Rationale: Oxidants are believed to play a major role in the development of emphysema.
Objectives: This study aimed to determine if the expression of human copper–zinc superoxide dismutase (CuZnSOD) within the lungs of mice protects against the development of emphysema.
Methods: Transgenic CuZnSOD and littermate mice were exposed to cigarette smoke (6 h/d, 5 d/wk, for 1 yr) and compared with nonexposed mice. A second group was treated with intratracheal elastase to induce emphysema.
Measurements: Lung inflammation was measured by cell counts and myeloperoxidase levels. Oxidative damage was assessed by immunofluorescence for 3-nitrotyrosine and 8-hydroxydeoxyguanosine and lipid peroxidation levels. The development of emphysema was determined by measuring the mean linear intercept (Lm).
Main Results: Smoke exposure caused a fourfold increase in neutrophilic inflammation and doubled lung myeloperoxidase activity. This inflammatory response did not occur in the smoke-exposed CuZnSOD mice. Similarly, CuZnSOD expression prevented the 58% increase in lung lipid peroxidation products that occurred after smoke exposure. Most important, CuZnSOD prevented the onset of emphysema in both the smoke-induced model (Lm, 68 exposed control vs. 58 exposed transgenic; p < 0.04) and elastase-generated model (Lm, 80 exposed control vs. 63 exposed transgenic; p < 0.03). These results demonstrate for the first time that antioxidants can prevent smoke-induced inflammation and can counteract the proteolytic cascade that leads to emphysema formation in two separate animal models of the disease.
Conclusions: These findings indicate that strategies aimed at enhancing or supplementing lung antioxidants could be effective for the prevention and treatment of this disease.
Cigarette smoke is the major etiologic factor associated with the development of chronic obstructive pulmonary disease (COPD). This condition affects 24 million Americans and, in 1998, it accounted for 110,000 deaths (1), making it the fourth leading cause of mortality nationwide. Although smoking prevalence rates have declined over the past 20 yr, current surveys indicate that 22.5% of the adult population continues to smoke and 82% of those individuals smoke on a daily basis (2). Despite the importance and rising prevalence of COPD, little progress has been made toward developing effective drug therapies. For a treatment strategy to work, it will need to suppress the inflammatory and destructive processes that underlie this disease. Antioxidants are a class of compounds with both antiinflammatory (3) and antiproteolytic (4) properties that have attracted significant interest as a potential therapy for emphysema (5–7).
The high concentration of oxidant molecules in cigarette smoke is believed to play a major role in the functional decline of individuals with COPD (8). It is estimated that each cigarette puff contains 1014 free radicals (9). Although the oxidants in the gas phase are short lived and affect primarily the upper respiratory tract, the oxidants in the tar phase contain relatively stable semiquinone radicals that may be particularly harmful to the lung (10). These radicals are capable of damaging the epithelial cells of the lower respiratory tract by causing oxidative injury to membrane lipids, proteins, carbohydrates, and DNA. Such injury may significantly impair cellular function, enhance inflammation, induce apoptosis, and stimulate dysfunctional matrix remodeling (10). The respiratory tract lining contains antioxidants that protect the lung from such free-radical injury (11–13). Cigarette smoke causes the activity of some antioxidants to decrease in the epithelial lung fluid and plasma of smokers (14, 15). Other antioxidants, however, are actually induced in the presence of cigarette smoke (16, 17). However, given the evidence of systemic oxidative stress in smokers (18–20), the antioxidant response that occurs is inadequate to prevent the development of free-radical injury.
All cells are exposed to oxidative stress during the course of normal metabolism. The primary sites of oxidant production are the electron transport chain of the mitochondria and oxidant-generating enzymes present in the cell (e.g., xanthine oxidase, nitric oxide synthase, and nicotinamide adenine dinucleotide phosphate reduced oxidase). The lung, given its direct exposure to oxygen in the ambient air, experiences enhanced oxidant stress compared with other organs. Cigarette smoke contributes additional oxidants and stimulates inflammation, further augmenting free-radical production. The first reactive oxygen species produced in the reduction pathway of oxygen to water is superoxide anion (O2·−). This free radical participates in the generation of other potent oxidants such as hydrogen peroxide, hydroxyl radical, and peroxynitrite (21). Thus, it plays a critical role in oxidative metabolism in the lung. Superoxide dismutases (SODs) are the primary class of enzymes that initiate the process of detoxifying superoxide anion by converting it into hydrogen peroxide. There are three mammalian SODs. SOD1, copper–zinc SOD (CuZnSOD) (22), is predominantly located in the cytosol of cells (22); mitochondrial manganese SOD (MnSOD) (23), or SOD2, is located in the mitochondria as well as the cytosol of cells (23); and SOD3, or extracellular SOD (ECSOD) (24), is located mainly outside of the cells where it exists bound to matrix proteins such as collagen and elastin (24). CuZnSOD accounts for approximately 80% of all SOD activity within the lung (25). This enzyme is located primarily in the bronchial epithelium, alveolar epithelium, mesenchymal cells, arterioles, and capillary endothelial cells of the lung (26, 27). Exogenous oxidants have been shown to activate cytosolic redox-sensitive transcriptional regulators such as nuclear factor-κB (28) and mitogen-activated protein kinases (29). On activation, these factors can induce the transcription of proinflammatory genes (28, 30). Thus, given its location and activity, CuZnSOD is uniquely positioned to defend the lung against the proinflammatory effects of oxidants present in cigarette smoke.
The aim of this current study was to determine whether enhanced expression of CuZnSOD within the lung could protect against the development of emphysema in two separate animal models. To evaluate this, transgenic mice expressing human CuZnSOD were exposed to cigarette smoke for 1 yr. In addition, a separate group of mice were treated with intratracheal elastase to induce emphysema. Comparative analyses were made with exposed littermates and nonexposed age-matched transgenic and littermate mice. This study identified the impact of SOD expression on lung inflammation, oxidative injury, and the subsequent development of emphysematous changes in the lung in response to these insults. The results of this study have been presented in abstract form (31, 32).
The generation of transgenic mice with the human CuZnSOD gene in the C57BL/6xCBA/J background was previously reported (33). A full description is provided in the online supplement.
Mice were exposed to cigarette smoke in a specially designed chamber for 6 h/d 5d/wk for 1 yr at a total particulate matter concentration of 250 mg/m3. Groups of 15 mice were used for these studies. A full description of the protocol is provided in the online supplement.
Mice were sedated with an intraperitoneal, injection of avertin. Then, 1.2 U of porcine pancreatic elastase (Sigma, St. Louis, MO) dissolved in 100 μl of sterile saline were injected into the airway using a microsprayer (Penn Century, Philadelphia, PA) (34) as per established protocol (35). After 3 wk, the mice were killed and the lungs were harvested for histologic analysis. Six mice from each group were used for these studies.
Paraffin-embedded lung tissues were prepared, and sections (3–4 μm) mounted onto slides were stained with hematoxylin and eosin (H&E) for histologic analysis. Specific staining for macrophages was conducted using MAC-3 antibodies (BD Biosciences, Pharmingen, San Diego, CA) as per established protocol (36).
Morphometric analyses were conducted in a standard fashion as previously reported by our laboratory (37, 38). For the smoking experiment, 15 mice were used, and for the elastase experiment, six mice were used. Details are available in the online supplement.
Lung lavage was obtained following established protocols of the lab (38). From each group, 10 mice were used. Full details are provided in the online supplement.
H&E and MAC-3 stained lung-tissue sections were examined for neutrophils and macrophages present within the lung-tissue parenchyma. For these studies 10 mice from each group were used. Full details of the analysis are provided in the online supplement.
Immunofluorescent studies were performed on lung tissue-sections using commercially available antibodies for 3-nitrotyrosine and 8-hydroxydeoxyguanosine. From each group six mice were used for these studies. Quantification of signal was performed using commercially available software (ImagePro; Media Cybernetics, Silver Spring, MD). A complete description of the protocol is provided in the online supplement.
Level of lipid peroxidation was measured in tissue homogenates by using the Lipid Peroxidation Assay Kit II (Calbiochem, San Diego, CA) following the manufacturer's instructions. From each group, 10 mice were used for these studies.
The lung extracts were assayed for myeloperoxidase activity by a spectrophotometric assay according to published methods (39). From each group, 10 mice were used for these studies.
Equal amounts of protein were loaded and gelatin zymography was performed on lung homogenate as previously described (40).
The culture media collected from A549 cells and peritoneal macrophages was tested in a neutrophil chemotaxis assay. Comparative analyses were made between untreated cells and cells treated with extracellular SOD (100 μg/ml). Full details are available in the online supplement. In a separate series of experiments, A549 cells were transfected using LipofectAMINE reagent (Invitrogen, Carlsbad, CA), 16 μg/ml, with plasmid containing the same DNA construct (CuZnSOD) that was used to create the transgenic mice. At 24 h after the transfection, the media from transfected and control A549 cells was collected and then tested for chemotactic ability using a neutrophil chemotaxis assay. All experiments were performed in triplicate and repeated on at least two occasions.
A neutrophil chemotaxis assay was performed on the cell-culture supernatants using neutrophils isolated from human blood. A full description is provided in the online supplement.
Lung RNA was extracted with Trizol (Invitrogen), according to the manufacturer's instructions, and expression of mRNA for matrix metalloproteinases (MMPs) was assessed using the RPA system from Pharmingen (Becton Dickinson, San Diego, CA). Four samples from each group were used for this analysis.
Data are expressed as the mean ± SEM. Two-way analysis of variance measures were conducted using commercially available software (Microsoft Excel, Seattle, WA). A difference was considered statistically significant at p = 0.05.
CuZnSOD transgenic mice and wild-type littermates were exposed to cigarette smoke as described in Methods. Inflammatory changes were examined in the lung lavage and tissue sections and compared with corresponding material from nonexposed wild-type littermates and nonexposed CuZnSOD transgenic mice. The sample size was n = 10 for each of the four groups for all the lung lavage and lung parenchyma measurements. The presence of the transgene markedly decreased lung neutrophilia in response to cigarette-smoke exposure. In wild-type littermate mice, an increase in neutrophil numbers within the lung tissue (3.6 cells/20 fields baseline vs. 9.2 cells/20 fields smoke exposed; p < 0.002; Figure 1A) was seen on smoke exposure. Similarly, in these mice there was an impressive increase in neutrophils in the lung lavage (0 baseline vs. 818 smoke exposed; p < 0.05; Figure 1B) in response to smoke. The smoke-exposed transgenic mice, however, did not demonstrate an increase in the number of neutrophils detected in the lung tissue (3.8 cells/20 fields baseline vs. 3.7 cells/20 fields smoke exposed; Figure 1A) and there was only a small increase in lung lavage neutrophils (0 baseline vs. 178 smoke exposed; Figure 1B) after smoke exposure. To further quantify the effect of SOD on lung neutrophils, the myeloperoxidase activity in the lung was measured. The activity of the lung tissue homogenate of the wild-type littermate control mice more than doubled in response to cigarette-smoke exposure (190 U/mg vs. 550 U/mg; n = 10 for each group; Figure 1C), consistent with the increase in neutrophil influx. However, the CuZnSOD transgenic mice did not demonstrate an increase in myeloperoxidase activity after smoke exposure. The histologic and lavage data demonstrate that CuZnSOD decreases the influx of neutrophils into the lung in response to cigarette smoke exposure.
Cigarette smoke caused a significant increase in the number of macrophages in the lung parenchyma of the wild-type littermate control mice (31 ± 8 cells/10 fields baseline vs. 56 ± 3 cells/10 fields smoke exposed; p < 0.05; Figure 2A). Similarly, there was a significant increase in macrophages in the lung lavage (28 × 104 baseline vs. 78 × 104 smoke exposed; p < 0.002; Figure 2B) in these mice. These inflammatory changes after smoke exposure were blunted in the transgenic mice. The smoke-exposed transgenic mice had a small increase in macrophages in the lung parenchyma (20 ± 6 cells/10 fields baseline vs. 33 ± 4 cells/10 fields smoke exposed; p value was not significant; Figures 2A). Likewise, there was a smaller increase in the number of macrophages in the lung lavage of the transgenic mice (16 ± 4 × 104 cells baseline vs. 42 ± 4 × 104 cells smoke exposed; p value was not significant; Figure 2B). The average macrophage cell size measured from the lung sections was identical in the smoke-exposed and nonexposed mice (37 μ2). Thus, the increase in macrophages that was detected cannot be attributed to an “edge effect” due to an increase in macrophage cell size (41). It is important to note, however, that the transgenic mice had fewer macrophages in their lungs at baseline. This may account for the lower levels of macrophages in the smoke-exposed transgenic mice compared with the littermates.
Staining for the oxidative markers 8-hydroxydeoxyguanosine (8[OH]-dG) and 3-nitrotyrosine can be seen in the smoke-exposed wild-type littermate mice when compared with nonexposed mice (Figures 3A and 3B; n = 6 for all groups for both markers). The staining was primarily detected within airway epithelial cells in large and medium-sized airways with none detected in the alveolar regions of the lung. There was a significant increase in signal intensity in the airway regions of the smoke-exposed littermate mice (40 mean fluorescent pixel intensity/airway area vs. 6 mean fluorescent pixel intensity/airway area for 8[OH]-dG, p < 0.04; and 35 mean fluorescent pixel intensity/airway area vs. 1 mean fluorescent pixel intensity/airway area for 3-nitrotyrosine, p < 0.02). In contrast, the airway epithelium of the smoke-exposed transgenic mice did not exhibit staining for these oxidative markers (Figures 3A and 3B, panel D). Signal intensity for both markers was markedly decreased in the smoke-exposed transgenic versus smoke-exposed control mice (11 mean fluorescent pixel intensity/airway area vs. 40 mean fluorescent pixel intensity/airway area for 8[OH]-dG, p < 0.05; and 5 mean fluorescent pixel intensity/airway area vs. 35 mean fluorescent pixel intensity/airway area for 3-nitrotyrosine, p < 0.01). To further quantify the oxidative damage present in the lung, lung lipid peroxidation measurements were performed. As seen in Figure 3C, cigarette-smoke exposure led to a significant increase in lipid peroxidation in wild-type littermate mice (n = 10 for each group tested). The CuZnSOD transgenic mice, however, exhibited no increase in lung lipid peroxidation in response to smoke exposure. These data provide evidence that CuZnSOD expression can prevent oxidative damage in the smoke-exposed lung.
Given the importance of proteases in the pathogenesis of emphysema, an RNAse protection assay was conducted to determine whether cigarette smoke exposure and the CuZnSOD transgene impacted on the expression of proteases within the lung. Four mice from each group were tested. The expression of MMP-12 and MMP-13 was significantly up-regulated in the smoke-exposed littermate mice (see Table 1). This induction did not occur in the smoke-exposed SOD mice. The expression of MMP-9 appeared to be unchanged from its baseline levels in both littermate and transgenic mice exposed to cigarette smoke (Table 1). Gelatin zymograms were also performed on lung tissue homogenates (data not shown). Consistent with the RNA data, no significant change in the level of MMP-9 was detected between wild-type littermate control mice and SOD transgenic mice.
Nonexposed Littermate Mice
Nonexposed SOD Mice
Smoke-exposed Littermate Mice
Smoke-exposed SOD Mice
|MMP-13||0.29 ± 0.04||0.28 ± 0.02||0.42 ± 0.04*||0.30 ± 0.03|
|MMP-12||0.32 ± 0.02||0.29 ± 0.02||0.50 ± 0.08*||0.29 ± 0.02|
|MMP-3||0.45 ± 0.02||0.56 ± 0.02||0.60 ± 0.06||0.46 ± 0.03|
|MMP-9||0.48 ± 0.10||0.53 ± 0.06||0.51 ± 0.04||0.45 ± 0.09|
We then examined if the increase in SOD could protect mice from the development of smoke-induced emphysema. Morphometric analyses were conducted on 10 mice in each group. There was a significant increase in mean linear intercept (Lm) in smoke-exposed littermate mice compared with nonexposed littermate mice (Figure 4 and Table 2) comparable to the published literature (42, 43). The expression of CuZnSOD prevented the formation of smoke-induced emphysema in the transgenic mice (Table 2; p < 0.04).
To determine if the expression of SOD protected from inflammation and subsequent emphysema formation in an animal model of emphysema generated through protease imbalance, the SOD transgenic mice were examined after intratracheal instillation of elastase and compared with wild-type littermates. Six mice in each group were used for this experiment. SOD conferred significant albeit not complete protection against the development of emphysema (Figure 4) as measured by changes in Lm (Table 2).
SOD expression decreased the number of inflammatory cells present in the lung after smoke exposure. The most striking effect of SOD, however, was its ability to completely blunt the smoke-induced increase in lung neutrophils. Macrophages and lung epithelial cells mediate the influx of neutrophils in response to cigarette-smoke exposure through chemotaxis. Therefore, we sought to determine in vitro if SOD treatment could directly affect the chemotactic potential of these cell types. A549 cells and peritoneal macrophages were treated with extracellular SOD and the neutrophil chemotactic activity of the media was measured using a modified Boyden chamber (44). At least six samples from each study group were tested. SOD treatment significantly decreased neutrophil chemotaxis in the media from both macrophages (directed/random ratio, 1.25 vs. 1.08; p < 0.02; Figure 5A) and A549 cells (directed/random ratio, 1.38 vs. 1.08; p < 0.009; Figure 5B). SOD transfection also decreased neutrophil chemotaxis in A549 cells (directed/random ratio, 1.73 vs. 1.42; p < 0.03; Figure 5C). These data demonstrate that SOD is able to act directly upon macrophages and lung epithelial cells and to inhibit neutrophil chemotaxis.
Oxidative stress has been implicated in the pathogenesis of COPD (8). Free radicals from inhaled cigarette smoke and oxidants endogenously formed by inflammatory cells expose the lung to an increased oxidant burden. Despite these observations, studies assessing the benefits of antioxidants for COPD have yielded mixed results. Antioxidants have been shown to decrease smoke-induced oxidative damage in animal (45, 46) and human studies (47). In addition, numerous epidemiologic studies have found a positive correlation between dietary intake of antioxidants and lung function (6, 48). However, clinical trials evaluating the efficacy of antioxidants have been less impressive. N-acetylcysteine, a precursor of glutathione, did not affect disease progression or exacerbation rate in a large-scale, randomized trial (49). These variable results may in part be explained by difficulties in obtaining sufficient levels of antioxidants within the lung and also dependent on the specific antioxidant utilized. This study demonstrates that a fourfold increase in CuZnSOD activity within the lung decreases lung inflammation, oxidant injury, protease expression, and emphysema formation in response to chronic smoke exposure and elastase administration. These results suggest that superoxide is a key mediator of the pathophysiologic responses that lead to the development of emphysema.
The formation of superoxide is the prime event in the development of oxidative injury. The main site of production is the mitochondria where an electron is transferred to a recipient oxygen molecule during oxidative metabolism. SOD is the primary enzyme to defend the lung from the damaging effects of superoxide. It does this by converting superoxide into hydrogen peroxide, which can then be broken down into water by antioxidants such as catalase and glutathione. If SOD activity is inadequate, superoxide can interact with nitric oxide (NO·) to form peroxynitrite (ONOO·−). This oxidant can react to form the potent and toxic hydroxyl (OH·) and nitrogen dioxide (NO2·) radicals, which are highly damaging to cell protein, lipids, and DNA. At baseline, the transgenic mice in the present study have a fourfold increase in CuZnSOD activity within their lungs (33) and cigarette smoke did not further induce this expression (data not shown). This enhanced antioxidant activity sufficed to protect the lungs of the mice from smoke-induced free-radical injury. The oxidative markers examined, 8-hydroxydeoxyguanosine, 3-nitrotyrosine, and lipid peroxidation, have all been detected in patients with COPD (50–52). In fact, the levels of these markers have been shown to correlate negatively with lung function (51, 52) and positively with lung inflammation (51). Given the positive association between inflammation and free-radical markers, it is conceivable that the lack of oxidative damage in the transgenic mice may have prevented the induction of inflammatory responses following cigarette smoke exposure.
An intriguing finding of the present study is the ability of CuZnSOD to prevent the increase in neutrophils after chronic cigarette smoke exposure and elastase treatment. Oxidants can stimulate neutrophil influx by stimulating the release of cytokines (53–55) and the expression of vascular and intercellular adhesion molecules (56). This influx is critical as the neutrophil plays a central role in the pathogenesis of emphysema (57, 58). Aside from the direct proteolytic lung injury they cause (59, 60), neutrophils can work together with other cell types to impact on the development of emphysema. In this regard, interactions with macrophages are particularly important. Macrophage-derived proteases such as MMP-1, MMP-9, and MMP-12 are key effectors of emphysema formation (61–63). Neutrophil elastase can inactivate tissue inhibitors of metalloproteinases (TIMPs), thus rendering the lung more susceptible to MMP-induced tissue damage (64). In addition, neutrophil elastase cleaves elastin peptides and complement factors that serve as potent chemoattractants for macrophages (65, 66). By preventing the influx of neutrophils into the lung, SOD blocked the inflammatory cascade that results in the formation of emphysema. This suggests that smoke-derived oxidants have a critical role in the initiation of the disease.
Our results demonstrate that SOD also protects against the formation of emphysema in the elastase-induced model of emphysema. Although the elastase- and smoke-induced models are distinct, they share similar mechanisms (i.e., proteolytic damage, inflammation, and matrix remodeling). The Lm method that we used to measure airspace enlargement is a relatively insensitive technique that fails to detect emphysema in up to 32% of patients (67, 68). Despite its lack of sensitivity, the marked reduction in Lm that we observed in the SOD elastase-treated mice demonstrates the protective effect of this antioxidant. This finding underscores the important interactions that occur between proteases and oxidants in the development of this disease. Elastase has been previously shown to induce reactive oxygen species in the lung (69). These free radicals can augment proteolytic damage by inactivating protease inhibitors (70), activating the latent form of proenzymes (71), and inducing the expression and release of proteases from both inflammatory cells and resident lung epithelial cells (72). In addition, oxidants are potent stimulators of cytokines (73), which further drive the inflammatory response generated by elastase. Once they are drawn to the lung, inflammatory cells release proteases that can lead to dysfunctional matrix remodeling. Antioxidants such as SOD can interrupt this self-perpetuating cycle and prevent the processes that ultimately lead to emphysema formation.
This study provides important insights of the role of antioxidants on the development of emphysema. The expression of CuZnSOD effectively counteracted the smoke-induced increase in oxidative damage, protease expression, and inflammation that occurred in exposed mice. Most important, SOD expression prevented the formation of emphysema in two separate animal models of the disease. This protective effect of SOD correlated with a marked decrease in neutrophil influx into the lung. Thus, these results not only denote the importance of superoxide dismutase but also affirm the central role of neutrophils in the pathogenesis of emphysema.
The authors thank Tina Zelonina and Jincy Thankachen for their technical assistance.
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