Maintaining redox balance in the lung is a dynamic process. It is especially challenging within the air passageways and alveolar spaces, where surface epithelial cells and resident phagocytes are exposed to ─ and provide the first line of defense against ─ a wide range of inhaled biologic (e.g., bacteria, viruses, allergens) and environmental agents (e.g., ozone, PM). In the present investigation, we used relatively simplistic in vitro and in vivo murine models of cytokine-induced epithelial and lung inflammation, respectively, to demonstrate the potential for NO (increased during inflammatory conditions) and ROS (increased as a consequence of traffic PM exposure) to “"co-operate”" to produce reactive oxidative as well as reactive nitrosative species (RNS) within PM-exposed lung cells. Specifically, we show that epithelial cells exposed to OC-rich DEP within an inflammatory microenvironment incur greater ROS/RNS burden and corresponding epithelial cytotoxicity; and that cytomix + DEP-exposed mice incur greater ROS/RNS production in lung phagocytes.
What evidence exists that respiratory inflammation is associated with increased NO production in people? Asthma, COPD, bronchitis, and rhinitis all represent a spectrum of respiratory disorders in which the clinical manifestations are orchestrated by, or in large part the result of, underlying inflammatory processes. Whereas all three nitric oxide synthase isoforms are present in the respiratory tract, clinical studies show that exhaled NO (eNO) levels are higher in patients with asthma [31, 32], COPD  and seasonal rhinitis . The magnitude of eNO increase is often proportionate to the degree of symptomatology, inflammation, and aeroallergen sensitization. Elevations are, in large part, derived from iNOS localized within the inflamed epithelium [35, 36] ─ with highest iNOS expression found in epithelial cells of patients with severe asthma . Additional studies reveal eNO increases in association with childhood exposure to traffic emissions , ambient air pollution , or early life exposure to PAHs . Moreover, recent studies suggest that such exposures may influence genetic and epigenetic variations in the iNOS promoters [41, 42].
How could these changes contribute to the adverse respiratory outcomes associated with traffic emissions in humans? Lung epithelial dysfunction is considered central to development of asthma; with insults such as air pollutants serving not simply as triggers for disease exacerbation, but also as playing critical roles in the origin and progression of airway and lung pathology . A growing body of literature further implicates impaired antioxidant defenses and disturbances in oxidation/reduction (redox) balance as risk factors for asthma development and asthma severity [44, 45]. Accumulating in vitro and in vivo experimental studies have shown that traffic PM exposure is associated with increased lung oxidant burden related to increased ROS such as O2˙-
[46–49]. Other studies of DEP-exposed rodents reveal concomitant increases in NO and ONOO- in BAL fluid cells [50, 51].
In the present investigation, data demonstrated that despite ROS increases in DEP-exposed epithelial cells, significant cytotoxicity was not observed unless cells had been exposed within an inflammatory microenvironment ─ suggesting a cooperative role of particle-induced ROS with existing lung inflammatory mediators. We clearly show how DEP effects were mediated by: (1) increased ROS (including O2˙-) production related to increased XdH expression and reduced CuZn SOD activity; and (2) increased RNS production owing to interaction of O2˙- with cytokine-induced, NO, to generate peroxynitrite. We confirm that DEP-induced epithelial effects could be partially ameliorated by providing additional SOD or blocking iNOS induction. Epithelial cell and in vivo phagocyte effects were, by and large, prevented by accelerating catalysis of the longer-lived, peroxynitrite radical, through administration of the iron porphyrin, FeTMPyP.
Normally, in health, respiratory tract epithelial cells and lung phagocytes work in concert to provide protection against inhaled microorganisms. The ability to greatly increase production of NO (and related RNS) is key to respiratory system innate immunity. In neutrophils, for example, cooperative action of NO and O2˙- imparts their ability to kill ingested microbial pathogens . Likewise, after microbial phagocytosis by alveolar macrophages, iNOS activation and respiratory “"burst”" activity similarly mediate pathogen clearance . A variation on this theme occurs in airway epithelial cells during whooping cough (pertussis) infection as infected epithelial cells respond to IL-1 by increasing iNOS. Excess NO induces epithelial autotoxicity and shedding of infected cells, thereby limiting spread of pertussis organisms to adjacent healthy cells . Consequently, the lung has developed an extensive capacity to withstand oxidative and nitrosative insult, at least on a short-term basis, as required during acute inflammatory response to infectious agents.
Respiratory pathogens, on the other hand, have evolved intricate NO-sensing capabilities and defense mechanisms of their own . Murine models of viral infection reveal major shifts in the cellular and temporal distribution of lung antioxidant enzymes during, for example, influenza pneumonia . RSV infection can similarly induce significant down-regulation of host airway antioxidant processes (e.g., SOD activity), which in infants (possibility owing to immature antioxidant defense mechanisms), can result in extensive oxidative epithelial damage and severe bronchiolitis .
Air pollution PM has inherent oxidant properties that are highly correlated with OC (e.g., PAH) and metal content . While such characteristics no doubt contribute to the overall oxidant effects of traffic PM, it is likely that ─ owing to their resemblance to inhaled microorganisms ─ in vivo toxicity and health effects are mediated largely by generation of ROS/RNS within exposed cells during futile attempts by innate host defenses to respond to inhaled fine PM as if they were potential pathogens. As such, cellular redox modulation, whether related to infection or PM exposure, appears to be deeply entangled with host inflammatory responses. We focused, therefore, on DEP-induced changes in glutathione because it is the most abundant intracellular antioxidant thiol, and is central to redox defense during oxidative/nitrosative stress . The diverse functions of GSH (γ-glu-cys-gly) originate from the sulfhydryl group in cysteine, enabling GSH to participate in redox cycling. Glutathione redox changes regulate not only signal transduction and airway inflammation, but also airway reactivity and hyperresponsiveness .
However, if and when simultaneous production of NO and O2˙- occur, excess RNS levels can exceed available cellular, and even tissue antioxidant capacity. Excessive RNS ─ not unlike excess ROS ─ can induce DNA damage, modify lipids [27, 28, 61], and cause protein misfolding and dysfunction . In response to misfolded protein, the unfolded protein response (UPR) triggers a series of intracellular events aimed at either eliminating rogue (damaged) cells by inducing apoptosis  or allowing cells to overcome the consequences of the stress by altering expression of anti-oxidant response genes, cell cycle progression, or inflammatory cascades . Our data similarly showed that significant epithelial damage occurred only if DEP exposure was associated with decreased GSH:GSSG ratios. This occurred only in the DEP-exposed “"inflamed”" cells in vitro. It appeared that as cumulative oxidative/nitrosative stress exceeded LA-4 cell capacity to maintain adequate redox status (i.e., GSH:GSSG ratios decreased), epithelial function became progressively impaired, resulting in cellular apoptosis and/or necrosis.
There were several limitations to this investigation. First, alveolar epithelial cell responses to PM may differ from that of airway epithelial cells, and further, particle effects in cell lines may or may not reflect that of their corresponding primary cells of origin. To this end, we previously showed that both LA-4 cells and primary murine airway epithelial cultures (established at an air-liquid interface) were more susceptible to DEP-induced effects when exposed within this cytokine-induced inflammatory microenvironment . Henceforth, we used the LA-4 cells as general surrogates to assess particle effects on surface cells of the respiratory tract. These generic in vitro and in vivo inflammatory models may also fail to recapitulate key features of disease processes occurring in asthmatics (i.e., TH2- or eosinophil-mediated effects). However, if traffic PM exposure was to only influence allergen-specific processes, effects would be unlikely to explain the health associations noted in patients with COPD, chronic bronchitis, or 50% of adult asthmatics who are not overtly atopic . It was because augmented health effects are associated with a broad range of inflammatory conditions, that we intentionally developed this cytokine combination to model a generic inflammatory state. Nonetheless, efforts are in progress to refine the in vivo cytokine protocol to better simulate longer-lived, lower-level, inflammation in the mice.
Although expression of XdH was increased during DEP exposure, XdH (normally involved in the metabolism of purines), can also be converted to xanthine oxidase, thus contributing to still greater O2˙- production. DEP influences on the mitochondrial respiratory chain or other enzyme systems (e.g., NADPH oxidases, P450 enzymes) cannot be ruled out as important sources of O2˙- production . Likewise, other oxidant (e.g., H2O2, ∙OH) or nitrosative [e.g., nitrosonium (NO+), nitroxyl (NO-), HNO] species may have contributed to DEP-induced redox changes.
Herein, relatively high-dose, short-term exposures to DEP were utilized. In so doing, this study was primarily designed to examine differential effects of DEP in healthy vs. diseased states, and as such, showed that under inflammatory conditions, LA-4 cells were 10-fold more susceptible to DEP-induced epithelial damage . Although real world exposures to urban air pollution are lower-level, they are also chronic and involve multiple pollutants. It may be relevant that school buses contribute substantially to DEP exposure in children, with onboard PM2.5 levels being four-fold higher than ambient levels, and two-fold higher than roadway levels . Aerosol particle counts at schools are 2.3- to 4.7-fold higher than areas without bus-related traffic . Likewise, in urban schools serviced by diesel buses, ambient near- and in-school PM2.5 fluctuations correspond temporally with bus drop-off hours and are highest in schools with the greatest number of buses in operation .
We further acknowledge that DEP composition can vary, and hence we used select DEP samples that resembled tunnel traffic PM (in terms of relative EC and OC content) to represent traffic-based PM. Other components of near-road emissions, for example, gaseous and semivolatile compounds  and metals from tire, brake, and rotor wear  reportedly have pro-oxidant properties and also participate redox cycling. It is possible, therefore, that similar to these diesel-derived particles, under inflammatory conditions, exposure to other traffic-related oxidants may similarly enhance ROS/RNS production.
Results of the present investigation provide biologic plausibility for the ever increasing epidemiological database associating traffic exposure with adverse health impact, especially in individuals with pre-existing respiratory diseases [1, 8, 71]. Relatedly, in asthmatics, airway redox balance appears to be shifted toward a more oxidized state  and the epithelial barrier is already somewhat compromised . In poorly controlled asthma, alveolar macrophages are prone to apoptosis and phagocytosis is impaired . Experimental studies implicate glutathione depletion and redox imbalance in these phagocyte deficits . Our data similarly implicates cellular redox imbalance as a precursor to both: (1) phagocyte and epithelial cell signaling and associated inflammatory processes and (2) epithelial injury and barrier dysfunction. These results support novel therapeutic approaches designed to increase the airways’ resistance against the inhaled environment agents rather than focusing solely on suppression of inflammation . Ancillary medical and nutritional interventions may be warranted, particularly in children and at-risk populations [75–77].
Based on the above experimental and clinical findings, we put forth the supposition that in at-risk populations, traffic-associated ROS/RNS production further compromises epithelial barrier and phagocytic cell function, thereby allowing penetration of inhaled pathogens and allergens deeper into lung tissue. In so doing, chronic traffic exposure would predispose individuals to repeated respiratory infection and immune cell antigen exposure, respectively; which overtime would promote and elicit end organ expression of atopic asthma . This scenario is supported by epidemiologic reports worldwide associating early life or childhood exposures to air pollution from traffic with development of respiratory infections and asthmatic and allergic symptoms [2–4, 6, 15, 20, 22, 78].