David I. Kasahara

Chronic Exposure to Ambient Levels of Urban Particles Affects Mouse Lung Development

RATIONALE Chronic exposure to air pollution has been associated with adverse effects on childrens lung growth. OBJECTIVES We analyzed the effects of chronic exposure to urban levels of particulate matter (PM) on selected phases of mouse lung development. METHODS The exposure occurred in two open-top chambers (filtered and nonfiltered) placed 20 m from a street with heavy traffic in São Paulo, 24 hours/day for 8 months. There was a significant reduction of the levels of PM(2.5) inside the filtered chamber (filtered = 2.9 +/- 3.0 microg/m(3), nonfiltered = 16.8 +/- 8.3 microg/m(3); P = 0.001). At this exposure site, vehicular sources are the major components of PM(2.5) (PM <or= 2.5 microm). Exposure of the parental generation in the two chambers occurred from the 10th to the 120th days of life. After mating and birth of offspring, a crossover of mothers and pups occurred within the chambers, resulting in four groups of pups: nonexposed, prenatal, postnatal, and pre+postnatal. Offspring were killed at the age of 15 (n = 42) and 90 (n = 35) days; lungs were analyzed by morphometry for surface to volume ratio (as an estimator of alveolization). Pressure-volume curves were performed in the older groups, using a 20-ml plethysmograph. MEASUREMENTS AND MAIN RESULTS Mice exposed to PM(2.5) pre+postnatally presented a smaller surface to volume ratio when compared with nonexposed animals (P = 0.036). The pre+postnatal group presented reduced inspiratory and expiratory volumes at higher levels of transpulmonary pressure (P = 0.001). There were no differences among prenatal and postnatal exposure and nonexposed animals. CONCLUSIONS Our data provide anatomical and functional support to the concept that chronic exposure to urban PM affects lung growth.

ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli

Activation of the NADPH oxidase homolog dual oxidase 1 (DUOX1) within the airway epithelium represents a key mechanism of innate airway host defense, through enhanced production of H2O2, which mediates cellular signaling pathways that regulate the production of various inflammatory mediators. Production of the CXC chemokine interleukin (IL)-8/CXCL8 forms a common epithelial response to many diverse stimuli, including bacterial and viral triggers, environmental oxidants, and other biological mediators, suggesting the potential involvement of a common signaling pathway that may involve DUOX1-dependent H2O2 production. Following previous reports showing that DUOX1 is activated by extracellular ATP and purinergic receptor stimulation, this study demonstrates that airway epithelial IL-8 production in response to several bacterial stimuli involves ATP release and DUOX1 activation. ATP-mediated DUOX1 activation resulted in the activation of ERK1/2 and NF-κB pathways, which was associated with epidermal growth factor receptor (EGFR) ligand shedding by ADAM17 (a disintegrin and metalloproteinase-17). Although ATP-mediated ADAM17 activation and IL-8 release were not prevented by extracellular H2O2 scavenging by catalase, these responses were attenuated by intracellular scavengers of H2O2 or related oxidants, suggesting an intracellular redox signaling mechanism. Both ADAM17 activation and IL-8 release were suppressed by inhibitors of EGFR/ERK1/2 signaling, which can regulate ADAM17 activity by serine/threonine phosphorylation. Collectively, our results indicate that ATP-mediated DUOX1 activation represents a common response mechanism to several environmental stimuli, involving H2O2-dependent EGFR/ERK activation, ADAM17 activation, and EGFR ligand shedding, leading to amplified epithelial EGFR activation and IL-8 production.

Inhibition of Arginase Activity Enhances Inflammation in Mice with Allergic Airway Disease, in Association with Increases in Protein S-Nitrosylation and Tyrosine Nitration

Pulmonary inflammation in asthma is orchestrated by the activity of NF-κB. NO and NO synthase (NOS) activity are important modulators of inflammation. The availability of the NOS substrate, l-arginine, is one of the mechanisms that controls the activity of NOS. Arginase also uses l-arginine as its substrate, and arginase-1 expression is highly induced in a murine model of asthma. Because we have previously described that arginase affects NOx content and interferes with the activation of NF-κB in lung epithelial cells, the goal of this study was to investigate the impact of arginase inhibition on the bioavailability of NO and the implications for NF-κB activation and inflammation in a mouse model of allergic airway disease. Administration of the arginase inhibitor BEC (S-(2-boronoethyl)-l-cysteine) decreased arginase activity and caused alterations in NO homeostasis, which were reflected by increases in S-nitrosylated and nitrated proteins in the lungs from inflamed mice. In contrast to our expectations, BEC enhanced perivascular and peribronchiolar lung inflammation, mucus metaplasia, NF-κB DNA binding, and mRNA expression of the NF-κB-driven chemokine genes CCL20 and KC, and lead to further increases in airways hyperresponsiveness. These results suggest that inhibition of arginase activity enhanced a variety of parameters relevant to allergic airways disease, possibly by altering NO homeostasis.

Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40–100 nm and less than 44 μm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 μm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 μM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Comparison of glucocorticoid and cysteinyl leukotriene receptor antagonist treatments in an experimental model of chronic airway inflammation in guinea‐pigs

Background Leukotriene receptor antagonists have been demonstrated in several studies to possess bronchodilating and anti‐inflammatory properties in asthma. However, there are few experimental studies performed to compare the effects of anti‐leukotrienes and glucocorticoids, most used anti‐inflammatory agents in asthma. In the present study, we evaluated the effects of treatment with dexamethasone or montelukast on eosinophil and mononuclear cell recruitment in an experimental model of allergen‐induced chronic airway inflammation in guinea‐pigs (GP).

Pulmonary Inflammation Induced by Subacute Ozone Is Augmented in Adiponectin-Deficient Mice: Role of IL-17A

Pulmonary responses to ozone, a common air pollutant, are augmented in obese individuals. Adiponectin, an adipose-derived hormone that declines in obesity, has regulatory effects on the immune system. To determine the role of adiponectin in the pulmonary inflammation induced by extended (48–72 h) low-dose (0.3 parts per million) exposure to ozone, adiponectin-deficient (Adipo−/−) and wild-type mice were exposed to ozone or to room air. In wild-type mice, ozone exposure increased total bronchoalveolar lavage (BAL) adiponectin. Ozone-induced lung inflammation, including increases in BAL neutrophils, protein (an index of lung injury), IL-6, keratinocyte-derived chemokine, LPS-induced CXC chemokine, and G-CSF were augmented in Adipo−/− versus wild-type mice. Ozone also increased IL-17A mRNA expression to a greater extent in Adipo−/− versus wild-type mice. Moreover, compared with control Ab, anti–IL-17A Ab attenuated ozone-induced increases in BAL neutrophils and G-CSF in Adipo−/− but not in wild-type mice, suggesting that IL-17A, by promoting G-CSF release, contributed to augmented neutrophilia in Adipo−/− mice. Flow cytometric analysis of lung cells revealed that the number of CD45+/F4/80+/IL-17A+ macrophages and γδ T cells expressing IL-17A increased after ozone exposure in wild-type mice and further increased in Adipo−/− mice. The IL-17+ macrophages were CD11c− (interstitial macrophages), whereas CD11c+ macrophages (alveolar macrophages) did not express IL-17A. Taken together, the data are consistent with the hypothesis that adiponectin protects against neutrophil recruitment induced by extended low-dose ozone exposure by inhibiting the induction and/or recruitment of IL-17A in interstitial macrophages and/or γδ T cells.

Impact of Adiponectin Deficiency on Pulmonary Responses to Acute Ozone Exposure in Mice

Obese mice have increased responses to acute ozone (O(3)) exposure. T-cadherin is a binding protein for the high-molecular weight isoforms of adiponectin, an anti-inflammatory hormone that declines in obesity. The objective of the present study was to determine whether adiponectin affects pulmonary responses to O(3), and whether these effects are mediated through T-cadherin. We performed bronchoalveolar lavage (BAL) and measured pulmonary responsiveness to methacholine after acute air or O(3) exposure (2 ppm for 3 h) in adiponectin-deficient (Adipo(-/-)) or T-cadherin-deficient (T-Cad(-/-)) mice. O(3) increased pulmonary responses to methacholine and increased BAL neutrophils and protein to a greater extent in wild-type than in Adipo(-/-) mice, whereas T-cadherin deficiency had no effect. O(3)-induced increases in BAL IL-6 and keratinocyte-derived chemokine (KC), which contribute to O(3)-induced pulmonary neutrophilia, were also greater in wild-type than in Adipo(-/-) mice. In contrast, responses to O(3) were not altered by transgenic overexpression of adiponectin. To determine which adiponectin isoforms are present in the lung, Western blotting was performed. The hexameric isoform of adiponectin dominated in serum, whereas BAL was dominated by the high-molecular weight isoform of adiponectin. Interestingly, serum adiponectin was greater in T-Cad(-/-) versus wild-type mice, whereas BAL adiponectin was lower in T-Cad(-/-) versus wild-type mice, suggesting that T-cadherin may be important for transit of high-molecular weight adiponectin from the blood to the lung. Our results indicate that adiponectin deficiency inhibits pulmonary inflammation induced by acute O(3) exposure, and that T-cadherin does not mediate the effects of adiponectin responsible for these events.

Acrolein Inhalation Suppresses Lipopolysaccharide-Induced Inflammatory Cytokine Production but Does Not Affect Acute Airways Neutrophilia

Acrolein is a reactive unsaturated aldehyde that is produced during endogenous oxidative processes and is a major bioactive component of environmental pollutants such as cigarette smoke. Because in vitro studies demonstrate that acrolein can inhibit neutrophil apoptosis, we evaluated the effects of in vivo acrolein exposure on acute lung inflammation induced by LPS. Male C57BL/6J mice received 300 μg/kg intratracheal LPS and were exposed to acrolein (5 parts per million, 6 h/day), either before or after LPS challenge. Exposure to acrolein either before or after LPS challenge did not significantly affect the overall extent of LPS-induced lung inflammation, or the duration of the inflammatory response, as observed from recovered lung lavage leukocytes and histology. However, exposure to acrolein after LPS instillation markedly diminished the LPS-induced production of several inflammatory cytokines, specifically TNF-α, IL-12, and the Th1 cytokine IFN-γ, which was associated with reduction in NF-κB activation. Our data demonstrate that acrolein exposure suppresses LPS-induced Th1 cytokine responses without affecting acute neutrophilia. Disruption of cytokine signaling by acrolein may represent a mechanism by which smoking contributes to chronic disease in chronic obstructive pulmonary disease and asthma.

Effects of neurokinins on airway and alveolar eosinophil recruitment.

The authors assessed the role of substance P (SP) and neurokinin A (NKA) and their receptor antagonists (RAs) SR140333 and SR48968 (respectively for NK 1 and NK 2 receptors) in pulmonary eosinophil influx induced by stimulation of capsaicin (CAP)-sensitive nerve terminals. The increase in respiratory system resistance after capsaicin infusion was attenuated by NK 2 RA and association of NK 1 NK 2 RA (P<.001). Respiratory system elastance (Ers) increase was attenuated with use of NK 1 NK 2 RA (P<.001). In alveolar wall, there was an increase in eosinophils after 30 minutes of CAP infusion (P<.001) and was attenuated after 24 hours. Pretreatment with NK 1 RA, NK 2 RA, and NK 1 NK 2 RA decreased eosinophils in alveolar wall (P<.001). SP induced an increase of eosinophils in alveolar wall (P<.001), although NKA may also contribute to this response. In airway wall, the authors observed an increase of eosinophils at 30 minutes (P=.006) till 24 hours after CAP infusion. They noticed a predominant influx of cells around airway wall after CAP and SP infusion. Pretreatment with NK 1 RA and NK 1 NK 2 RA reduced eosinophils (P<.001) in airway wall. Both SP and NKA contribute to eosinophil lung recruitment in distal airways and in alveolar wall, and these findings suggest that neurokinins may contribute to the development of eosinophilic inflammation in both allergic asthma and hypersensitivity pneumonitis. capsaicin lung inflammation neurokinin A receptor antagonists substance P

Pulmonary responses to subacute ozone exposure in obese vs. lean mice.

The purpose of this study was to determine whether obesity affects pulmonary responses following a 3-day ozone exposure. Obese db/db and lean wild-type mice were exposed to ozone (0.3 ppm) for 72 h. In wild-type mice, ozone exposure caused pulmonary injury and inflammation, and these events were associated with reduced pulmonary compliance. In db/db mice, ozone-induced neutrophil recruitment to the lung was reduced and no reduction in compliance was observed. Similar results were obtained in obese Cpe(fat) mice, indicating that loss of leptin signaling in db/db mice does not account for these obesity-related changes. To examine the role of interleukin (IL)-6 in this obesity-related difference in ozone responsiveness, wild-type and IL-6-deficient mice were raised on 10% or 60% fat diets. Compared with 10% fat-fed mice, wild-type 60% fat-fed mice were obese and had reduced neutrophil recruitment following ozone. IL-6 deficiency reduced ozone-induced neutrophil recruitment in 10% fat-fed mice. In contrast, in obese mice, no effect of IL-6 deficiency on neutrophil recruitment was observed. Obesity-related differences in the effect of ozone on compliance were observed in both wild-type and IL-6-deficient mice. Obesity-related differences in serum IL-6 were observed and may account for obesity-related differences in the effect of IL-6 deficiency on neutrophil recruitment. In summary, the neutrophilic inflammation induced by prolonged low level ozone exposure was attenuated in obese mice and appeared to result from an absence of IL-6-dependent neutrophil recruitment in the obese mice.