Site-specific dynamics of CD11b+ and CD103+ dendritic cell accumulations following ozone exposure.

Pulmonary dendritic cells (DCs) are among the first responders to inhaled environmental stimuli such as ozone (O(3)), which has been shown to activate these cells. O(3) reacts with epithelial lining fluid (ELF) components in an anatomically site-specific manner dictated by O(3) concentration, airway flow patterns, and ELF substrate concentration. Accordingly, the anatomical distribution of ELF reaction products and airway injury are hypothesized to produce selective DC maturation differentially within the airways. To investigate how O(3) affects regional airway DC populations, we utilized a model of O(3)-induced pulmonary inflammation, wherein C57BL/6 mice were exposed to 0.8 ppm O(3) 8 h/day for 1, 3, and 5 days. This model induced mild inflammation and no remarkable epithelial injury. Tracheal, but not more distant airway sites, and mediastinal lymph node (MLN) DC numbers were increased significantly after the third exposure day. The largest increase in each tissue was of the CD103(+) DC phenotype. After 3 days of exposure, fewer DCs expressed CD80, CD40, and CCR7, and, at this same time point, total MLN T cell numbers increased. Together, these data demonstrate that O(3) exposure induced site-specific and phenotype changes in the pulmonary and regional lymph node DC populations. Possibly contributing to ozone-mediated asthma perturbation, the phenotypic changes to DCs within pulmonary regions may alter responses to antigenic stimuli. Decreased costimulatory molecule expression within the MLN suggests induction of tolerance mechanisms; increased tracheal DC number may raise the potential for allergic sensitization and asthmatic exacerbation, thus overcoming O(3)-induced decrements in costimulatory molecule expression.

Desferrioxamine inhibits protein tyrosine nitration: mechanisms and implications.

Tissues are exposed to exogenous and endogenous nitrogen dioxide ((·)NO(2)), which is the terminal agent in protein tyrosine nitration. Besides iron chelation, the hydroxamic acid (HA) desferrioxamine (DFO) shows multiple functionalities including nitration inhibition. To investigate mechanisms whereby DFO affects 3-nitrotyrosine (3-NT) formation, we utilized gas-phase (·)NO(2) exposures, to limit introduction of other reactive species, and a lung surface model wherein red cell membranes (RCM) were immobilized under a defined aqueous film. When RCM were exposed to ()NO(2) covered by +/- DFO: (i) DFO inhibited 3-NT formation more effectively than other HA and non-HA chelators; (ii) 3-NT inhibition occurred at very low[DFO] for prolonged times; and (iii) 3-NT formation was iron independent but inhibition required DFO present. DFO poorly reacted with (·)NO(2) compared to ascorbate, assessed via (·)NO(2) reactive absorption and aqueous-phase oxidation rates, yet limited 3-NT formation at far lower concentrations. DFO also inhibited nitration under aqueous bulk-phase conditions, and inhibited 3-NT generated by active myeloperoxidase "bound" to RCM. Per the above and kinetic analyses suggesting preferential DFO versus (·)NO(2) reaction within membranes, we conclude that DFO inhibits 3-NT formation predominantly by facile repair of the tyrosyl radical intermediate, which prevents (·)NO(2) addition, and thus nitration, and potentially influences biochemical functionalities.

Postnatal episodic ozone results in persistent attenuation of pulmonary and peripheral blood responses to LPS challenge.

Early life is a dynamic period of growth for the lung and immune system. We hypothesized that ambient ozone exposure during postnatal development can affect the innate immune response to other environmental challenges in a persistent fashion. To test this hypothesis, we exposed infant rhesus macaque monkeys to a regimen of 11 ozone cycles between 30 days and 6 mo of age; each cycle consisted of ozone for 5 days (0.5 parts per million at 8 h/day) followed by 9 days of filtered air. Animals were subsequently housed in filtered air conditions and challenged with a single dose of inhaled LPS at 1 yr of age. After completion of the ozone exposure regimen at 6 mo of age, total peripheral blood leukocyte and polymorphonuclear leukocyte (PMN) numbers were reduced, whereas eosinophil counts increased. In lavage, total cell numbers at 6 mo were not affected by ozone, however, there was a significant reduction in lymphocytes and increased eosinophils. Following an additional 6 mo of filtered air housing, only monocytes were increased in blood and lavage in previously exposed animals. In response to LPS challenge, animals with a prior history of ozone showed an attenuated peripheral blood and lavage PMN response compared with controls. In vitro stimulation of peripheral blood mononuclear cells with LPS resulted in reduced secretion of IL-6 and IL-8 protein in association with prior ozone exposure. Collectively, our findings suggest that ozone exposure during infancy can result in a persistent effect on both pulmonary and systemic innate immune responses later in life.

Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys.

Children chronically exposed to high levels of ozone (O(3)), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O(3) [0.5 parts per million (ppm), 8 h/day; "1-cycle"] or filtered air (FA) or 11 biweekly cycles of O(3) (FA days 1-9; 0.5 ppm, 8 h/day on days 10-14; "11-cycle"). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH(2)), and uric acid (UA) concentration. Eleven-cycle O(3) induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O(3) also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments.

Pulmonary ozone exposure induces vascular dysfunction, mitochondrial damage, and atherogenesis.

More than 100 million people in the United States live in areas that exceed current ozone air quality standards. In addition to its known pulmonary effects, environmental ozone exposures have been associated with increased hospital admissions related to cardiovascular events, but to date, no studies have elucidated the potential molecular mechanisms that may account for exposure-related vascular impacts. Because of the known pulmonary redox and immune biology stemming from ozone exposure, we hypothesized that ozone inhalation would initiate oxidant stress, mitochondrial damage, and dysfunction within the vasculature. Accordingly, these factors were quantified in mice consequent to a cyclic, intermittent pattern of ozone or filtered air control exposure. Ozone significantly modulated vascular tone regulation and increased oxidant stress and mitochondrial DNA damage (mtDNA), which was accompanied by significantly decreased vascular endothelial nitric oxide synthase protein and indices of nitric oxide production. To examine influences on atherosclerotic lesion formation, apoE-/- mice were exposed as above, and aortic plaques were quantified. Exposure resulted in significantly increased atherogenesis compared with filtered air controls. Vascular mitochondrial damage was additionally quantified in ozone- and filtered air-exposed infant macaque monkeys. These studies revealed that ozone increased vascular mtDNA damage in nonhuman primates in a fashion consistent with known atherosclerotic lesion susceptibility in humans. Consequently, inhaled ozone, in the absence of other environmental toxicants, promotes increased vascular dysfunction, oxidative stress, mitochondrial damage, and atherogenesis.

Three-dimensional mapping of ozone-induced injury in the nasal airways of monkeys using magnetic resonance imaging and morphometric techniques.

Age-related changes in gross and microscopic structure of the nasal cavity may alter local tissue susceptibility as well as the dose of inhaled toxicant delivered to susceptible sites. This article describes a novel method for the use of magnetic resonance imaging, 3-dimensional airway modeling, and morphometric techniques to characterize the distribution and magnitude of ozone-induced nasal injury in infant monkeys. Using this method, we generated age-specific, 3-dimensional, epithelial maps of the nasal airways of infant Rhesus macaques. The principal nasal lesions observed in this primate model of ozone-induced nasal toxicology were neutrophilic rhinitis, along with necrosis and exfoliation of the epithelium lining the anterior maxilloturbinate. These lesions, induced by acute or cyclic (episodic) exposures, were examined by light microscopy, quantified by morphometric techniques, and mapped on 3-dimensional models of the nasal airways. Here, we describe the histopathologic, imaging, and computational biology methods developed to precisely characterize, localize, quantify, and map these nasal lesions. By combining these techniques, the location and severity of the nasal epithelial injury were correlated with epithelial type, nasal airway geometry, and local biochemical and molecular changes on an individual animal basis. These correlations are critical for accurate predictive modeling of exposure-dose-response relationships in the nasal airways, and subsequent extrapolation of nasal findings in animals to humans for determining risk.

Antioxidant-mediated augmentation of ozone-induced membrane oxidation.

The pulmonary epithelial lining fluid (ELF) contains substrates, e.g., ascorbic acid (AH2), uric acid (UA), glutathione (GSH), proteins, and unsaturated lipids, which undergo facile reaction with inhaled ozone (O3). Reactions near the ELF gas/liquid interface likely provide the driving force for O3 absorption ("reactive absorption") and constrain O3 diffusion to the underlying epithelium. To investigate the potential mechanisms wherein O3/ELF interactions may induce cellular damage, we utilized a red cell membrane (RCM) model intermittently covered by an aqueous film to mimic the lung surface compartmentation, and evaluated exposure-mediated loss of acetylcholinesterase activity (AChE) and TBARS accumulation. In the absence of aqueous reactants, O3 exposure induced no detectable changes in AChE or TBARS. AH2 and GSH preferentially induced oxidative damage in a dose-dependent fashion. AH2-mediated RCM oxidation was not inhibited by superoxide dismutase, catalase, mannitol, or Fe chelators. O3 reaction with UA, Trolox, or albumin produced no RCM oxidation but oxidation occurred when AH2 was combined with UA or albumin. Rat bronchoalveolar lavage fluid (BALF) also induced RCM oxidation. However, in vivo O3 exposure dampened the extent of BALF-mediated RCM oxidation. Although we cannot completely rule out O3 diffusion to the RCM, product(s) derived from O3 + AH2/GSH reactions (possibly O3*- or 1O2) likely initiated RCM oxidation and may suggest that in vivo, such secondary species account for O3 permeation through the ELF leading to cellular perturbations.

Interfacial phospholipids inhibit ozone-reactive absorption-mediated cytotoxicity in vitro.

The intrapulmonary distribution of inhaled ozone (O(3)) and induction of site-specific cell injury are related to complex interactions among airflow patterns, local gas-phase concentrations, and the rates of O(3) flux into, and reaction and diffusion within, the epithelial lining fluid (ELF). Recent studies demonstrated that interfacial phospholipid films appreciably inhibited NO(2) absorption. Because surface-active phospholipids are present on alveolar and airway interfaces, we investigated the effects of interfacial films on O(3)-reactive absorption and acute cell injury. Compressed films of dipalmitoyl-glycero-3-phosphocholine (DPPC) and rat lung lavage lipids significantly reduced O(3)-reactive absorption by ascorbic acid, reduced glutathione, and uric acid. Conversely, unsaturated phosphatidylcholine films did not inhibit O(3) absorption. We evaluated O(3)-mediated cell injury using a human lung fibroblast cell culture system, an intermittent tilting exposure regimen to produce a thin covering layer, and nuclear fluorochrome permeability. Exposure produced negligible injury in cells covered with MEM. However, addition of AH(2) produced appreciable (<50%) cell injury. Film spreading of DPPC monolayers necessitated the use of untilted regimens. Induction of acute cell injury in untilted cultures required both AH(2) plus very high O(3) concentrations. Addition of DPPC films significantly reduced cell injury. We conclude that acute cell injury likely results from O(3) reaction with ELF substrates. Furthermore, interfacial films of surface-active, saturated phospholipids reduce the local dose of O(3)-derived reaction products. Finally, because O(3) local dose and tissue damage likely correlate, we propose that interfacial phospholipids may modulate intrapulmonary distribution of inhaled O(3) and the extent of site-specific cell injury.

Influence of epithelial lining fluid lipids on NO(2)-induced membrane oxidation and nitration.

Within the pulmonary epithelial lining layer (ELF), antioxidants such as ascorbic acid (AH(2)) and glutathione (GSH) react with inhaled nitrogen dioxide ((*)NO(2)) to produce reactive oxygen species (ROS) that induce cellular oxidation. Because the ELF contains unsaturated fatty acids (UFA), which potentially react with (*)NO(2) and/or the antioxidant-derived ROS, we studied the influence of aqueous phase model UFA [egg phosphatidylcholine (EggPC) liposomes] on exposure-induced oxidation and nitration of membranes. Our lung surface model used gas phase (*)NO(2) exposures of immobilized red cell membranes (RCM) overlaid with defined aqueous phases. Acetyl cholinesterase (AChE) activity, TBARS, and 3-nitrotyrosine (3-NT) were used to assess protein and lipid oxidation and RCM nitration, respectively. During (*)NO(2) exposure, AH(2) and GSH induced AChE loss and TBARS, which were unchanged with buffer only. Exposures of EggPC generated extensive TBARS but not AChE loss; addition of AH(2)/GSH to EggPC resulted in smaller AChE declines and fewer TBARS. 3-NT formation occurred with or without EggPC, low concentration antioxidants, SOD, catalase, or DTPA, but was inhibitable by desferrioxamine or high antioxidant concentrations. The data suggest that reaction/diffusion limitations govern (*)NO(2) distribution, that (*)NO(2) per se directly nitrates tyrosine residues within hydrophobic regions, and that the induction of secondary oxidative processes is dependent on nonlinear relationships among (*)NO(2) flux rates, antioxidant concentrations, and diffusivity of secondary reactive species.