Ozone is an air pollutant that is known to have a variety of deleterious effects on the human lung [1–6]. These include inflammation, increased airway reactivity, and an increased susceptibility to infection. Ozone exposure has been reported to disrupt epithelial integrity, impair effective phagocytosis, and compromise mucociliary clearance . However, other studies where increased epithelial permeability and changes in ventilation are not observed indicate that these effects may be highly ozone dose-dependent . Ozone effects are more pronounced in asthmatics , especially children . Interestingly, ozone-induced inflammation, as measured by neutrophil influx and IL-8 levels, differs between normal subjects and asthmatics, but does not correlate with pulmonary function changes . Differences in the response to ozone among individuals having polymorphisms in genes related to oxidative stress implicate oxidative stress in these processes and provide a basis for varying susceptibility to ozone-induced symptoms .
Mechanisms involved in ozone-induced lung damage have been investigated in animal models [8–14]. In general, experimental animals require significantly higher doses of O3 exposure than humans  to reach comparable amounts of O3 concentration in the distal lung. Measurement of various parameters in bronchoalveolar lavage (BAL) revealed that resting rodents exposed to high O3 doses (2 ppm) were either comparable (polymorphonuclear leukocytes (PMNs), protein) or lower (macrophages) than the exercising human exposed to considerably lower O3 exposures (0.44 ppm). Therefore, it is necessary that rodents be exposed to high O3 concentrations to better enable extrapolation of findings from animal studies to human. Our laboratory has demonstrated ozone-dependent changes in mice in epithelial permeability, inflammatory mediators, and susceptibility to pneumonia [8, 9, 16]. The changes in epithelial permeability have been attributed to TLR-4-mediated changes in iNOS activity . A role for oxidative stress in ozone-induced pathophysiology has been postulated based on increases in F2-isoprostane , a lipid peroxidation product, as well as reductions in inflammatory mediators and allergen sensitivity by antioxidant treatment . The involvement of oxidative stress is further supported by studies in which genetic polymorphisms influence the response to ozone . Although the pathophysiology of ozone-induced lung damage is incompletely understood, these mechanistic and genetic association studies provide a strong rationale for oxidative stress  playing a key role in the response to ozone exposure.
Host defense function is one of the many processes that can be disrupted by oxidative stress. Ozone has been implicated in increasing susceptibility to infection in humans [18, 19] and in a number of animal studies (reviewed in ), as have other sources of oxidative stress such as sublethal hyperoxia . The basis for these effects is not known, but may relate to the oxidative modification of molecules involved in innate immune processes by reactive oxidant species, lipid peroxidation products, or other molecules generated by oxidative stress. Oxidation of protein molecules can interfere with their function and alter their metabolism by either promoting their degradation or causing the formation of protein aggregates that are not readily degraded [21, 22].
Surfactant protein-A (SP-A), a major component of BAL, is an example of an innate immune protein whose function is disrupted by oxidation. SP-A is known to play a variety of roles in innate immune function. These include serving as an opsonin for the recognition of some pathogens [23, 24], regulating the production of cell surface antigens and inflammatory mediator expression by some immune cells [25, 26], participating in the development of dendritic cells , regulating reactive oxidant production [28, 29], and others . However, a series of studies from our laboratory has shown that several of these functions are compromised when SP-A is oxidized)[9, 31–34]. A number of studies have explored the function of SP-A in vivo by subjecting SP-A-/- (SP-A knockout; KO) mice to various infectious or environmental challenges. These include studies of susceptibility to bacterial infection [35, 36], susceptibility to viral infection [37, 38], oxidant-mediated killing of mycoplasma , response to ozone exposure [8, 16], and the impact of ozone exposure on susceptibility to pneumonia . These in vivo studies have confirmed the diversity of SP-A's influence on innate immune function. Several studies from our laboratory have explored the role of SP-A in vivo in ozone exposure and innate immunity [8, 9, 16]. We have shown that the response of KO mice to acute ozone exposure, while similar in many respects to that of wild type (WT) mice, has some unique features including the influx of immune cells into the alveolar spaces. KO mice apparently sustain more tissue damage than WT mice, as indicated by BAL lactate dehydrogenase (LDH) levels detectable immediately after a 3 hr ozone exposure. However, at 4 hr after a 3 hr exposure to ozone lower relative numbers of neutrophils were observed in KO mice than WT mice , in part explaining the differences in lung mRNA levels for MIP-2, and to a lesser degree for MCP-1, between the two strains. Paradoxically however, no differences were observed in MIP-2 and MCP-1 protein levels between the two strains, underscoring, perhaps, the complexity of the processes involved. We have also shown that ozone exposure increases the susceptibility of mice to infection, at least in part due to the oxidation of SP-A , and that KO mice are more susceptible to infection than WT mice .
In this study, in order to gain insight into the mechanisms for the studies described above, we employed a discovery proteomic approach to investigate the effects of ozone exposure on the BAL proteome. We also utilized a strain of SP-A KO mice and compared them to WT mice on the same genetic background in order to elucidate the effect of SP-A on these processes. This type of unbiased approach is not dependent upon previously published studies and may be instrumental in generating specific novel hypotheses involving proteins and pathways that may not have been previously implicated in the process being studied. In the case of ozone-induced lung injury each of the studies described above has typically had a very narrow focus, and integrating all of these results into a unified understanding of the pathophysiology of ozone exposure has been difficult [8, 40–44].
Preliminary assessments of ozone-induced changes in rat and mouse BAL proteins have used conventional 2-D gel approaches to examine a small group of proteins [45, 46]. In one case, differences between an ozone-sensitive strain and an ozone-resistant strain in the response to ozone were explored , and in the other, the effects of ozone on 1-nitronaphthalene adduct formation were probed . In the present study we exposed WT and KO mice to ozone or filtered air and studied the resulting changes in the BAL proteome using two-dimensional difference gel electrophoresis (2D-DIGE), a discovery proteomics technique [47–49] for quantitation, coupled with Matrix Assisted Laser Desorption Ionization-Time-of-Flight/Time-of-Flight (MALDI-ToF/ToF) tandem mass spectrometry for identification of proteins. These techniques make it possible to simultaneously analyze hundreds of proteins in biological samples and have helped identify both pathways and additional proteins involved in these pathways in various experimental systems [50–52]. We recently employed a similar approach to examine age-related changes in the rat BAL proteome). This combination of methods for protein quantification and identification of proteins has proven useful in quantitative comparisons of protein expression and has not been previously applied to a comparison of this kind of SP-A KO mice with WT mice on the same genetic background.
In this study 2D-DIGE and MALDI-ToF/ToF were used to examine the impact of ozone on lung injury in the presence or absence of SP-A, a molecule with an important role in innate immune function. Using the PANTHER database and published literature we assigned many of the proteins identified to three major categories. By comparing the data obtained in WT and KO mice we have put forward a specific and novel hypothesis for the role of SP-A in redox balance and innate immunity in response to ozone-induced oxidative stress.