Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS) are frequent complications in critically ill patients and are responsible for significant morbidity and mortality (1, 2). Treatment of the underlying disease and excellent supportive care using “lung-protective” strategies of mechanical ventilation (3) contribute to successful clinical outcomes. However, aside from the use of activated protein C in the subset of ALI/ARDS patients with sepsis (4), specific therapies are lacking, and the cascade of events leading to ALI and ARDS, once initiated, is much less amenable to specific treatment modalities.
Regardless of the underlying illness, the clinical and pathologic manifestations of ALI/ARDS are very similar, indicating the existence of final common pathways that represent potential therapeutic targets (1, 5). In essence, these syndromes reflect severe injury leading to dysfunction and compromise of the barrier properties of the pulmonary endothelium and epithelium as a consequence of an unregulated acute inflammatory response (6). In this hypothetical construct, an initiating event (sepsis, shock, trauma, multiple transfusions, pancreatitis, etc.) leads to activation of the acute inflammatory response on a systemic level. One of the earliest manifestations is activation of pulmonary endothelium and macrophages (alveolar and interstitial), upregulation of adhesion molecules, and production of cytokines and chemokines that induce a massive sequestration of neutrophils within the pulmonary microvasculature. These cells transmigrate across the endothelium and epithelium into the alveolar space and release a variety of cytotoxic and proinflammatory compounds, including proteolytic enzymes, reactive oxygen species (ROS) and nitrogen species, cationic proteins, lipid mediators, and additional inflammatory cytokines (Figure 1)
Figure 1. Mechanisms of lung injury. Step 1: In response to an inciting event such as LPS production as a result of a bacterial infection, the pulmonary macrophages and endothelium become activated and upregulate surface expression of adhesion molecules. Step 2: This leads to neutrophil adhesion and subsequent transmigration from the intravascular space into the alveolus. Step 3: The activated neutrophil produces a plethora of inflammatory mediators that include reactive oxygen species (ROS) such as OH− and O2− and nitric oxide (NO), cytokines, chemokines, proteases, and cationic proteins. In addition to the activated neutrophil, the other cells that make up the alveolar unit, including the pulmonary monocytes and macrophages, as well as the pulmonary endothelial and epithelial cells also contribute significantly to the production of inflammatory mediators. The neutrophil, however, is the major source of ROS production in the context of acute lung injury and ARDS. In the current study, α-tocopherol (α-TOC) provides significant protection from lung injury lung following exposure to lipopolysaccharide (LPS) and correlates with a significant impairment of neutrophil recruitment into the alveolar airspace. The potential mechanisms by which α-TOC confers a protective effect are discussed in the text. These include (i) prevention of neutrophil adhesion by abrogating the expression of adhesion molecules on the surface of endothelial cells and neutrophils; (ii) impaired neutrophil binding to the endothelium due to changes in the topographic distribution or binding affinities of the surface adhesion molecules; and (iii) impaired neutrophil transmigration due to changes in the membrane stability of the migrating neutrophil, the endothelial, and/or the epithelial cell.
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Since the original description of ARDS more than 30 years ago (8), investigators have endeavored to elucidate the mechanisms regulating the pathways leading to lung injury, with the ultimate goal of developing therapeutic tools to ameliorate or prevent lung and multi-organ injury in patients at risk. One important facet of ALI/ARDS, and one that is the focus of a recent manuscript published in the AJRCMB (9), is oxidative injury to the lung mediated by ROS, partly reduced derivatives of molecular oxygen (10–13). Biologically important ROS include superoxide anion radical (O2−), hydrogen peroxide (H2O2), hydroxyl radical (OH−), and hypohalous acids such as HOCl (Table 1)
Stable Species | Reactive Species |
|---|---|
| O2 | O2− |
| OH− | |
| H2O2 | |
| HOCl | |
| O | |
| NO* | −OONO |
| NO· | |
| NO2− | |
| NO3− |
To neutralize free radicals and counteract the detrimental effects of ROS generated during normal metabolism, cells express a number of endogenous antioxidants such as superoxide dismutase, catalase, and glutathione peroxidase (reviewed in Ref. 21). However, these antioxidants are rapidly overwhelmed during an acute inflammatory response.
In the context of ALI/ARDS, there are many potential sources of ROS, including itinerant and resident leukocytes (neutrophils, monocytes, and macrophages), parenchymal cells (endothelial and epithelial cells, fibroblasts, and myocytes), circulating oxidant-generating enzymes (xanthine oxidase), and inhaled gases with high concentrations of oxygen that are often used during mechanical ventilation.
There is much experimental evidence supporting the role of oxidants and oxidative injury in the pathogenesis of ALI/ARDS. Evidence of oxidative stress and injury is common to most experimental models of lung injury and is corroborated in studies of patients with ALI/ARDS, underscoring the physiologic importance of these processes (11, 22–24). For example, patients with ARDS have increased levels of H2O2 in exhaled breath condensate (25, 26). Moreover, bronchoalveolar lavage fluid from these patients contains an excess of oxidatively modified proteins in combination with a relative deficiency in antioxidant molecules such as glutathione (27–30). Thus, although there are many checks and balances in this system in the form of antioxidant defenses, in ALI/ARDS there is extensive overproduction of ROS to the extent that endogenous antioxidants are overwhelmed, permitting oxidative cell damage.
Leukocytes, principally neutrophils and macrophages, are generally considered to be the most prodigious source of ROS in this setting (31, 32). Leukocytes express two enzyme systems, the NADPH oxidase (33) and nitric oxide synthases (NOS) (34, 35) that can generate reactive species in substantial amounts. The large numbers of activated neutrophils in the lung in ALI/ARDS has focused attention on these phagocytes as a major source of ROS. Diverse proinflammatory compounds including lipopolysaccharide (LPS), cytokines, chemokines, complement fragments, clotting fragments, and lipid mediators that are elevated in patients with ALI/ARDS, are capable of priming and/or activating neutrophils to generate ROS.
It is also evident that nonprofessional phagocytes including endothelium (36, 37), epithelium (38), fibroblasts (39), and smooth muscle cells (40) express oxidases capable of generating physiologically important amounts of ROS. Other sources of ROS include mitochondrial electron transport chain (41), cytochrome P450 (42), and xanthine oxidase (43, 44). Finally, inhaled oxidants including high concentrations of oxygen used in mechanical ventilation can contribute to formation of ROS.
As mentioned above, various cellular components can be subject to oxidative modifications including membrane, cytosolic, and nuclear lipids and proteins. Cellular membranes and especially plasma membranes are primary targets of ROS. The fatty acid side chains of membrane phospholipids undergo peroxidation under oxidative stress (45). Membrane fluidity is dependent on lipid composition of the plasma membrane, and alterations in this composition, including those by oxidation, profoundly influence diverse aspects of membrane function. In the context of acute inflammation, oxidation of components of the endothelial or epithelial plasma membrane could facilitate neutrophil recruitment into the lung by compromising the barrier function of these cells, thereby allowing leakage of chemokines and other chemoattractant molecules into the vascular space. Additionally, oxidant exposure can lead to enhanced leukocyte adhesion either by direct oxidative modification of components of the endothelial plasma membrane generating lipid mediators or by “inside-out” signaling leading to enhanced surface expression and affinity of adhesion molecules (46). Interestingly, the antioxidant α-tocopherol (α-TOC) decreases the expression of adhesion molecules on both neutrophils (47) and activated endothelial cells (48).
Cytosolic and nuclear events initiated by oxidants might also contribute to inflammatory injury and be amenable to pharmacologic intervention. For example, elevation of cytokines and chemokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-2, IL-6, and IL-8 is a feature common to lung injury of diverse etiologies. Cytokine expression is primarily regulated at a transcriptional level. NF-κB is a DNA-binding factor that stimulates transcription of many different cytokines involved in acute inflammation, and is activated in ALI and ARDS (49–51). NF-κB is normally a heterodimer that is sequestered in the cytosol in the quiescent state by association with the IκB family of inhibitors. Upon stimulation, IκB becomes phosphorylated and dissociates from NF-κB, allowing the free NF-κB to translocate to the nucleus, where it binds to promoter regions of specific genes and induces transcription. There is evidence that NF-κB is activated in the context of ALI/ARDS and may be regulated by changes in IκB expression that are in turn dependent in on oxidants produced by, for example, xanthine oxidase (49–51).
Attempts to attenuate lung injury have focused on modulation of the signaling pathways leading to increased inflammatory cytokine/chemokine production, and on restoration of the oxidant/antioxidant balance to limit the degree of oxidative cell damage. Because NF-κB is the final common step of transcriptional regulation of inflammatory cytokine expression, modulation of its activity has been the target of a number of anti-inflammatory drugs such as glucocorticoids. These agents inhibit NF-κB and also impair binding of another transcription regulatory factor, activator protein-1, to DNA and thus prevent cytokine-induced transcription of proinflammatory genes. Although glucocorticoids certainly dampen the inflammatory response and inhibit cytokine expression, administration of these drugs is often associated with untoward effects, such as a generalized depression of the immune response, that are clearly detrimental to acutely ill patients, many of whom have concomitant infections.
Another strategy to limit oxidative lung injury is to restore the oxidant/antioxidant balance by augmenting the intracellular pool of antioxidants. Early studies have attempted to augment intracellular glutathione stores by delivery of glutathione by various methods, including intravenous, intraperitoneal, and aerosol administration (52, 53). A major problem with this strategy is that glutathione must be metabolized extracellularly before being taken up by cells. Although aerosolized glutathione did increase glutathione levels in airway epithelial lining fluid, no increase in intracellular levels was achieved (11, 53, 54).
Another potential method to augment intracellular glutathione levels is to administer precursors such as N-acetylcysteine (NAC). NAC repletes intracellular cysteine, a glutathione precursor resulting in increased intracellular glutathione. NAC is known to protect cells from oxidative injury (55–57) and is standard therapy for acetaminophen overdose, where it presumably acts by preventing the free radical–induced hepatic injury. In small clinical trials of patients with ARDS, treatment with intravenous NAC (58, 59) and L-2-oxothiazolidine-4-carboxylate (OTZ), a related compound with a similar mechanism of action (60), was associated with improved cardiorespiratory physiologic parameters and better clinical outcomes. Although administration of NAC and OTZ did result in increased intracellular glutathione levels, the clinical effects of the drugs were modest and short-lived (60).
α-TOC (or vitamin E), the subject of a recent study published in the AJRCMB (9), is a naturally occurring antioxidant that is present in all cell membranes at low concentrations. It has cytoprotective properties that are largely attributed to its ability to scavenge highly reactive oxygen species, thus attenuating lipid peroxidation of cellular membranes (61, 62). Indeed, in a rat model of hypoxia-induced lung injury (63), administration of liposomal α-TOC inhibited lipid peroxidation of lung tissue and restored the phosopholipid composition of the lung to the normal control levels. These effects were associated with improved oxygenation and lung mechanics. α-TOC is reported to have additional properties including the ability to modulate the immune response by enhancing both cell-mediated and humoral immunity (47). Although the mechanisms of these activities have not been well defined, α-TOC inhibits protein kinase C, a ubiquitously expressed signaling molecule that is involved in the early steps of many proinflammatory signaling pathways (47, 64).
Rocksén and coworkers addresses the role of α-TOC as a therapeutic agent in the treatment of mice with ALI induced by aerosolized LPS (9). Administration of liposomal α-toc (given intraperitoneally 1 h after LPS exposure) resulted in significant protection against lung injury. There are several seminal observations reported in this manuscript. First, a beneficial therapeutic effect of α-TOC was noted even when administered following LPS exposure. Previous studies have primarily assessed the effect of agents given before or concomitant with the inciting injury, a paradigm that is often not applicable in the clinical setting of ALI/ARDS. Second, α-Toc appears to exert its protective function via a mechanism that is distinct from that of glucocorticoids. Third, the beneficial effects of α-Toc are independent of effects on cytokine and chemokine transcription or expression. The molecular mechanisms responsible for these beneficial effects remain to be clarified, but could involve direct affects on endothelial or epithelial membranes.
In this study, aerosolized LPS induced an inflammatory response manifest by neutrophil recruitment into the alveolar space accompanied by ROS production and inflammatory cytokine production. Treatment with α-Toc, even at low doses following LPS exposure, significantly decreased neutrophil transmigration into the alveolar space and diminished neutrophil ROS production. When the effect of α-Toc was compared with dexamethasone, a synthetic glucocorticoid, α-Toc was observed to exert its protective role via a distinct and additive mechanism. Unlike dexamethasone, α-Toc had no effect on the NF-κB translocation, transcriptional regulation or expression of inflammatory cytokines. An intriguing observation is that α-Toc treatment prevented LPS-induced neutrophil recruitment into the alveolar spaces and diminished oxidant production by alveolar neutrophils, although peripheral blood neutrophilia and pulmonary vascular sequestration of neutrophils were unaffected.
What, then, are the relevant targets of α-Toc and the mechanism(s) by which it prevents lung injury and pulmonary edema following endotoxin challenge? The data from the study by Rocksén and coworkers (9) strongly suggest that α-Toc inhibits neutrophil recruitment into the alveolar spaces and consequently prevents cytotoxic and oxidative cell injury to the endothelium and epithelium. Diminished neutrophil recruitment following α-TOC treatment could be a result of inhibition of neutrophil adhesion to endothelium. Indeed, α-TOC is known to suppress endothelial and leukocyte adhesion molecule expression (47, 65). In this study (9), neutrophil expression of CD11b/18 by intrapulmonary neutrophils was diminished following α-TOC treatment. The effect of α-TOC on endothelial cell activation and adhesion molecule expression was not addressed.
A second mechanism by which α-TOC could inhibit neutrophil adhesion to the endothelium is by altering the topographical distribution and/or binding affinities of surface adhesion molecules on either neutrophils or endothelial cells. Because α-TOC can attenuate lipid peroxidation (66, 67) and thus stabilize membrane lipids, it is possible that α-TOC prevents oxidant-induced alterations in membrane fluidity, thereby diminishing binding of neutrophils by endothelial adhesion molecules or entrapment of neutrophils in capillaries due to alterations in cellular biomechanical properties (68).
Stabilization of the plasma membrane by α-TOC could also affect the ability of neutrophils to transmigrate across the endothelium and epithelium. As discussed earlier, diapedesis of neutrophils through the endothelium and epithelium requires significant alterations of the cytoskeleton and intercellular junctions as the cell squeezes between endothelial and epithelial cells. Under normal circumstances, endothelial and epithelial cells form barriers that retard leakage of fluid and solutes from the intravascular to the extravascular and alveolar compartments. However, unregulated activation of neutrophils during transmigration may result in disruption of endothelial (69) and epithelial intercellular junctions (70) and alter endothelial and epithelial cell morphology to allow passage of the neutrophil. It is plausible that α-Toc prevents these membrane alterations, thus diminishing neutrophil transmigration.
To be certain, there are limitations to the study by Rocksén and colleagues (9). First, although the use of areosolozed LPS induces a vigorous pulmonary inflammatory response with resultant lung injury, this model does not reflect the complex spectrum of human ALI/ARDS that occurs in association with sepsis and shock. Second, there are important differences between murine and human neutrophils, such as the prominence of NO production and lack of defensins in the former, which might be important in the pathogenesis of inflammatory tissue injury. For these and other reasons, therapeutic interventions that have shown great promise in animal studies have yielded mixed results in human trials including the use of antioxidants in ARDS (57–59, 71). It is important to bear these limitations in mind before extrapolating the results of the current study, no matter how promising, to human ALI/ARDS.
Regardless of the targets of α-Toc and the mechanisms by which it exerts its protective effects in lung injury, the results of the study by Rocksén and coworkers (9) offer an exciting potential new therapeutic approach to the management of ALI/ARDS. Furthermore, the questions that this manuscript raises about the potential mechanisms of α-Toc activity will provide an exciting basis for future research into the mechanisms of oxidant-induced cellular and organ injury, not only in the context of ALI but also in other inflammatory diseases, including arthritis, ischemia–reperfusion injury, transplant rejection, atherosclerosis, and congestive heart failure.
This article was supported by funds from the Canadian Institutes of Health Research and the National Sanatorium Association.
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