Rationale: Ozone is a common environmental air pollutant that contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced airway hyperresponsiveness, remain poorly understood. We have previously reported that toll-like receptor 4 (TLR4)–deficient animals are protected against ozone-induced airway hyperresponsiveness (AHR) and that hyaluronan (HA) mediates ozone-induced AHR. However, the relation between TLR4 and hyaluronan in the airway response to ozone remains unexplored.
Objectives: We hypothesized that HA acts as an endogenous TLR4 ligand for the development of AHR after ozone-induced environmental airway injury.
Methods: TLR4-deficient and wild-type C57BL/6 mice were exposed to either inhaled ozone or intratracheal HA and the inflammatory and AHR response was measured.
Measurements and Main Results: TLR4-deficient mice have similar levels of cellular inflammation, lung injury, and soluble HA levels as those of C57BL/6 mice after inhaled ozone exposure. However, TLR4-deficient mice are partially protected from AHR after ozone exposure as well as after direct intratracheal instillation of endotoxin-free low molecular weight HA. Similar patterns of TLR4-dependent cytokines were observed in the bronchial alveolar lavage fluid after exposure to either ozone or HA. Exposure to ozone increased immunohistological staining of TLR4 on lung macrophages. Furthermore, in vitro HA exposure of bone marrow–derived macrophages induced NF-κB and production of a similar pattern of proinflammatory cytokines in a manner dependent on TLR4.
Conclusions: Our observations support the observation that extracellular matrix HA contributes to ozone-induced airways disease. Furthermore, our results support that TLR4 contributes to the biological response to HA by mediating both the production of proinflammatory cytokines and the development of ozone-induced AHR.
environmental airways injury; asthma; toll-like receptor; macrophage; TNF-α
Ozone is an environmentally reactive oxidant, and pycnogenol is a mixture of flavonoid compounds extracted from pine tree bark that have antioxidant activity. We investigated the effects of pycnogenol on reactive nitrogen species, antioxidant responses, and airway responsiveness in BALB/c mice exposed to ozone.
Antioxidant levels were determined using high performance liquid chromatography with electrochemical detection. Nitric oxide (NO) metabolites in bronchoalveolar lavage (BAL) fluid from BALB/c mice in filtered air and 2 ppm ozone with pycnogenol pretreatment before ozone exposure (n = 6) were quantified colorimetrically using the Griess reaction.
Uric acid and ascorbic acid concentrations were significantly higher in BAL fluid following pretreatment with pycnogenol, whereas γ-tocopherol concentrations were higher in the ozone exposed group but were similar in the ozone and pycnogenol pretreatment groups. Retinol and γ-tocopherol concentrations tended to increase in the ozone exposure group but were similar in the ozone and pycnogenol pretreatment groups following ozone exposure. Malonylaldehyde concentrations increased in the ozone exposure group but were similar in the ozone and pycnogenol plus ozone groups. The nitrite and total NO metabolite concentrations in BAL fluid, which parallel the in vivo generation of NO in the airways, were significantly greater in the ozone exposed group than the group exposed to filtered air, but decreased with pycnogenol pretreatment.
Pycnogenol may increase levels of antioxidant enzymes and decrease levels of nitrogen species, suggesting that antioxidants minimize the effects of acute ozone exposure via a protective mechanism.
Antioxidants; Nitric oxide; Reactive nitrogen species; Ozone
Exposure to ozone induces airway hyperresponsiveness (AHR) mediated partly by SP released from nerve terminals of intrinsic airway neurons. Our recent studies showed that IL-1, an important multifunctional proinflammatory cytokine, increases synthesis and release of SP from intrinsic airway neurons. The purpose of this study is to investigate the possible involvement of endogenous IL-1 in modulating neural responses associated with ozone-enhanced airway responsiveness. Ferrets were exposed to 2 ppm ozone or filtered air for 3 hrs. IL-1 in the bronchoalveolar lavage (BAL) fluid was significantly increased in ozone-exposed animals and responses of tracheal smooth muscle to methacholine (MCh) and electrical field stimulation (EFS) were elevated significantly. Both the SP nerve fiber density in tracheal smooth muscle and the number of SP-containing neurons in airway ganglia were significantly increased following ozone exposure. Pretreatment with IL-1 receptor antagonist (IL-1 Ra) significantly diminished ozone-enhanced airway responses to EFS as well as ozone-increased SP in the airway. To selectively investigate intrinsic airway neurons, segments of ferret trachea were maintained in culture conditions for 24 hrs to eliminate extrinsic contributions from sensory nerves. The segments were then exposed to 2 ppm ozone in vitro for 3 hrs. The changes of ozone-induced airway responses to MCh and EFS, and the SP levels in airway neurons paralleled those observed with in vivo ozone exposure. The ozone-enhanced airway responses and neuronal SP levels were inhibited by pretreatment with IL-1 Ra. These findings show that IL-1 is released during ozone exposure enhances airway responsiveness by modulating SP expression in airway neurons.
airway smooth muscle contraction; muscarinic agonists; neurokinin receptor; airway innervation
Acute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5'-AMP-activated kinase (AMPK), which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.
Control and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance (AFC) was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage (BAL) and lung homogenates (LH). Levels of AMPK-Thr172 phosphorylation and basolateral membrane Na(+)-K(+)-ATPase abundance were determined by Western blot.
In control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels (3 vs 4.4 nM, p = 0.02) and malondialdehyde concentrations (110 vs 230 μmole/g wet tissue) were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr172 to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na(+)-K(+)-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.
Our results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.
Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR). Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease.
We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh) to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN-γ in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy.
The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF-γ increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks.
These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR.
IL-17A induces the release of pro-inflammatory cytokines and of reactive oxygen species which could lead to neutrophilic inflammation. We determined the role of IL-17 receptor (IL-17R) signalling in oxidant-induced lung emphysema and airway hyperresponsiveness. IL-17R−/− and wild-type C57/BL6 mice were exposed to ozone (3 ppm; 3 hours) for 12 times over 6 weeks. Bronchial responsiveness to acetylcholine was measured, and lungs were retrieved. Mean linear intercept (Lm) and isometric contractile responses of intrapulmonary airways to acetylcholine were determined. In wild-type mice but not in IL-17R−/−, chronic ozone exposure caused airway hyperresponsiveness. The increase in Lm after chronic ozone exposure of wild-type mice was also observed in IL-17R−/− mice. The increased maximal contractile response to acetylcholine seen in airways of wild-type mice exposed to ozone was abolished in IL-17R−/− mice. p38-mitogen-activated protein kinase (MAPK) and dexamethasone-dependent increase in contractile response was reduced in airways from IL-17R−/− ozone-exposed mice. Lung inflammation scores were not altered in IL-17R−/− mice exposed to ozone compared to wild-type mice. The increased release of IL-17 and IL-1β, and the activation of p38 MAPK in the lungs of ozone-exposed mice was reduced in IL-17R−/− mice. IL-17R signalling underlies the increase in airway hyperresponsiveness seen after ozone exposure, mediated by the increased contractility of airway smooth muscle. The emphysema and lung inflammation induced by ozone is not dependent on IL-17.
Background: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored.
Objectives: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone.
Methods: We exposed C57BL/6J and mindin-deficient (–/–) mice to aerosolized LPS (0.9 μg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline
exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies.
Results: After inhalation of LPS, mindin–/– mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin–/– mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin–/– mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin–/– mice demonstrated reduced constriction in response to carbachol.
Conclusions: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of mindin in airway smooth muscle contractility after exposure to ozone.
airway smooth muscle; endotoxin; innate immunity; lipopolysaccharide; LPS; lung; mindin; ozone; Tlr4; toll-like receptor
Ozone exposure is associated with exacerbation of reactive airways disease. We have previously reported that the damage-associated molecular pattern, hyaluronan, is required for the complete biological response to ambient ozone and that hyaluronan fragments signal through toll-like receptor 4 (TLR4). In this study, we further investigated the role of TLR4 adaptors in ozone–induced airway hyperresponsiveness (AHR) and the direct response to hyaluronan fragments (HA). Using a murine model of AHR, C57BL/6J, TLR4−/−, MyD88−/−, and TIRAP−/− mice were characterized for AHR after exposure to either ozone (1 ppm×3 h) or HA fragments. Animals were characterized for AHR with methacholine challenge, cellular inflammation, lung injury, and production of pro-inflammatory cytokines. Ozone-exposed C57BL/6J mice developed cellular inflammation, lung injury, pro-inflammatory cytokines, and AHR, while mice deficient in TLR4, MyD88 or TIRAP demonstrated both reduced AHR and reduced levels of pro-inflammatory cytokines including TNFα, IL-1β, MCP-1, IL-6 and KC. The level of hyaluronan was increased after inhalation of ozone in each strain of mice. Direct challenge of mice to hyaluronan resulted in AHR in C57BL/6J mice, but not in TLR4−/−, MyD88−/−, or TIRAP−/− mice. HA-induced cytokine production in wild-type mice was significantly reduced in TLR4−/−, MyD88−/−, or TIRAP−/− mice. In conclusion, our findings support that ozone-induced airway hyperresponsiveness is dependent on the HA-TLR4-MyD88-TIRAP signaling pathway.
Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A+ CD45+ cells in the lungs and these effects were abolished in TCRδ−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A+ CD45+ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A+ γδ T cells. Il17a mRNA and IL-17A+ γδ T cells were also lower in obese Cpefat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A+ γδ T cells to the lung.
Surfactant protein–D (Sftpd) is a pulmonary collectin important in down-regulating macrophage inflammatory responses. In these experiments, we analyzed the effects of chronic macrophage inflammation attributable to loss of Sftpd on the persistence of ozone-induced injury, macrophage activation, and altered functioning in the lung. Wild-type (Sftpd+/+) and Sftpd−/− mice (aged 8 wk) were exposed to air or ozone (0.8 parts per million, 3 h). Bronchoalveolar lavage (BAL) fluid and tissue were collected 72 hours later. In Sftpd−/− mice, but not Sftpd+/+ mice, increased BAL protein and nitrogen oxides were observed after ozone inhalation, indicating prolonged lung injury and oxidative stress. Increased numbers of macrophages were also present in BAL fluid and in histologic sections from Sftpd−/− mice. These cells were enlarged and foamy, suggesting that they were activated. This conclusion was supported by findings of increased BAL chemotactic activity, and increased expression of inducible nitric oxide synthase in lung macrophages. In both Sftpd+/+ and Sftpd−/− mice, inhalation of ozone was associated with functional alterations in the lung. Although these alterations were limited to central airway mechanics in Sftpd+/+ mice, both central airway and parenchymal mechanics were modified by ozone exposure in Sftpd−/− mice. The most notable changes were evident in resistance and elastance spectra and baseline lung function, and in lung responsiveness to changes in positive end-expiratory pressure. These data demonstrate that a loss of Sftpd is associated with prolonged lung injury, oxidative stress, and macrophage accumulation and activation in response to ozone, and with more extensive functional changes consistent with the loss of parenchymal integrity.
ozone; surfactant protein–D; macrophages; iNOS; lung function
Exposure to ozone, which is a major component of air pollution, induces a form of asthma that occurs in the absence of adaptive immunity. Although ozone-induced asthma is characterized by airway neutrophilia, and not eosinophilia, it is nevertheless associated with airway hyperreactivity (AHR), which is a cardinal feature of asthma. Because AHR induced by allergens requires the presence of natural killer T (NKT) cells, we asked whether ozone-induced AHR had similar requirements. We found that repeated exposure of wild-type (WT) mice to ozone induced severe AHR associated with an increase in airway NKT cells, neutrophils, and macrophages. Surprisingly, NKT cell–deficient (CD1d−/− and Jα18−/−) mice failed to develop ozone-induced AHR. Further, treatment of WT mice with an anti-CD1d mAb blocked NKT cell activation and prevented ozone-induced AHR. Moreover, ozone-induced, but not allergen-induced, AHR was associated with NKT cells producing interleukin (IL)-17, and failed to occur in IL-17−/− mice nor in WT mice treated with anti–IL-17 mAb. Thus, ozone exposure induces AHR that requires the presence of NKT cells and IL-17 production. Because NKT cells are required for the development of two very disparate forms of AHR (ozone- and allergen-induced), our results strongly suggest that NKT cells mediate a unifying pathogenic mechanism for several distinct forms of asthma, and represent a unique target for effective asthma therapy.
Ozone exposure causes airway hyperreactivity and increases hospitalizations resulting from pulmonary complications. Ozone reacts with the epithelial lining fluid and airway epithelium to produce reactive oxygen species and lipid peroxidation products, which then activate cell signaling pathways, including the mitogen activated protein kinase (MAPK) pathway. Both p38 and c-Jun NH2 terminal kinase (JNK) are MAPK family members that are activated by cellular stress and inflammation. To test the contribution of both p38 and JNK MAPK to ozone-induced airway hyperreactivity, guinea pigs were pretreated with dual p38 and JNK MAPK inhibitors (30 mg/kg, ip) 60 minutes before exposure to 2 ppm ozone or filtered air for 4 hours. One day later airway reactivity was measured in anesthetized animals. Ozone caused airway hyperreactivity one day post-exposure, and blocking p38 and JNK MAPK completely prevented ozone-induced airway hyperreactivity. Blocking p38 and JNK MAPK also suppressed parasympathetic nerve activity in air exposed animals, suggesting p38 and JNK MAPK contribute to acetylcholine release by airway parasympathetic nerves. Ozone inhibited neuronal M2 muscarinic receptors and blocking both p38 and JNK prevented M2 receptor dysfunction. Neutrophil influx into bronchoalveolar lavage was not affected by MAPK inhibitors. Thus p38 and JNK MAPK mediate ozone-induced airway hyperreactivity through multiple mechanisms including prevention of neuronal M2 receptor dysfunction.
Hyaluronan is a high molecular weight component of pulmonary extracelluar matrix and lung injury can generate low molecular weight hyaluronan fragment (HA) that functions as endogenous ligand to cell surface receptors CD44 and toll like receptor 4 (TLR4). This leads to activation of intracellular NFκB signaling and pro-inflammatory cytokine production. Based on previous information that ozone exposure causes increased HA in bronchial alveolar lavage fluid (BALF) and ozone pre-exposure primes immune response to inhaled LPS, we hypothesized that HA production during ozone exposure augments the inflammatory response to LPS. We demonstrate that acute ozone exposure at 1ppm for 3 hours primes the immune response to low dose aerosolized LPS in C57BL/6J mice, resulting in increased neutrophil recruitment into the airspaces, increased levels of protein and pro-inflammatory cytokines in the BALF, and increased airway hyperresponsiveness (AHR). Intratracheal instillation of endotoxin-free HA (25 μg) enhances the biological response to inhaled LPS in a manner similar to ozone pre-exposure. In vitro studies using bone marrow-derived macrophages indicate that HA enhances LPS responses measured by TNFα production while immunofluorescence staining of murine alveolar macrophages demonstrates that HA induces TLR4 peripheralization and lipid raft co-localization. Collectively, our observations support that ozone primes macrophage responsiveness to low dose LPS, in part, due to hyaluronan-induced TLR4 peripheralization in lung macrophages.
environmental; ozone; lipopolysaccharide; hyaluronan; TLR4; toll-like receptor; innate immunity; macrophage; lung injury
The functional role of nitric oxide (NO) and various nitric oxide synthase (NOS) isoforms in asthma remains unclear.
This study investigated the effects of ozone and ovalbumin (OVA) exposure on NOS isoforms.
The expression of inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) in lung tissue was measured. Enhanced pause (Penh) was measured as a marker of airway obstruction. Nitrate and nitrite in bronchoalveolar lavage (BAL) fluid were measured using a modified Griess reaction.
The nitrate concentration in BAL fluid from the OVA-sensitized/ozone-exposed/OVA-challenged group was greater than that of the OVA-sensitized/saline-challenged group. Methacholine-induced Penh was increased in the OVA-sensitized/ozone-exposed/OVA-challenged group, with a shift in the dose-response curve to the left, compared with the OVA-sensitized/saline-challenged group. The levels of nNOS and eNOS were increased significantly in the OVA-sensitized/ozone-exposed/OVA-challenged group and the iNOS levels were reduced compared with the OVA-sensitized/saline-challenged group.
In mice, ozone is associated with increases in lung eNOS and nNOS, and decreases in iNOS. None of these enzymes are further affected by allergens, suggesting that the NOS isoforms play different roles in airway inflammation after ozone exposure.
Nitric oxide synthase; Ozone; Asthma
Background: The role of the Nlrp3 inflammasome in nonallergic airway hyperresponsiveness (AHR) has not previously been reported. Recent evidence supports both interleukin (IL) 1β and short fragments of hyaluronan (HA) as contributors to the biological response to inhaled ozone.
Objective: Because extracellular secretion of IL-1β requires activation of the inflammasome, we investigated the role of the inflammasome proteins ASC, caspase1, and Nlrp3 in the biological response to ozone and HA.
Methods: C57BL/6J wild-type mice and mice deficient in ASC, caspase1, or Nlrp3 were exposed to ozone (1 ppm for 3 hr) or HA followed by analysis of airway resistance, cellular inflammation, and total protein and cytokines in bronchoalveolar lavage fluid (BALF). Transcription levels of IL-1β and IL-18 were determined in two populations of lung macrophages. In addition, we examined levels of cleaved caspase1 and cleaved IL-1β as markers of inflammasome activation in isolated alveolar macrophages harvested from BALF from HA-treated mice.
Results: We observed that genes of the Nlrp3 inflammasome were required for development of AHR following exposure to either ozone or HA fragments. These genes are partially required for the cellular inflammatory response to ozone. The expression of IL-1β mRNA in alveolar macrophages was up-regulated after either ozone or HA challenge and was not dependent on the Nlrp3 inflammasome. However, soluble levels of IL-1β protein were dependent on the inflammasome after challenge with either ozone or HA. HA challenge resulted in cleavage of macrophage-derived caspase1 and IL-1β, suggesting a role for alveolar macrophages in Nlrp3-dependent AHR.
Conclusions: The Nlrp3 inflammasome is required for the development of ozone-induced reactive airways disease.
asthma; environment; extracellular matrix; innate immunity; ozone; toll-like receptor
Evidence suggests inhibition of leukocyte trafficking mitigates, in part, ozone-induced inflammation. In the present study, the authors postulated that inhibition of myristoylated alanine-rich C kinase substrate (MARCKS), an 82-kDa protein with multiple biological roles, could inhibit ozone-induced leukocyte trafficking and cytokine secretions. BALB/c mice (n = 5/cohort) were exposed to ozone (100 ppb) or forced air (FA) for 4 hours. MARCKS-inhibiting peptides, MANS, BIO-11000, BIO-11006, or scrambled control peptide RNS, were intratracheally administered prior to ozone exposure. Ozone selectively enhanced bronchoalveolar lavage (BAL) levels of killer cells (KCs; 6 ± 0.9-fold), interleukin-6 (IL-6; 12.7 ± 1.9-fold), and tumor necrosis factor (TNF; 2.1 ± 0.5-fold) as compared to cohorts exposed to FA. Additionally, ozone increased BAL neutrophils by 21% ± 2% with no significant (P > .05) changes in other cell types. MANS, BIO-11000, and BIO-11006 significantly reduced ozone-induced KC secretion by 66% ± 14%, 47% ± 15%, and 71.1% ± 14%, and IL-6 secretion by 69% ± 12%, 40% ± 7%, and 86.1% ± 11%, respectively. Ozone-mediated increases in BAL neutrophils were reduced by MANS (86% ± 7%) and BIO-11006 (84% ± 2.5%), but not BIO-11000. These studies identify for the first time the novel potential of MARCKS protein inhibitors in abrogating ozone-induced increases in neutrophils, cytokines, and chemokines in BAL fluid. BIO-11006 is being developed as a treatment for chronic obstructive pulmonary disorder (COPD) and is currently being evaluated in a phase 2 clinical study.
asthma; BIO-11006; COPD; cytokines; inflammation; MANS peptide
Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that sub-populations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm × 3h) or filtered air. 24 h after the exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone exposed mice, which were distinct from resident alveolar macrophages (AM) and identified by enhanced Gr-1+ expression (Gr-1 Macs). Further analysis identified that Gr-1+ Macs exhibited high expression of MARCO, CX3CR1, and NQO1. Gr-1+ Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness (AHR), exacerbated neutrophil influx, accumulation of 8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1β, IL-6, CCL2, and TNF-α). Our results identify a novel subset of lung macrophages, which are derived from a resident intermediate, dependent upon CX3CR1, and appear to protect the host from the biological response to ozone.
Air pollutant exposure is linked with childhood asthma incidence and exacerbations, and maternal exposure to airborne pollutants during pregnancy increases airway hyperreactivity (AHR) in offspring. To determine if exposure to diesel exhaust (DE) during pregnancy worsened postnatal ozone-induced AHR, timed pregnant C57BL/6 mice were exposed to DE (0.5 or 2.0 mg/m3) 4 hours daily from Gestation Day 9–17, or received twice-weekly oropharyngeal aspirations of the collected DE particles (DEPs). Placentas and fetal lungs were harvested on Gestation Day 18 for cytokine analysis. In other litters, pups born to dams exposed to air or DE, or to dams treated with aspirated diesel particles, were exposed to filtered air or 1 ppm ozone beginning the day after birth, for 3 hours per day, 3 days per week for 4 weeks. Additional pups were monitored after a 4-week recovery period. Diesel inhalation or aspiration during pregnancy increased levels of placental and fetal lung cytokines. There were no significant effects on airway leukocytes, but prenatal diesel augmented ozone-induced elevations of bronchoalveolar lavage cytokines at 4 weeks. Mice born to the high-concentration diesel–exposed dams had worse ozone-induced AHR, which persisted in the 4-week recovery animals. Prenatal diesel exposure combined with postnatal ozone exposure also worsened secondary alveolar crest development. We conclude that maternal inhalation of DE in pregnancy provokes a fetal inflammatory response that, combined with postnatal ozone exposure, impairs alveolar development, and causes a more severe and long-lasting AHR to ozone exposure.
diesel; fetal inflammation; ozone; airway hyperreactivity
Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury.
To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma) were exposed to clean air (0ppb), medium (100ppb), and high (200ppb) ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL) 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG) in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay].
Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner.
Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.
Ozone exposure in the lab and environment causes airway hyperreactivity lasting at least 3 days in humans and animals. In guinea pigs 1 day after ozone exposure, airway hyperreactivity is mediated by eosinophils that block neuronal M2 muscarinic receptor function, thus increasing acetylcholine release from airway parasympathetic nerves. However, mechanisms of ozone-induced airway hyperreactivity change over time, so that depleting eosinophils 3 days after ozone makes airway hyperreactivity worse rather than better. Ozone exposure increases IL-1β in bone marrow, which may contribute to acute and chronic airway hyperreactivity. To test whether IL-1β mediates ozone-induced airway hyperreactivity 1 and 3 days after ozone exposure, guinea pigs were pretreated with an IL-1 receptor antagonist (anakinra, 30 mg/kg, intraperitoneally) 30 minutes before exposure to filtered air or to ozone (2 ppm, 4 h). One or three days after exposure, airway reactivity was measured in anesthetized guinea pigs. The IL-1 receptor antagonist prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone exposure. Ozone-induced airway hyperreactivity was vagally mediated, since bronchoconstriction induced by intravenous acetylcholine was not changed by ozone. The IL-1 receptor antagonist selectively prevented ozone-induced reduction of eosinophils around nerves and prevented ozone-induced deposition of extracellular eosinophil major basic protein in airways. These data demonstrate that IL-1 mediates ozone-induced airway hyperreactivity at 3 days, but not 1 day, after ozone exposure. Furthermore, preventing hyperreactivity was accompanied by decreased eosinophil major basic protein deposition within the lung, suggesting that IL-1 affects eosinophil activation 3 days after ozone exposure.
asthma; eosinophils; cytokines; parasympathetic nerves; lungs
Chronic exposure to high levels of ozone induces emphysema and chronic inflammation in mice. We determined the recovery from ozone-induced injury and whether an antioxidant, N-acetylcysteine (NAC), could prevent or reverse the lung damage.
Mice were exposed to ozone (2.5 ppm, 3 hours/12 exposures, over 6 weeks) and studied 24 hours (24h) or 6 weeks (6W) later. Nac (100 mg/kg, intraperitoneally) was administered either before each exposure (preventive) or after completion of exposure (therapeutic) for 6 weeks.
After ozone exposure, there was an increase in functional residual capacity, total lung volume, and lung compliance, and a reduction in the ratio of forced expiratory volume at 25 and 50 milliseconds to forced vital capacity (FEV25/FVC, FEV50/FVC). Mean linear intercept (Lm) and airway hyperresponsiveness (AHR) to acetylcholine increased, and remained unchanged at 6W after cessation of exposure. Preventive NAC reduced the number of BAL macrophages and airway smooth muscle (ASM) mass. Therapeutic NAC reversed AHR, and reduced ASM mass and apoptotic cells.
Emphysema and lung function changes were irreversible up to 6W after cessation of ozone exposure, and were not reversed by NAC. The beneficial effects of therapeutic NAC may be restricted to the ASM.
γδ T cells regulate airway reactivity, but their role in ozone (O3)-induced airway hyperresponsiveness (AHR) is not known. Our objective was to determine the role of γδ T cells in O3-induced AHR. Different strains of mice, including those that were genetically manipulated or antibody-depleted to render them deficient in total γδ T cells or specific subsets of γδ T cells, were exposed to 2.0 ppm of O3 for 3 hours. Airway reactivity to inhaled methacholine, airway inflammation, and epithelial cell damage were monitored. Exposure of C57BL/6 mice to O3 resulted in a transient increase in airway reactivity, neutrophilia, and increased numbers of epithelial cells in the lavage fluid. TCR-δ−/− mice did not develop AHR, although they exhibited an increase in neutrophils and epithelial cells in the lavage fluid. Similarly, depletion of γδ T cells in wild-type mice suppressed O3-induced AHR without influencing airway inflammation or epithelial damage. Depletion of Vγ1+, but not of Vγ4+ T cells, reduced O3-induced AHR, and transfer of total γδ T cells or Vγ1+ T cells to TCR-δ−/− mice restored AHR. After transfer of Vγ1+ cells to TCR-δ−/− mice, restoration of AHR after O3 exposure was blocked by anti–TNF-α. However, AHR could be restored in TCR-δ−/−mice by transfer of γδ T cells from TNF-α–deficient mice, indicating that another cell type was the source of TNF-α. These results demonstrate that TNF-α and activation of Vγ1+ γδ T cells are required for the development of AHR after O3 exposure.
ozone; airway responsiveness; γδ T cells; TNF-α
Ozone concentrations are predicted to increase over the next 50 years due to global warming and the increased release of precursor chemicals. It is therefore urgent that good, reliable biomarkers are available to quantify the toxicity of this pollutant gas at the population level. Such a biomarker would need to be easily performed, reproducible, economically viable, and reflective of ongoing pathological processes occurring within the lung.
We examined whether blood neutrophilia occurred following a controlled ozone challenge and addressed whether this could serve as a biomarker for ozone-induced airway inflammation. Three separate groups of healthy subjects were exposed to ozone (0.2 ppm, 2h) and filtered air (FA) on two separate occasions. Peripheral blood samples were collected and bronchoscopy with biopsy sampling and lavages was performed at 1.5h post exposures in group 1 (n=13), at 6h in group 2 (n=15) and at 18h in group 3 (n=15). Total and differential cell counts were assessed in blood, bronchial tissue and airway lavages.
In peripheral blood, we observed fewer neutrophils 1.5h after ozone compared with the parallel air exposure (-1.1±1.0x109 cells/L, p<0.01), at 6h neutrophil numbers were increased compared to FA (+1.2±1.3x109 cells/L, p<0.01), and at 18h this response had fully attenuated. Ozone induced a peak in neutrophil numbers at 6h post exposure in all compartments examined, with a positive correlation between the response in blood and bronchial biopsies.
These data demonstrate a systemic neutrophilia in healthy subjects following an acute ozone exposure, which mirrors the inflammatory response in the lung mucosa and lumen. This relationship suggests that blood neutrophilia could be used as a relatively simple functional biomarker for the effect of ozone on the lung.
Mechanisms of cardiovascular injuries from exposure to gas and particulate air pollutants are unknown.
We sought to determine whether episodic exposure of rats to ozone or diesel exhaust particles (DEP) causes differential cardiovascular impairments that are exacerbated by ozone plus DEP.
Methods and results
Male Wistar Kyoto rats (10–12 weeks of age) were exposed to air, ozone (0.4 ppm), DEP (2.1 mg/m3), or ozone (0.38 ppm) + DEP (2.2 mg/m3) for 5 hr/day, 1 day/week for 16 weeks, or to air, ozone (0.51 or 1.0 ppm), or DEP (1.9 mg/m3) for 5 hr/day for 2 days. At the end of each exposure period, we examined pulmonary and cardiovascular biomarkers of injury. In the 16-week study, we observed mild pulmonary pathology in the ozone, DEP, and ozone + DEP exposure groups, a slight decrease in circulating lymphocytes in the ozone and DEP groups, and decreased platelets in the DEP group. After 16 weeks of exposure, mRNA biomarkers of oxidative stress (hemeoxygenase-1), thrombosis (tissue factor, plasminogen activator inhibitor-1, tissue plasminogen activator, and von Willebrand factor), vasoconstriction (endothelin-1, endothelin receptors A and B, endothelial NO synthase) and proteolysis [matrix metalloprotease (MMP)-2, MMP-3, and tissue inhibitor of matrix metalloprotease-2] were increased by DEP and/or ozone in the aorta, but not in the heart. Aortic LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) mRNA and protein increased after ozone exposure, and LOX-1 protein increased after exposure to ozone + DEP. RAGE (receptor for advanced glycation end products) mRNA increased in the ozone + DEP group. Exposure to ozone or DEP depleted cardiac mitochondrial phospholipid fatty acids (DEP > ozone). The combined effect of ozone and DEP exposure was less pronounced than exposure to either pollutant alone. Exposure to ozone or DEP for 2 days (acute) caused mild changes in the aorta.
In animals exposed to ozone or DEP alone for 16 weeks, we observed elevated biomarkers of vascular impairments in the aorta, with the loss of phospholipid fatty acids in myocardial mitochondria. We conclude that there is a possible role of oxidized lipids and protein through LOX-1 and/or RAGE signaling.
air pollution; aorta; cardiovascular; diesel exhaust particles; inhalation; LOX-1; ozone; vascular
The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. However, it is not known if GSTM1 modulates these ozone responses in vivo in humans
The purpose of this study was to determine if the GSTM1 null genotype modulates ozone responses in humans.
Thirty-five normal volunteers were genotyped for the GSTM1 null mutation and underwent a standard ozone exposure protocol to determine if lung function and inflammatory responses to ozone were different between the 19 GSTM1 normal and 16 GSTM1 null volunteers.
GSTM1 did not modulate lung function responses to acute ozone. Granulocyte influx 4 hours after challenge was similar between GSTM1 normal and null volunteers. However, GSTM1 null volunteers had significantly increased airway neutrophils 24 hours after challenge, as well as increased expression of HLA-DR on airway macrophages and dendritic cells.
The GSTM1 null genotype is associated with increased airways inflammation 24 hours following ozone exposure, consistent with the lag time observed between increased ambient air ozone exposure and exacerbations of lung disease.
These observations suggest that the GSTM1 null genotype likely confers increased risk for exacerbation of ozone-induced lung disease through promoting an enhanced neutrophilic and monocytic inflammatory response to ozone.
The GSTM1 null genotype is associated with increased risk for ozone-induced lung disease. We report the GSTM1 genotype modulates ozone-induced inflammation but not lung function, and may predict persons at risk for environmental lung disease.
Glutathione-S-Transferase Mu 1; Ozone; Pollution; Inflammation; Polymorphonuclear Neutrophil; Macrophage; Dendritic cell