American Journal of Respiratory and Critical Care Medicine

Previously we reported that alcohol abuse increases the incidence of the acute respiratory distress syndrome (ARDS) in septic patients, and that chronic ethanol ingestion in rats depletes alveolar epithelial glutathione and increases endotoxin-mediated lung edema. In this study we examined a potential mechanism by which ethanol-induced glutathione depletion could predispose to acute lung injury. We hypothesized that glutathione depletion activates matrix metalloproteinases (MMPs), thereby increasing degradation of the alveolar extracellular matrix (ECM) during sepsis. Ethanol-fed rats (20% vol/vol in water for 6 wk) were given endotoxin (2 mg/kg, intraperitoneally) followed 2 h later by lung isolation and ex vivo perfusion with n-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 7 M). Ethanol ingestion increased (p < 0.05) MMP-9 and MMP-2 activity, as determined by zymography, in the lung tissue and lavage fluid compared with control-fed rats, and increased (p < 0.05) levels of the 7S fragment of type IV collagen in the lung lavage fluid. Ethanol ingestion increased activation, but not production, of the MMP-9 and MMP-2 zymogens. Finally, although concomitant ingestion of N-acetylcysteine had no effect (p > 0.05) on MMP production, it increased (p > 0.05) lung glutathione levels, blocked (p < 0.05) MMP-9 and MMP-2 activation, and decreased (p < 0.05) levels of the 7S fragment of type IV collagen. We conclude that chronic ethanol ingestion, via glutathione depletion, activates MMPs during sepsis, thereby increasing degradation of the alveolar epithelial ECM. Lois M, Brown LAS, Moss IM, Roman J, Guidot DM. Ethanol ingestion increases activation of matrix metalloproteinases in rat lungs during acute endotoxemia.

The acute respiratory distress syndrome (ARDS) is a common form of acute lung injury with a high mortality (approximately 50%) and for which there are no effective therapies. One hallmark of ARDS is disruption of the alveolar epithelial extracellular matrix (ECM) (1). This abnormality appears to contribute to the alveolar flooding with proteinaceous fluid that characterizes the syndrome. However, the mechanisms by which the ECM is altered during acute lung injury are largely unknown.

Recent evidence suggests that degradation of the ECM by specific enzymes known as matrix metalloproteinases (MMPs) may contribute to the development of acute lung injury. A subgroup of these enzymes known as type IV collagenases including MMP-9 (also known as gelatinase A) and MMP-2 (also known as gelatinase B), can degrade basement membrane components including (type IV) collagen, fibronectins, and gelatin. Their role in the pathogenesis of ARDS has been suggested by the observation that these enzymes are increased in the lung lavage fluid of ARDS patients (2-4). In addition, patients with ARDS have increased levels of the 7S fragment of type IV collagen in their serum (5) and in their lung lavage fluid (6), suggesting that the increased levels of MMP-9 and MMP-2 lead to increased degradation of the alveolar epithelial ECM during acute lung injury.

Until recently, the alveolar ECM was considered to be a static structural component of the lung tissue. However, it is now recognized that in addition to its structural function, the ECM serves as a modulator of cell growth and development, inflammation, angiogenesis, cell migration, tissue differentiation, and repair. As these processes occur, they influence both the cellular and matrix composition and the ultimate fate and functionality of the newly formed tissue. There is growing evidence that aberrant remodeling of the extracellular matrix (ECM) contributes both to the early inflammatory phase as well as to the later fibroproliferative phase of the syndrome (1, 7-9).

Although diverse insults, including sepsis, trauma, and aspiration, place patients at risk for ARDS, we cannot predict which patients will develop the syndrome. Recently we identified that alcohol abuse significantly increases the incidence and severity of ARDS in at-risk patients (10). Based on this observation, we developed an animal model of acute lung injury in rats chronically fed with ethanol in order to study the pathophysiological mechanisms involved in the development of this syndrome. Using this model, we determined that chronic ethanol ingestion decreased the levels of the critical antioxidant glutathione in the alveolar epithelial cells and lining fluid, and increased edematous lung injury from endotoxin (11). This study suggested that ethanol-mediated disruptions in glutathione homeostasis could play a key role in the development of ARDS. However, the precise mechanism by which ethanol ingestion induces edematous lung injury, and the role of glutathione in this process, are unknown. One possibility is that low glutathione levels in the alveolar epithelial lining fluid may create a milieu that favors tissue remodeling, a process that is central to the morphological and physiological consequences of ARDS. Because the tissue-based activity of MMPs appears to be influenced by glutathione homeostasis (12-15), we hypothesized that chronic ethanol ingestion, via depletion of glutathione, could increase MMP activity within the alveolar epithelial space and thereby increase damage to the alveolar ECM during acute inflammatory stresses such as sepsis.

In this report, we tested the aforementioned hypothesis by examining the effects of chronic ethanol ingestion on one aspect of tissue remodeling, namely ECM degradation. We tested whether chronic ethanol ingestion induces the production and/or activation of matrix-degrading members of the MMP family. In particular, we focused on the production and activity of MMP-9 and MMP-2, also known as type IV collagenases, because their levels are increased in the lung lavage fluids of ARDS patients (2-4). We then tested whether or not glutathione replacement, even in the setting of chronic ethanol ingestion, could mitigate the effects of ethanol on MMP-9 and MMP-2 activity during endotoxemia.

Ethanol Feeding of Rats

Young adult male Sprague-Dawley rats (150 to 200 g; Charles River Laboratories, Inc., Wilmington, MA) were fed a standard chow diet with or without ethanol added to their drinking water which they consumed ad libitum. In the first set of experiments, we determined the effects of ethanol ingestion on lung MMP activity. Ethanol-fed rats (n = 8) were given ethanol (5% vol/vol in water) for 1 wk, then 10% vol/vol for 1 wk, and then 20% vol/vol for 4 wk. Control-fed rats (n = 8) received only water. In a second set of experiments in which the role of glutathione homeostasis in MMP activation and lung injury was assessed, another group of ethanol-fed rats (n = 6), N-acetylcysteine (0.163 mg/ml; Sigma, St. Louis, MO) was added to the ethanol/water mixture throughout the feeding period. A separate group of control-fed rats (n = 6) and ethanol-fed rats (n = 6) were used in these experiments.

Endotoxin Treatment and Ex Vivo Lung Perfusion

Ethanol-fed and control-fed rats were given endotoxin (2 mg/kg in saline of Salmonella typhimurium, antigenic properties 4.5.12:1 1.2; Sigma, St. Louis, MO) or saline intraperitoneally. This treatment with endotoxin produces acute endotoxemia and sequestration of circulating neutrophils in the pulmonary capillaries (16). After 2 h, rats were anesthetized with pentobarbital (60 mg/kg, intraperitoneally), a tracheostomy cannula was placed and secured with 2-O ligature, and the lungs and heart were excised en bloc. We employed the identical ex vivo ventilation and perfusion protocol that we published previously (11). Lungs were ventilated with a tidal volume of 3 ml at a rate of 60/ min with 2.5 cm of H2O positive end-expiratory pressure with a gas mixture containing 5% CO2, 21% O2, and 74% N2. Lungs were perfused at a rate of 40 ml/kg body weight/min with Earle's balanced salt solution (Sigma) to which was added sodium bicarbonate (2.2 g/L) and Ficoll-70 (40 mg/ml; Sigma), and the final pH adjusted to 7.40. After a 20-min equilibration period, n-formyl-methionyl-leucyl-phenylalanine (fMLP, 10−7 M; Sigma) was added to the perfusate and the perfusion was maintained for 60 min. Lung weight gain was monitored continuously with a force transducer, with the scale set for zero to 8 g. At the end of the perfusion period, the lungs were lavaged with saline (5 ml × 3), and the lung tissue was dissected free of extrapulmonary tissue, placed in 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma) buffer, and stored at −70° C until samples were processed. The lavage fluid was centrifuged at 500 × g for 5 min and the supernatants and cellular fractions were separated. The cellular fraction was placed in a CHAPS buffer and both the lung lavage supernatant and the cellular fractions were stored at −70° C until samples were processed.

Determination of Glutathione Levels

For determining the levels of reduced glutathione (GSH) and the predominant form of oxidized glutathione (glutathione disulfide or GSSG) in lung lavage fluid, we used a variation of the high-performance liquid chromatography (HPLC) method presented by Martin and White (17). Briefly, each sample was extracted in 5% perchloric acid with 0.2 M boric acid and γ-glutamyl-glutamate (10 μM) as an internal standard. Iodoacetic acid was added and the pH adjusted to 9.0 ± 0.2. After incubation for 20 min to obtain S-carboxymethyl derivatives of thiols, dansyl chloride was added and the samples were incubated for 24 h in the dark. Samples were then separated on an amine column with the solvents described by Reed and coworkers (18). Fluorescence detection was used for separation and quantification of the dansyl derivatives. The dilution of the lung epithelial lining fluid by the saline lavage was estimated by concomitant measurements of urea in the plasma and lavage fluid (19).

Protein Determination

The protein concentrations of the lung tissue homogenates, lung lavage fluids, and the cellular fractions of the lung lavage fluids were determined by a standard assay (20) using a kit from Bio-Rad (Richmond, CA).

Gelatinase Zymography

Sodium dodecyl sulfate–polyacrylamide gels containing 1 mg/ml of gelatin were used to identify proteins with gelatinolytic activity from bronchoalveolar lavage (BAL) fluid, BAL cells, and lung tissue (the same protein content is loaded in each well). Prestained molecular weight markers (Bio-Rad) were included to estimate the molecular weight of the gelatinolytic bands. Purified MMP-2 and MMP-9 standards were run in each gel. Electrophoresis was performed with 25 mA per gel. After electrophoresis, gels were incubated for 60 min in 2.5% Triton X-100, washed and then incubated overnight at 37° C in 50 mM Tris, 10 mM CaCl2. The gel was stained with 0.5% Coomassie Blue R-250 in water/methanol/acetic acid (45:45:10) and then destained with 50% methanol in water. Activity was determined by densitometric analysis with a Molecular Dynamics densitometer using ImageQuant software, version 3.3 (Molecular Dynamics, Sunnyvale, CA). The background staining of the gel was arbitrarily defined as zero, and each band of gelatinase activity was quantitated and compared with each other within the same gels.

In order to confirm that the enzymatic activity obtained was caused by MMPs and not by other enzymes that can degrade gelatin such as serine proteases, gels were also incubated with ethylenediaminetetraacetic acid (EDTA) (10 mM). MMPs are completely inhibited under these conditions, thereby distinguishing them from other gelatinases (21). In order to evaluate the role of MMP activation, samples were activated with 1 mM p-amino-phenylmercuric acetate (APMA) (Sigma Chemical Co., St. Louis, MO) and incubated for 2 h at 37° C after which zymography was performed. APMA activates latent MMPs (zymogens) that have either been synthesized and are still within the cell, or that have been synthesized and secreted into the extracellular environment (21).

Quantitation of 7S Collagen Fragment

An ELISA for the 7S fragment was established using a published protocol (22), with a monoclonal antibody directed against the 7S fragment of type IV collagen (BioDesign, Kennebunk, ME).

Statistical Analyses

Values shown represent mean ± SEM. Values were compared by analysis of variance and corrected by Student-Newman-Keuls test for differences between groups. A p value of < 0.05 was considered significant.

Effect of Ethanol Ingestion on MMP Activity in Isolated Perfused Lungs

To examine the effects of chronic ethanol ingestion on the activity of MMPs in the lungs of rats exposed to endotoxin, we employed the identical model that we used previously (11). In this study, lungs that were isolated from ethanol-fed rats that were treated with endotoxin and then perfused with fMLP had more (p < 0.05) weight gain than lungs isolated from comparably treated control-fed rats (5.1 ± 1.0 versus 1.9 ± 0.5 g). This is consistent with our previous results in this same model (11).

We next examined lung tissue, lung lavage fluid, and the cells from the lung lavage fluid for gelatinase activity using the zymography techniques described previously. We found bands of degradation at 72k and 92k that are consistent with MMP-2 and MMP-9, and these bands ran the same distance in each gel as the purified MMP-2 and MMP-9 (used as positive controls), respectively. In addition, incubation with EDTA completely eliminated these bands of gelatinase activity, consistent with the known properties of MMP-2 and MMP-9.

We determined that lungs isolated from ethanol-fed, endotoxin-treated rats that were perfused with fMLP had higher (p < 0.05) levels of MMP-9 (gelatinase A) and MMP-2 (gelatinase B) activity in the lung tissue compared with lungs isolated from comparably treated, control-fed rats (Figure 1A). Similar results were obtained in the lung lavage fluid supernatant (Figure 1B), and cells from the lung lavage fluid (Figure 1C) of ethanol-fed, endotoxin-treated rats that were perfused with fMLP (p < 0.05).

Lung tissue from control-fed rats had 397 ± 276 relative densitometry units of MMP-9 activity and 415 ± 330 relative densitometry units of MMP-2 activity, compared with 1,743 ± 597 relative densitometry units of MMP-9 activity and 1,593 ± 332 relative densitometry units of MMP-2 activity in the lung tissue from ethanol-fed rats (n = 8 in both groups). In fact, six of eight lungs from control-fed rats had no demonstrable MMP-9 or MMP-2 activity, whereas all eight lungs from ethanol-fed rats had activity of MMP-9 (> 850 relative densitometry units) and MMP-2 (> 650 relative densitometry units). A representative zymography gel is shown in Figure 2. In contrast, lungs isolated from ethanol-fed and control-fed rats that were treated with endotoxin but perfused without fMLP, or were treated only with saline intraperitoneally and then perfused with fMLP, had no demonstrable MMP-9 or MMP-2 activity (not shown). In the lung lavage fluid, three of the eight control-fed rats had no demonstrable MMP-9 or MMP-2 activity (364 ± 250 relative densitometry units for MMP-9 and 693 ± 346 relative densitometry units for MMP-2). By comparison, all of the ethanol-fed rats had MMP-9 and MMP-2 activity in the lung lavage fluid (2,234 ± 241 relative densitometry units for MMP-9 and 2,232 ± 326 relative densitometry units for MMP-2). In contrast, lung lavage fluid obtained from ethanol-fed and control-fed rats that were treated with endotoxin but perfused without fMLP, or were treated only with saline intraperitoneally and then perfused with fMLP, had no demonstrable MMP-9 or MMP-2 activity (not shown).

Finally, in the cells from the lavage fluid (which were > 95% alveolar macrophages in all cases), none of the control-fed rats had any demonstrable MMP-9 or MMP-2 activity, whereas six of eight ethanol-fed rats had demonstrable MMP-9 and MMP-2 activity (1,267 ± 369 relative densitometry units for MMP-9 and 608 ± 233 relative densitometry units for MMP-2). In contrast, cells obtained from the lung lavage fluid from ethanol-fed and control-fed rats that were treated with endotoxin but perfused without fMLP, or were treated only with saline intraperitoneally and then perfused with fMLP, had no demonstrable MMP-9 or MMP-2 activity (not shown).

Effect of Ethanol on Production versus Activation of MMP-9 and MMP-2

To assess if the increased MMP-9 and MMP-2 activity could be accounted for by increased production of the zymogens or increased activation of zymogens already present, gel zymography was performed on lung tissue, lung lavage fluid, and the cells from the lung lavage fluid after treatment with APMA. APMA is an organomercurial compound that activates the zymogens, or latent MMPs, that are present within cells (synthesized) as well as any latent MMPs that are present in extracellular compartments (synthesized and secreted but not activated). We determined that after treatment with APMA, ethanol-fed and control-fed rats had the same (p > 0.05) activity of MMP-9 and MMP-2 in the lung tissue, lung lavage fluid, and cells from the lung lavage fluid (Figure 3).

Effects of N-acetylcysteine in Ethanol-fed Rats

To test the hypothesis that ethanol ingestion increased MMP-9 and MMP-2 activation by depleting the alveolar epithelium of glutathione, we performed another set of experiments with the synthetic glutathione precursor N-acetylcysteine added to the ethanol:water mixture.

Lung edema formation. Lungs from ethanol-fed rats that were treated with N-acetylcysteine had less (p < 0.05) acute edema than lungs from untreated, ethanol-fed rats (lung weight gain 2.9 ± 0.9 g versus 5.8 ± 0.8 g), after endotoxin priming and ex vivo perfusion with fMLP.

Lung lavage fluid levels of glutathione and oxidized glutathione. To examine the effects of chronic ethanol ingestion ± N-acetylcysteine treatment on lung glutathione homeostasis, we measured the levels of GSH and GSSG in the lung lavage fluid following the experimental protocol. We determined that lung lavage fluid from ethanol-fed rats had lower (p < 0.05) levels of GSH than lung lavage fluid from control-fed rats (Figure 4). In contradistinction, lung lavage fluid from ethanol-fed rats that were supplemented with N-acetylcysteine had higher (p < 0.05) levels of GSH than lung lavage fluid from control-fed rats (Figure 4). In contrast, lung lavage fluid from ethanol-fed rats had the same (p > 0.05) levels of GSSG as lung lavage fluid from control-fed rats (Figure 4). However, lung lavage fluid from ethanol-fed rats that were supplemented with N-acetylcysteine had higher (p < 0.05) levels of GSSG than lung lavage fluid from control-fed rats and ethanol-fed rats (Figure 4).

Activation of MMP-9 and MMP-2. In parallel to the levels of GSH, there was no detectable MMP-9 or MMP-2 activity in the lung tissue, lung lavage fluid, or cells from the lung lavage fluid in the control-fed rats or in ethanol-fed rats that had been supplemented with N-acetylcysteine (not shown). In contrast, there was significant MMP-9 and MMP-2 activity in the lung tissue, lung lavage fluid, and cells from the lung lavage fluid in all six of the untreated, ethanol-fed rats. Shown in Figure 5 is a representative zymography gel of lung tissue MMP-9 and MMP-2 activity. In contradistinction, after treatment with APMA there was no difference (p > 0.05) in MMP-9 or MMP-2 activity in the lung tissue, lung lavage fluid, or cells from the lung lavage fluid in the N-acetylcysteine-treated, ethanol-fed rats compared with the untreated, ethanol-fed rats and the control-fed rats. Shown in Figure 6 are the MMP-9 and MMP-2 relative activities (as reflected by densitometry) in the lung tissue from all three experimental groups.

Effect of Ethanol Ingestion with and without Glutathione Replacement on Lavage Fluid Levels of 7S Collagen

In parallel to MMP-9 and MMP-2 activity, the lung lavage fluid from ethanol-fed rats had higher (p < 0.05) levels of the 7S fragment of type IV collagen compared with the lung lavage fluid from control-fed rats (Figure 7). In contrast, the lung lavage fluid from ethanol-fed rats that had been supplemented with N-acetylcysteine had the same (p < 0.05) levels of the 7S fragment of type IV collagen as the lung lavage fluid from control-fed rats (Figure 7).

In this study we determined that chronic ethanol ingestion activated the type IV collagenases MMP-9 and MMP-2 in the lungs of endotoxin-treated rats. Gelatinase (i.e., type IV collagenase) activity was markedly increased in the lung tissue, the lung lavage fluid, and the cells (predominantly macrophages) from the lung lavage fluid, of ethanol-fed rats during acute inflammation. Although MMP-9 and MMP-2 activity was increased, production of these enzymes appeared to be unchanged. Specifically, the enzyme activity of MMP-9 and MMP-2 after treatment with APMA was the same in the lungs of the ethanol-fed and control-fed rats, indicating that the production of the MMP-9 and MMP-2 zymogens was not altered by ethanol ingestion. Importantly, MMP-9 and MMP-2 activation was associated with a significant elevation in the 7S fragment of type IV collagen in the lung lavage fluid, suggesting that there was increased degradation of the alveolar ECM. In parallel, and consistent with our previous finding (11), ethanol ingestion markedly decreased the levels of the antioxidant glutathione in the lung lavage fluid. Finally, although N-acetylcysteine, a commonly used synthetic glutathione precursor, had no apparent effect on MMP-9 or MMP-2 production, it increased the levels of glutathione in the lung lavage fluid, inhibited activation of MMP-9 and MMP-2, and decreased acute lung edema in ethanol-fed rats during acute inflammation. Taken together, these findings indicate that ethanol ingestion, via perturbations in alveolar lining fluid glutathione, activates MMP-9 and MMP-2 during endotoxin-mediated inflammation, thereby increasing degradation of the alveolar ECM.

These results are important because they provide new insights into a potential mechanism by which chronic alcohol abuse predisposes to acute lung injury in at-risk patients. We previously reported that chronic alcohol abuse increased the incidence and mortality of ARDS in critically ill patients (10). This susceptibility was most significant in those patients with sepsis (10). Furthermore, the effect of alcohol abuse was independent of the presence of liver disease (10). More recently, we determined that chronic ethanol ingestion in rats dramatically decreased concentrations of glutathione in the alveolar epithelial cells and lung lavage fluid, and increased acute edematous lung injury in this same model of endotoxin treatment and ex vivo perfusion (11). In this model (11, 16), endotoxin (2 mg/kg intraperitoneally) produces intense neutrophil sequestration in the pulmonary capillaries and acute edema formation after perfusion with fMLP, a potent activator of neutrophils and other inflammatory cells. We used this model because it enabled us to separate the lung from the systemic effects of shock and thereby examine lung-specific activation of MMP-9 and MMP-2. We cannot exclude the possibility that ethanol-mediated effects on the liver or other organs did not somehow alter the pulmonary response to endotoxin before lung isolation. In fact, such effects are certainly possible, and would be consistent with the evolving concept that ARDS is not a lung-specific entity but rather that interactions between the lung and other organs, including the liver, influence the pathophysiology of the syndrome. Although our previous study provided the novel observation that ethanol ingestion has profound effects on alveolar epithelial glutathione homeostasis, it did not identify a specific mechanism by which ethanol predisposes the lung to endotoxin-mediated injury. Ethanol ingestion alone did not produce any lung edema during ex vivo perfusion, but markedly increased lung edema in endotoxin-primed lungs that were perfused with fMLP (11). In the current study, we hypothesized that ethanol-mediated glutathione depletion could increase activation of MMPs, and that the increased collagenolytic activity could degrade the alveolar ECM and exacerbate edema formation during acute inflammation. Although to our knowledge this has not been studied in the lung, there is evidence that alterations in glutathione can increase MMP activity in myocardial fibroblasts (12), and can modulate MMP-dependent metastatic activity of tumor cells (14). Importantly, we determined that supplementing the ethanol diet with N-acetylcysteine completely inhibited MMP-9 and MMP-2 activation, and decreased acute edema formation, after endotoxin treatment and ex vivo perfusion. This argues that glutathione depletion, and not ethanol ingestion per se, increases MMP activation during sepsis.

The role of MMPs in the pathogenesis of ARDS has been suggested by multiple studies. MMPs and neutrophil elastase are elevated in the lung lavage fluid of patients with ARDS (2-4). In animal studies, MMPs are elevated in the lungs of endotoxin-treated guinea pigs (21) and in the lungs of rats exposed to hyperoxia (23). Other studies have shown that patients with ARDS have elevated levels of collagen fragments including procollagen III and the 7S fragment of type IV collagen in their lung lavage fluid (1, 6, 24), indicating degradation of the ECM during acute lung injury.

The MMPs are a family of zinc- and calcium-dependent endopeptidases that specifically degrade the ECM. They share common amino acid sequences, and are synthesized by diverse cell types including mesenchymal cells, macrophages/monocytes, polymorphonuclear cells, alveolar type II epithelial cells, and tumor cells (25-27). In our current study, we found evidence for MMP activation in alveolar macrophages as well as the lung parenchyma. These enzymes are synthesized and secreted as zymogens, and require cleavage of an N-terminal propeptide for proteolytic activity to be apparent (25). MMPs are tightly regulated, with control at the levels of expression, synthesis, and activation (28). In addition, their activity in vivo is balanced by unique proteins known as tissue inhibitors of metalloproteinases (TIMPs) (28-30). The activation of MMPs can be achieved by proteolytic and nonproteolytic mechanisms (31). The most important proteolytic activator in vivo appears to be plasmin (28), although they can also be activated by serine proteases. Multiple oxidants, particularly reactive oxygen species, can activate MMPs via a nonproteolytic mechanism. A critical cysteine in the N-terminal propeptide interacts with the zinc molecule of the catalytic site, thereby displacing the water molecule that is required for catalytic activity (28). It is postulated that this cysteine is oxidized and unable to protect the catalytic site, thereby activating the enzyme (28, 32, 33). Therefore, glutathione could prevent or limit nonproteolytic activation by providing alternative sulfhydryl moieties for oxidation by reactive oxygen species, particularly during acute inflammation. Our current study supports this hypothesis. Although the lungs of control-fed and ethanol-fed rats had similar levels of latent MMP-9 and MMP-2, there was no evidence of enzyme activation in either group in the absence of inflammation, and very little activation of these enzymes in the control-fed animals after endotoxin treatment and perfusion with fMLP. In contrast, in the lungs of the ethanol-fed rats, in which the glutathione levels were extremely low, acute inflammation was associated with marked activation of MMP-9 and MMP-2 and increased levels of the 7S fragment of type IV collagen, both of which were completely blocked by treatment with N-acetylcysteine.

Importantly, although N-acetylcysteine treatment increased the levels of glutathione in the lung lavage fluid, the levels of oxidized glutathione (glutathione disulfide or GSSG) were actually higher in this group. We previously determined that the lung lavage fluid levels of GSSG in control-fed and ethanol-fed rats at baseline (i.e., before endotoxin-priming and ex vivo perfusion with fMLP) were < 10 μM (11). By comparison, in the current study we determined that these levels increased approximately 10-fold during endotoxin-mediated inflammation, consistent with an acute oxidative stress in the alveolar epithelial space. Somewhat surprisingly, the levels of GSSG were even more markedly increased in the ethanol-fed rats that were supplemented with N-acetylcysteine, even though this treatment prevented activation of MMP-2 and MMP-9. However, this was in parallel to an 8-fold increase in glutathione levels in this group. Therefore, it appears from this study that the availability of glutathione may be more important than the absolute levels of oxidized glutathione in determining whether or not latent MMPs are activated in the alveolar lining fluid during acute inflammation. These data indicate that glutathione is an important regulator of MMP activity in the alveolar space during acute inflammation, and that chronic alcohol abuse disrupts this regulation. Whether the absolute or relative levels of glutathione and its oxidized forms directly influence MMP activation or indirectly inhibit activation by maintaining a reducing environment within the alveolar space is at present unknown.

Although this study supports the hypothesis that alcohol abuse predisposes patients to ARDS by increasing degradation of the alveolar ECM, additional studies in both animal models and humans are necessary to provide more definitive evidence. There are undoubtedly other mechanisms by which alcohol ingestion renders the lung susceptible to acute injury. For example, in our previous study we determined that chronic ethanol ingestion impaired synthesis and secretion of surfactant phospholipids by alveolar epithelial type II cells in vitro (11). Because surfactant dysfunction is a hallmark of ARDS, it is plausible that chronic alcohol abuse could disrupt surfactant homeostasis in vivo. Furthermore, although clinical trials of N-acetylcysteine therapy in ARDS have suggested some potential benefit (34, 35), it is not clear how impairments in glutathione homeostasis contribute to acute lung injury. In our current study, MMP activation was minimal in rats that did not consume ethanol, raising the possibility that glutathione replacement with drugs such as N-acetylcysteine may only be efficacious in the subset of ARDS patients with a significant history of alcohol abuse.

In summary, our current study indicates that ethanol ingestion, via glutathione depletion, increases activation of matrix-degrading enzymes in the alveolar space during acute inflammation. Concomitant ingestion of the synthetic glutathione precursor N-acetylcysteine completely inhibited MMP activation in the lungs of ethanol-fed rats, and decreased endotoxin-mediated acute edematous injury. The observation that alcohol abuse predisposes to ARDS is a recent one (10), and presents important new questions about the pathophysiology of acute lung injury. This study provides a potential mechanism by which chronic alcohol abuse predisposes critically ill patients to developing ARDS. In addition, it raises the possibility that glutathione replacement could limit activation of MMPs in the lungs of at-risk patients with a history of alcohol abuse, thereby reducing the severity of their acute lung injury. Future clinical trials of glutathione replacement or other new agents for the treatment of ARDS should take into account potential differences in this subgroup of patients.

The authors thank Robert Raynor, Frank Harris, and William and Kelli Schuyler for their technical support.

Supported by a grant from the Georgia Chapter of the American Lung Association for Dr. Guidot, and a national Research Grant from the American Lung Association for Dr. Moss.

1. Matsubara O., Tamura A., Ohdama S., Mark E. J.Alveolar basement membrane breaks down in diffuse alveolar damage: an immunohistochemical study. Pathol. Int.451995473482
2. Delclaux C., D'Ortho M. P., Delacourt C., Lebargy F., Brun-Buisson C., Brochard L., Lemaire F., Lafuma C., Harf A.Gelatinases in epithelial lining fluid of patients with adult respiratory distress syndrome. Am. J. Physiol.2721997L442451
3. Torii K., Iida K., Miyazaki Y., Saga S., Kondoh Y., Taniguchi H., Taki F., Takagi K., Matsuyama M., Suzuki R.Higher concentrations of matrix metalloproteinases in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome. Am. J. Respir. Crit. Care Med.15519974346
4. Ricou B., Nicod L., Lacraz S., Welgus H. G., Suter P. M., Dayer J. M.Matrix metalloproteinases and TIMP in acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med.1541996346352
5. Kawamura M., Yamasawa F., Ishizaka A., Kato R., Kikuchi K., Kobayashi K., Aoki T., Sakamaki F., Hasegawa N., Kawashiro T., Ishihara T.Serum concentrations of 7S collagen and prognosis in patients with the adult respiratory distress syndrome. Thorax491994144146
6. Kondoh Y., Taniguchi H., Taki F., Takagi K., Satake T.7S collagen in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome. Chest101199210911094
7. Hayashi T., Stetler-Stevenson W. G., Fleming M. V., Fishback N., Koss M. N., Liotta L. A., Ferrans V. J., Travis W. D.Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis. Am. J. Pathol.149199612411256
8. Bitterman P. B.Pathogenesis of fibrosis in acute lung injury. Am. J. Med.92199239S43S
9. Campbell E. J., Senior R. M., Welgus H. G.Extracellular matrix injury during lung inflammation. Chest921987161167
10. Moss M., Bucher B., Moore F. A., Moore E. E., Parsons P. E.The role of chronic alcohol abuse in the development of acute respiratory distress syndrome in adults. J.A.M.A.27519965054
11. Holguin F., Moss I. M., Brown L. S., Guidot D. M.Chronic ethanol ingestion impairs alveolar type II cell glutathione homeostasis and function and predisposes to endotoxin-mediated acute edematous lung injury in rats. J. Clin. Invest.1011998761768
12. Tyagi S. C., Kumar S., Borders S.Reduction-oxidation (redox) state regulation of extracellular matrix metalloproteinases and tissue inhibitors in cardiac normal and transformed fibroblast cells. J. Cell. Biochem.611996139151
13. Tyagi S. C., Ratajska A., Weber K. T.Myocardial matrix metalloproteinase(s): localization and activation. Mol. Cell. Biochem.12619934959
14. Albini A., D'Agostini F., Giunciuglio D., Paglieri I., Balansky R., De Flora S.Inhibition of invasion, gelatinase activity, tumor take and metastasis of malignant cells by N-acetylcysteine. Int. J. Cancer611995121129
15. Ferguson D. M., Gores G. J., Bronk S. F., Krom R. A.An increase in cytosolic protease activity during liver preservation: inhibition by glutathione and glycine. Transplantation551993627633
16. Voelkel N. F., Czartolomna J., Simpson J., Murphy R. C.FMLP causes eicosanoid-dependent vasoconstriction in lungs from endotoxin-primed rats. Am. Rev. Respir. Dis.1451992701711
17. Martin J., White J. M.Fluorimetric determination of oxidized and reduced glutathione in cells and tissues by high-performance liquid chromatography following derivatization with dansyl chloride. J. Chromatogr.5681991221225
18. Reed D. J., Babson J. R., Beatty P. W., Brodie A. E., Ellis W. W., Potter D. W.High performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide and related thiols and disulfides. Anal. Biochem.10619805562
19. Rennard S. I., Basset G., Lecossier D., O'Donnell K. M., Pinkston P., Martin P. G., Crystal R. G.Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J. Appl. Physiol.601986532538
20. Pittet J. F., Brenner T. J., Modelska K., Matthay M. A.Alveolar liquid clearance is increased by endogenous catecholamines in hemorrhagic shock in rats. J. Appl. Physiol.811996830837
21. D'Ortho M. P., Jarreau P. H., Delacourt C., Macquin-Mavier I., Levame M., Pezet S., Harf A., Lafuma C.Matrix metalloproteinase and elastase activities in LPS-induced acute lung injury in guinea pigs. Am. J. Physiol.2661994L209216
22. Bartlett W. C., Noelle R. J.A cell surface ELISA to detect interleukin 4-induced class II MHC expression on murine B cells. J. Immunol. Methods10519877985
23. Pardo A., Selman M., Ridge K., Barrios R., Sznajder J. I.Increased expression of gelatinases and collagenase in rat lungs exposed to 100% oxygen. Am. J. Respir. Crit. Care Med.154199610671075
24. Clark J. G., Milberg J. A., Steinberg K. P., Hudson L. D.Type III procollagen peptide in the adult respiratory distress syndrome: association of increased peptide levels in bronchoalveolar lavage fluid with increased risk for death. Ann. Intern. Med.12219951723
25. Baramova E., Foidart J. M.Matrix metalloproteinase family. Cell Biol. Int.191995239242
26. Matrisian L. M.The matrix degrading metalloproteinases. BioEssays141992455463
27. Emonard H., Grimaud J.-A.Matrix metalloproteinases: a review. Cell. Mol. Biol.361990131153
28. Murphy G., Willenbrock F., Crabbe T., O'Shea M., Ward R., Atkinson S., O'Connell J., Docherty A.Regulation of matrix metalloproteinase activity. Ann. N.Y. Acad. Sci.73219943141
29. Murphy G., Dochery J. P.The matrix metalloproteinases and their inhibitors. Am. J. Respir. Cell Mol. Biol.71992120125
30. Overall C. M.Regulation of tissue inhibitor of metalloproteinase expression. Ann. N.Y. Acad. Sci.73219945164
31. Sang Q. X., Birkedal-Hansen H., Van Wart H. E.Proteolytic and non-proteolytic activation of human neutrophil progelatinase B. Biochim. Biophys. Acta1251199599108
32. Grams F., Huber R., Kress L. F., Moroder L., Bode W.Activation of snake venom metalloproteinases by a cysteine switch-like mechanism. FEBS Lett.33519937680
33. Rajagopalan S., Meng X. P., Ramasamy S., Harrison D. G., Galis Z. S.Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro: implications for atherosclerotic plaque stability. J. Clin. Invest.98199625722579
34. Jepsen S., Herlevsen P., Knudsen P. X., Bud M. I., Klausen N.-O.Antioxidant treatment with n-acetylcysteine during adult respiratory distress syndrome: a prospective, randomized, placebo controlled study. Crit. Care Med.201992918923
35. Suter P. M., Domenighrtti G., Schaller M., Laverriere M., Ritz R., Perret C.N-acetylcysteine enhances recovery from acute lung injury in man: a randomized, double blind, placebo-controlled clinical study. Chest1051994190194
Correspondence and requests for reprints should be addressed to David M. Guidot, M.D., Atlanta VAMC (151-P), 1670 Clairmont Road, Decatur, GA 30033. E-mail:

Related

No related items
American Journal of Respiratory and Critical Care Medicine
160
4

Click to see any corrections or updates and to confirm this is the authentic version of record