wk after elastase administration. After SS treatment, Lm decreased by 52% and PCNA-positive alveolar epithelial cells increased compared with the SS- group. Concentrations of vascular endothelial growth factor in BALF and endothelial nitric oxide synthase protein expression in pulmonary vessels tended to be higher in the SS+ group vs. the SS- group in this protocol. In conclusion, SS inhibited the development of elastase-induced pulmonary emphysema in mice. This therapeutic effect was due not only to anti-inflammation but also to the promotion of alveolar epithelial cell regeneration, partly mediated by restoring endothelial cell functions.

Limitation of cigarette consumption by CYP2A6*4, *7 and *9 polymorphisms

The whole gene deletion CYP2A6*4, the defect of the main nicotine oxidase, contributes to limiting lifelong and daily cigarette consumption. However, the effects on smoking habits of CYP2A6*7 and *9, two major functional polymorphisms common in Asian populations, have not been reported. The present study examined the relationship between polymorphisms *4, *7 and *9 with the smoking habits of 200 Japanese smokers who visited the Keio University Hospital (Tokyo, Japan). The allele frequencies of *1 (wild type), *4, *7 and *9 were 52, 17, 11 and 20%, respectively. When the three polymorphisms were considered simultaneously, the percentages of homozygous wild type, heterozygote, and homozygous mutants and compound heterozygotes were 26.0, 52.5 and 21.5%, respectively. Homozygous mutants and compound heterozygotes (nu200a=u200a43) smoked fewer cigarettes daily than heterozygotes (nu200a=u200a105) and homozygous wild-type individuals (nu200a=u200a52). Smokers with *7/*7, *9/*9 or *7/*9 had lower daily cigarette consumption than smokers with *1/*1. In conclusion, polymorphisms *4, *7 and *9 of CYP2A6 were detected in approximately three out of four Japanese smokers, and their daily cigarette consumption was genetically modulated by these functional polymorphisms.

Adam8 Limits the Development of Allergic Airway Inflammation in Mice

To determine whether a disintegrin and metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyperresponsiveness (AHR), we compared AAI and AHR in wild-type (WT) versus Adam8−/− mice in different genetic backgrounds sensitized and challenged with OVA or house dust mite protein extract. OVA- and house dust mite–treated Adam8−/− mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some Th2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8’s anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8−/− mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8−/− macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients, but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.

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

Background and objective:u2003 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.

Interleukin-18: The Master Regulator Driving Destructive and Remodeling Processes in the Lungs of Patients with Chronic Obstructive Pulmonary Disease?

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal lung inflammatory response to inhaled noxious particles or gas including those present in cigarette smoke. Patients with COPD can have destructive processes (airspace enlargement) in their lungs alongside seemingly disparate chronic remodeling processes in their airways and pulmonary vasculature (1, 2). However, it has not been clear whether similar or distinct signaling pathways drive the development of the destructive versus remodeling lung pathologies in COPD lungs. A common feature of the pulmonary lesions associated with COPD is inflammation, and exaggerated T helper type 1 (Th1), Th2 cytokine, and Th17 responses have been linked to COPD pathogenesis (3–7). However, it has not been clear to what extent Th1, Th2, and Th17 cytokine responses contribute to individual pathologies in COPD lungs, or whether they interact synergistically or otherwise to promote the development of COPD. The article by Kang and colleagues in this issue of the Journal (pp. 1205–1217) addresses these knowledge gaps by showing for the first time that interleukin-18 (IL-18) is an upstream, master regulator that promotes a complex pattern of Th1 and Th17/Th2 cytokine responses in the lungs of mice (8). The IL-18–driven increases in Th1 cytokine responses are responsible for the airspace enlargement developing in the IL-18 transgenic mice, whereas the IL-18–driven increases in Th17/Th2 cytokine responses cause chronic remodeling in the airways and pulmonary vessels in the IL-18 transgenic mice. The authors also identify novel reciprocal regulation of the Th1 versus Th17/Th2 cytokine responses in the lungs and the development of the distinct lung pathologies associated with each cytokine response. n nIL-18 is produced by myeloid leukocytes and lung epithelial cells (9) and binds to its receptor (IL-18R), which is expressed at low levels on naive T cells. In the presence of IL-12, IL-18 acts as a Th1 cytokine because IL-12 increases IL-18R expression by Th1 cells, which enables IL-18 to promote Th1-cell polarization and proliferation, secretion of interferon-γ (IFN-γ), and macrophage and polymorphonuclear neutrophil accumulation in tissues (9). However, in the absence of IL-12, IL-18 induces the release of Th2 and Th17 cytokines from activated Th1 cells and other leukocytes (9). Prior studies have linked IL-18 to COPD pathogenesis because IL-18 levels are increased in blood and lung samples from patients with COPD and correlate negatively with lung function (10–12), and IL-18R-α−/− mice are protected from cigarette smoke–induced airspace enlargement (10). However, until now, it has not been clear whether IL-18 contributes to other COPD lung pathologies. To address this issue, Kang and coworkers generated and evaluated dual construct transgenic mice that overexpress IL-18 in a doxycycline-inducible manner in the adult murine lung using the CC10 promoter. After 4 months of IL-18 overexpression in the lung, the mice developed robust lung inflammation and airspace enlargement along with impressive small fibrosis, airway mucus metaplasia, and vascular remodeling associated with pulmonary hypertension and right ventricular hypertrophy. The authors genetically deleted IFN-γ, IL-13, and IL-17A in IL-18 transgenic mice and thereby identified activities for IFN-γ in promoting cytotoxic lymphocyte responses in the lung, alveolar septal cell apoptosis, and airspace enlargement (Figure 1). Additionally, they found that: (1) IL-17A is required for IL-18-induced increases in lung IL-13 levels; and (2) IL-17A and IL-13 together promote lung inflammation, airway mucus metaplasia, and airway and vascular remodeling in IL-18 transgenic mice (Figure 1). Kang and colleagues also found that IFN-γ reduces lung levels of IL-17A and IL-13 to thereby abrogate IL-17A/IL-13–driven lung remodeling processes, and IL-17A and IL-13 both lower lung levels of IFN-γ to reduce airspace enlargement in IL-18 transgenic mice (Figure 1). n n n nFigure 1. n nIL-18 drives both the destructive and remodeling processes in the lungs of IL-18 transgenic mice. Novel pathways identified by Kang and coworkers in IL-18 transgenic mice are highlighted with red arrows and lines. Inducible overexpression of IL-18 in ... n n n nThe article by Kang and colleagues is noteworthy for several reasons. First, the investigators identify a novel master cytokine regulator that can drive all of the key pathologies found in COPD lungs. Second, the authors demonstrate for the first time that Th1 and Th17/Th2 cytokine responses counterregulate both each other and the lung pathologies associated with each response in IL-18 transgenic mice. Third, IL-18 transgenic mice represent a new murine model of COPD characterized by robust airspace enlargement as well as impressive COPD-like remodeling processes in their airways and vasculature, unlike C57BL/6 wild-type mice exposed to smoke, which is the mostly commonly used murine model of COPD (13). Other strengths of the article are the use of the inducible transgenic system to avoid potential effects of IL-18 overexpression on lung development and the targeting overexpression of IL-18 to lung epithelial cells, which endogenously produce IL-18 in the COPD lung (11). n nFuture studies could address whether the increased lung levels of IL-18 in IL-18 transgenic mice are of similar magnitude to those occurring in the lungs of patients with COPD to assess whether the results of this study are relevant to human COPD. It would be of interest to determine whether IL-18 overexpression in macrophages would produce similar results given that macrophages are an important source of IL-18 in COPD lungs (11). Although this study highlights the activities of CD4+ T cells in COPD-like lung pathologies in IL-18 transgenic mice, the mice were housed under specific pathogen-free conditions and thus not stimulated by pathogen-derived antigens. It would be interesting to determine whether the lung pathologies in IL-18 transgenic mice would be exacerbated by infection of their respiratory tracts with bacteria or viruses that have been linked to exacerbations in human patients with COPD. For reasons that are not clear, human patients with COPD vary considerably in the extent to which they develop different lung lesions. Future studies could address whether the latter is related to genetically or environmentally determined differences in levels of IL-18 expression or signaling in different cells in different compartments of the lung. n nThe results of the study of Kang and coworkers could have therapeutic implications for COPD. First, they provide evidence that monotherapy targeting either a Th1 or a Th2 cytokine might have both deleterious and beneficial effects. Monotherapy targeting IFN-γ might limit emphysema progression but worsen airway and vascular remodeling, whereas monotherapy directed against IL-13 might limit the progression of chronic remodeling processes in the lungs but accelerate the progression of emphysema. Second, the studies identify IL-18 as a potential upstream target for future COPD therapeutics to limit both the destructive and remodeling processes occurring in COPD lungs. In this respect, it is noteworthy that neutralizing antibodies to IL-18 have efficacy in preclinical models of inflammation and tissue injury in other organ systems (14, 15). However, IL-18 has crucial host defense and antitumor activities (16), and gene therapy to increase IL-18 levels in tissues protects experimental animals from infection and tumor growth and metastasis (17, 18). Given that patients with COPD can have infective disease exacerbations and are at increased risk from developing lung cancer (19), it would be important to determine the safety as well as the efficacy of novel therapeutics targeting IL-18 in the lungs of patients with COPD.

Common functional polymorphisms in the cathepsin S promoter in Japanese subjects: Possible contribution to pulmonary emphysema

Background and objective:u2003 Cathepsin S is involved in the pathogenesis of COPD in murine models overexpressing interferon (IFN)‐γ and IL‐13. It is widely accepted that genetic factors partly influence susceptibility to COPD; however, the association of genetic polymorphisms in the cathepsin S gene with COPD has not been reported previously. In this study, functional polymorphisms in the 5′‐flanking region of the human cathepsin S gene were identified and their association with COPD phenotypes was investigated.

Plasma Cathepsin S and Cathepsin S/Cystatin C Ratios Are Potential Biomarkers for COPD

Purpose. This study aimed to examine whether plasma levels of cathepsin S or its inhibitor, cystatin C, may serve as biomarkers for COPD. Patients and Methods. We measured anthropometrics and performed pulmonary function tests and chest CT scans on 94 patients with COPD and 31 subjects with productive cough but no airflow obstruction (“at risk”; AR). In these subjects and in 52 healthy nonsmokers (NS) and 66 healthy smokers (HS) we measured plasma concentrations of cathepsin S and cystatin C using an ELISA. Data were analyzed using simple and logistic regression and receiver operating characteristic analyses. Results. Cathepsin S and cystatin C plasma levels were significantly higher in the COPD and AR groups than in the NS and HS groups (p < 0.01). Among the COPD patients and AR subjects, plasma cathepsin S levels and cathepsin S/cystatin C ratios, but not cystatin C levels, were negatively related to severe airflow limitation (% FEV1 predicted < 50%; p = 0.005) and severe emphysema as assessed by low attenuation area (LAA) score on chest CT scans (LAA ≥ 8.0; p = 0.001). Conclusion. Plasma cathepsin S and cathepsin S/cystatin C ratios may serve as potential biomarkers for COPD.

Plasma cytokine profiles related to smoking-sensitivity and phenotypes of chronic obstructive pulmonary disease

Abstract Chronic obstructive pulmonary disease (COPD) develops only in smoking-sensitive smokers and manifests heterogeneous phenotypes, including emphysema and non-emphysema types. We aimed to identify biomarkers related to the smoking-sensitivity and phenotypes of COPD. Among 240 smokers suggestive of COPD, we studied on four groups defined by % forced expiratory volume in one second (FEV1) and computed tomography-based pulmonary emphysema. Plasma concentrations of 33 inflammatory markers were measured in four groups as well as Non-smokers using multiplex protein arrays. IL-5, IL-7 and IL-13 were identified to be associated with smoking sensitivity and IL-6 and IL-10 were candidate biomarkers for airway-lesion dominant COPD.

Evaluation of a new fiber-grating vision sensor for assessing pulmonary functions in healthy and COPD subjects

Spirometry is practically the only tool to evaluate pulmonary functions. Other automatic systems comparable to spirometry are expected. A fiber-grating (FG) vision sensor is a non-contact respiratory monitoring system to detect changes in volumes by measuring the movement of laser spots on the body surface. We examined the contributions of the FG sensor to evaluating pulmonary functions. The FG sensor showed a linear correlation with spirometry in tidal volumes (TV) obtained from five controls (R = 0.98, P < 0.0001). We also showed agreement of TV between the two devices using Bland-Altman analysis. TV measured by the FG sensor were reproducible and applicable to distinct subjects. To detect airway obstruction, we performed forced expiration in controls (n = 16) and chronic obstructive pulmonary disease (COPD) patients (n = 18) with the FG sensor and spirometry. Forced expiratory volume in 1 s (FEV(1)) and FEV(1)/forced vital capacity in COPD patients were lower than those in controls by the FG sensor. In addition, prolonged expiration in natural breathing by the FG sensor was related to airflow limitation by spirometry. The FG sensor was helpful to measure volume changes and to evaluate pulmonary functions in controls and patients with COPD. Its upcoming clinical applications are promising for simplicity and feasibility.

Polymorphonuclear Neutrophils Move into the Fast Lane in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal inflammatory response of the lung to inhaled noxious particles and gases. The main risk factor for COPD in developed countries is cigarette smoke exposure. However, for unclear reasons not all smokers develop COPD. One of the first inflammatory cell types implicated in the pathogenesis of COPD was the polymorphonuclear neutrophil (PMN). PMNs and their products were linked to COPD in the 1960s when severe early-onset emphysema was found to be associated with inherited deficiency of α1-antitrypsin (AAT) (1), which is an important physiologic inhibitor of the PMN-derived proteinase neutrophil elastase (NE), in the lower respiratory tract. Subsequent studies showed that PMNs are the most abundant inflammatory cell type present in the airways and lung parenchyma of patients with COPD. In addition, airway PMN counts were shown to correlate positively with disease progression and their number increases further during acute exacerbations (2). PMNs contribute to the pathogenesis of COPD by releasing proteinases (especially NE), reactive oxygen species, and proinflammatory mediators that promote lung inflammation and destruction in COPD. n nAlthough most studies have focused on enumerating PMNs or their products in the lung and airways of human patients with COPD and experimental animals with emphysema, studies dating back to the late 1980s reported that circulating PMNs in patients with COPD have functional abnormalities that could potentially contribute to COPD pathogenesis. These studies reported that smokers and patients with COPD have increased lung retention of PMNs due to increased PMN margination and reduced PMN deformability within the pulmonary vasculature, and this process increases during acute exacerbations of COPD (3, 4). It has been shown that PMNs from patients with COPD have increased extracellular proteolytic activity and increased capacity to migrate to formyl-methionyl-leucyl-phenylalanine (fMLP) in Boyden Chamber assays when compared with cells from healthy smokers and nonsmokers (5). Other studies reported that PMNs from patients with COPD have dysregulated levels of cell adhesion molecules, chemokine receptors, and intracellular tyrosine kinases that participate in PMN adhesion and migration (6, 7). However, the abnormalities in PMN migratory responses were not fully characterized and the aberrant signaling pathways responsible for them were not identified in these earlier studies. n nThe article by Sapey and coworkers in this issue of the Journal (pp. 1176–1186) builds upon this prior literature by using novel technologies to more completely characterize the abnormal migratory responses of PMNs from patients with COPD (8). This article also provides novel insights into the aberrant intracellular signaling pathway that contributes to the abnormal migratory responses of PMNs from patients with COPD. The authors used shallow chemotaxis gradients in modified Dunn Chambers and time lapse video microscopy to compare the real-time migratory velocity, migration accuracy, and morphology of PMNs from patients with COPD with normal plasma levels of AAT (“common garden variety” COPD) to that of cells from age-matched healthy nonsmokers and healthy smokers to various chemoattractants in vitro. The authors also studied cells from patients with AAT deficiency having similar airflow obstruction and smoking histories and taking similar medications (including inhaled corticosteroids) as the patients with COPD to control for the potentially confounding effects of these variables on PMN function. They showed that PMNs from patients with “common garden variety” COPD migrated with much greater speed than cells from the control groups, but the migration accuracy of COPD PMNs to chemoattractant gradients was impaired. The PMNs of patients with COPD took more circuitous and less efficient routes toward the stimuli. This migratory abnormality was associated with reduced PMN pseudopodia formation (reflective of the reduced migratory accuracy) but normal expression of key chemokine receptors, including CXCR1, CXCR2, and the fMLP receptor, FPR1. This PMN migration defect did not occur in patients with AAT deficiency having similar pulmonary function as the COPD group. Surprisingly, the migratory abnormalities of COPD PMNs did not correlate with disease severity (as assessed by GOLD stage) and were present even in patients with GOLD 1 stage disease. The authors implicated abnormal phosphatidyl inositol-3-kinase (PI3K) signaling in the abnormal migration pattern of PMNs from patients with COPD since incubating COPD PMNs in vitro with low concentrations (1 nM) of a synthetic PI3K inhibitor (Ly294002) reversed the migratory defect in COPD cells but had no effect on the migratory responses of PMNs from the other subject groups. The authors concluded that in patients with COPD there is excessive but highly inaccurate migration of PMNs to chemotactic stimuli, which may not only hinder clearance of bacteria from the lung but may also promote excessive lung destruction in patients with COPD. This latter concept is supported by observations that when chemoattractants activate PMNs to migrate, PMNs translocate serine proteinases and matrix metalloproteinases from their intracellular granules to the cell surface in proteinase inhibitor–resistant forms and also release high concentrations of proteinases into the pericellular environment; these proteinases transiently overwhelm the local proteinase inhibitor defense (9–11). n nThe concept that excessive and inaccurate PMN migration is linked to COPD pathogenesis and might be amenable to therapeutic intervention is novel. One limitation of the article is that the authors implicated the PI3K signaling pathway in this migratory defect exclusively by testing the efficacy of a synthetic PI3K inhibitor on PMN migration in vitro (8). PI3Ks are a family of proteins that catalyze the phosphorylation of the 3-OH position of phosphoinositides and generate lipids that control a wide variety of intracellular signaling pathways involved in cell growth, proliferation, motility, and survival. PMNs express several PI3K isoforms, including PI3Kδ and PI3Kγ, which have different biological activities. Not only is LY294002 not selective for different PI3K isoforms, it also has activities on other lipid kinases and other unrelated proteins such as NF-κB, which is activated in leukocytes in patients with COPD (12). PI3K-independent motility responses have also been reported in human PMNs (13). Future studies could confirm that the PI3K pathways are contributing to the migratory defect observed in COPD PMNs and identify the isoforms involved. n nIt is also still unclear whether the PMN migratory defect identified in this study occurs exclusively in patients with COPD, since patients with lung diseases other than COPD and AAT deficiency were not studied (8). In this respect it is noteworthy that stimulated PMNs from both patients with COPD and patients with asthma release increased amounts of reactive oxygen species when compared with cells from healthy subjects (14). Furthermore, PMNs from patients with respiratory diseases other than AAT deficiency should be studied in light of a recent finding that PMNs from AAT-deficient patients have abnormal chemotactic responses due, in part, to loss of AAT-mediated regulation of the biological activity of IL-8 (15). It is not clear whether the defect in COPD PMN migration responses to chemoattractants is a primary defect in PMN function in a subset of smokers at risk from progression to COPD or secondary to systemic abnormalities occurring in COPD, since patients with COPD have increased circulating levels of regulators of leukocyte function such as TNF-α, IL-8, IL-1β, and IL-6 (16). However, the lack of correlation between COPD severity and the PMN migratory defect argues against the latter possibility (8). The contribution of the excessive and inaccurate migration of COPD PMNs to COPD progression also is not clear. Patients with COPD have other PMN functional abnormalities, including defective phagocytosis of bacteria (17) and increased production of oxidants (14), and also have functional abnormalities in other leukocytes germane to COPD pathogenesis, including macrophages and T lymphocytes (18, 19). Nevertheless, the study by Sapey and colleagues confirms the utility of studying the functional characteristics of peripheral blood PMNs as a surrogate for airway and lung PMNs. n nThe study by Sapey and coworkers (8) is timely, since it identifies PI3K inhibition as a novel therapeutic strategy for COPD. Components of the PI3K pathway play a crucial role in the expression of inflammatory mediators, inflammatory cell recruitment, and immune cell function in COPD (20). In addition, the PI3K pathway has been successfully targeted to reverse corticosteroid insensitivity in leukocytes from patients with COPD and in the lungs of experimental animals with COPD (21, 22). The study by Sapey and colleagues thus adds weight to the argument that selective PI3K inhibition might have antiinflammatory potential in patients with COPD.