COPD is a devastating and increasingly prevalent disease that is closely associated with oxidative stress induced by chronic exposure to environmental stressors, especially cigarette smoke. Although tobacco smoke is highly concentrated in oxidants, not all individuals exposed to this hazard develop lung disease. Therefore, studying the endogenous enzymes that regulate cigarette smoke-induced oxidative stress may provide important clues to understand why some people are more susceptible to development of COPD than others. We hypothesize here that ECSOD may function to protect individuals from cigarette smoke exposure. This theory is substantiated by the observation that individuals with a particular ECSOD polymorphism (R213G) are 50% less likely to be diagnosed with COPD than control patients [14
]. In the past 2 years there have been five publications implicating SOD3 polymorphisms as risk factors for lung disease [14
]. There are several potential mechanisms that have been suggested to explain how ECSOD might protect individuals from diseases such as COPD. For example, ECSOD has been shown to modulate lung inflammation by preventing oxidative fragmentation of the extracellular matrix of the lung [17
]. We were therefore interested to investigate the role of ECSOD in both inflammation and oxidative stress induced by cigarette smoke. NOX enzymes are also potential endogenous candidates for smoke-induced oxidative stress [39
]. We therefore investigated the potential role of these enzymes in cigarette smoke exposure.
In this study, we show that exposure to cigarette smoke induces oxidative stress as measured by the fluorogenic indicator dyes DCFDA and DHE (). Although these dyes have been used extensively to detect H2
, respectively, there is evidence to suggest that other ROS are capable of oxidizing the compounds, resulting in a fluorescent signal [40
]. Therefore, we are careful to report the increased fluorescence observed here as a marker of global, cellular oxidative stress rather than an indication of the explicit species of free radical. Because ECSOD and NOX enzymes regulate O−2
specifically, and we show here that manipulation of these enzymes modulates oxidative stress, we hypothesize that O−2
, in particular, is responsible for cigarette smoke-induced oxidative stress and inflammation that occur in our model. However, because of the highly reactive nature of O−2
, we recognize the possibility that other ROS may be formed quickly and may also play an important role. Fluorogenic spin traps or other more specific methods will be employed in future studies to determine the actual species of ROS responsible for signaling sustained oxidative stress and inflammation within this smoking model. In addition, measuring reduced GSH levels provides a sensitive indication of changes in the redox environment, both intracellularly and extracellularly. Interestingly, GSH levels were increased in response to prolonged (24 h) CSE exposure in primary mouse macrophage cultures (). This increase is probably a compensatory mechanism to offset the marked increase in ROS that are generated upon CSE exposure.
Discrepancies in the literature exist related to total TSP concentrations used in in vivo smoking experiments [43
]. TSP concentrations were therefore assessed to determine an appropriate level to evoke an inflammatory response in the 2-week exposure time frame used in our model (). Total cell counts in the BALF of mice exposed to 0, 25, or 100 μg/m3
TSP were measured and it was determined that a TSP of 100 μg/m3
was required to induce a measurable response (). All subsequent experiments were performed with this TSP concentration (100 μg/m3
). The percentage of neutrophils in the total cell count (BALF cell pellet) was increased in mice exposed to cigarette smoke, indicating that inflammatory cell recruitment signals were turned on. This suggests a potential role for NOX enzymes because they are abundantly expressed in neutrophils ().
Because ECSOD is substantially expressed in the lung, its role as a protective antioxidant in smoking-induced lung disease is of primary interest in this field of research. Incubation with a SOD mimetic or overexpression of ECSOD sufficiently inhibited cigarette smoke-induced oxidative stress (). These data suggest that ECSOD activity may be an important host defense mechanism associated with prevention of COPD, despite exposure to the concentration of oxidants in cigarette smoke.
In addition to understanding the inherent antioxidant defense mechanisms in place to offset smoke-induced oxidative stress, the source of sustained ROS production is also of interest. Clearly, cigarette smoke exposure induces the recruitment of neutrophils to epithelial lining fluid in vivo (). Because of its high expression in this cell type, NOX activity is a primary candidate for the source of chronic oxidative stress associated with COPD. Inhibition of NOX with inhibitors (DPI and apocynin) showed protection against CSE-induced oxidative stress in primary mouse macrophages (). Apocynin protected more completely than did DPI and this may be due, in part, to documented scavenging properties of apocynin and/or slight toxicity observed with DPI [45
]. We proceeded to assess the effect of CSE in a NOX-deficient system, as superfluous activity of pharmacologic inhibitors (DPI and apocynin) could potentially have an effect on the measurable oxidative stress response [46
]. Primary macrophages isolated from NOX knockout mice also exhibited decreased CSE-induced oxidative stress (). These data show that NOX enzyme complexes play an important role in cigarette smoke-induced oxidative stress and suggest a role for the enzymes in COPD.
Interestingly, we did not find ECSOD overexpression to be protective against the formation of free 8-isoprostanes in primary BMMs (). Based upon this observation, we conclude that cigarette smoke may induce oxidative stress initially at the level of the membrane via a pathway that is not mediated by ECSOD. However, additional data presented here suggest that subsequent oxidative signals are mediated via NOX and are protected against by ECSOD.
Although in vitro studies are useful to elucidate the specific molecules associated with cigarette smoke-induced oxidative stress, there are several changes observed in intact mice exposed to cigarette smoke which cannot necessarily be replicated in cell culture. Therefore, the effects of ECSOD and NOX were measured in vivo by exposing transgenic mice to cigarette smoke and comparing their response to that of wild-type mice. MIP2 is a proinflammatory cytokine that has been shown to increase upon exposure to cigarette smoke [47
]. As expected, this cytokine was increased in response to cigarette smoke exposure in wild-type mice, but interestingly, was not altered by smoke exposure in ECSOD overexpressers or NOX KO mice (). In addition, IFNγ was increased in wild-type mice exposed to CS but not in either of the transgenics (). These data suggest that the inflammatory signals that regulate cytokine release are triggered by smoke-induced O−2
In concordance with data previously reported [48
], mice exposed to cigarette smoke showed increased levels of reduced GSH in their BALF. However, ECSOD-overexpressing and NOX KO mice did not exhibit the significant increase observed in wild-type mice (). These data suggest that there is a O−2
-dependent signal that induces increased GSH production in mice exposed to cigarette smoke.
4-HNE, an aldehyde by-product formed by free radical-induced lipid peroxidation, can react with proteins and is therefore utilized as a reliable marker of oxidative stress by immunoblot. Lung tissues from human patients with varying diagnoses of COPD severity were assessed for lipid peroxidation using this method (). 4-HNE levels correlated with severity of COPD. Interestingly, smoking status alone did not predict lipid peroxidation, which suggests that there is something inherently different in COPD patients that makes them susceptible to oxidative lung damage.
These data suggest that the mechanisms underlying the host defense against cigarette smoke-induced oxidative damage and subsequent development of COPD may include endogenous oxidases and antioxidant enzymes. Because not all chronic smokers develop disease, it is essential to understand the mechanisms of endogenous enzymes that regulate oxidative lung damage. Understanding why some individuals continue to produce free radicals and suffer oxidative damage even after smoking cessation, whereas others do not, can provide important insights and potential therapeutic targets for this devastating disease.