Activating the Nrf2-mediated antioxidant response element restores barrier function in the alveolar epithelium of HIV-1 transgenic rats.

The master transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the expression of antioxidant and phase II-metabolizing enzymes by activating the antioxidant response element (ARE) and thereby protects cells and tissues from oxidative stress. Pulmonary complications remain the leading cause of death in human immunodeficiency virus (HIV)-1-infected individuals, who display systemic oxidative stress and glutathione deficiency that can be modeled in transgenic rats where HIV-1-related viral proteins decrease glutathione levels and cause epithelial barrier dysfunction within the alveolar space by as yet unknown mechanisms. We hypothesized that HIV-1-related proteins inhibit Nrf2-mediated antioxidant defenses and thereby disrupt the normally tight alveolar epithelial barrier. Nrf2 RNA silencing dampened Nrf2/ARE activity, decreased the expression of the tight junction proteins zonula occludens-1, occludin, and claudin-18, increased paracellular permeability of alveolar epithelial monolayers derived from wild-type rats, and therefore reproduced the effects of HIV-1 transgene expression on the epithelial barrier that we had previously described. In contrast, upregulating Nrf2 activity, either by plasmid-mediated overexpression or treatment with the Nrf2 activator sulforaphane, increased the expression of ARE-dependent antioxidants, including NAD(P)H dehydrogenase, quinone 1 and glutathione, improved the expression of tight junction proteins, and restored the ability to form tight barriers in alveolar epithelial cells from HIV-1 transgenic rats. Taken together, these new findings argue that HIV-1-related proteins downregulate Nrf2 expression and/or activity within the alveolar epithelium, which in turn impairs antioxidant defenses and barrier function, thereby rendering the lung susceptible to oxidative stress and injury. Furthermore, this study suggests that activating the Nrf2/ARE pathway with the dietary supplement sulforaphane could augment antioxidant defenses and lung health in HIV-1-infected individuals.

Alcohol ingestion disrupts alveolar epithelial barrier function by activation of macrophage-derived transforming growth factor beta1.

BACKGROUND: Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Experimentally, alcohol-induced oxidative stress increases the expression of transforming growth factor ß1 (TGFß1) in the lung; however, we do not know the precise contribution of various alveolar cells in this process. In the present study, we focused on cell-cell interactions between alveolar macrophages and epithelial cells and the potential mechanisms by which TGFß1 may become activated in the alveolar space of the alcoholic lung. METHODS: Primary alveolar macrophages and epithelial cells were isolated from control- and alcohol-fed Sprague-Dawley rats. Expression of TGFß1 and the epithelial integrin αvß6 were examined by real time PCR and either immunocytochemistry or flow cytometry. Alveolar epithelial cells were cultured on transwell supports in the presence of macrophage cell lysate from control- or alcohol-fed rats or in the presence of viable macrophages ± alcohol. Epithelial barrier function was assessed by transepithelial resistance (TER) and paracellular flux of Texas Red dextran. RESULTS: TGFß1 expression was increased in alveolar macrophages from alcohol-fed rats, and TGFß1 protein was predominantly membrane-bound. Importantly, alveolar macrophage cellular lysate from alcohol-fed rats decreased TER and increased paracellular dextran flux in primary alveolar epithelial cell monolayers as compared to the lysates from control-fed rats. Alcohol-induced epithelial barrier dysfunction was prevented by anti-TGFß1 antibody treatment, indicating the presence of bioactive TGFß1 in the macrophage lysate. In addition, co-culturing macrophages and epithelial cells in the presence of alcohol decreased epithelial barrier function, which also was prevented by anti-TGFß1 and anti-αvß6 treatment. In parallel, chronic alcohol ingestion in vivo, or direct treatment with active TGFß1 in vitro, increased the expression of αvß6 integrin, which is known to activate TGFß1, in alveolar epithelial cells. CONCLUSIONS: Taken together, these data suggest that interactions between alveolar epithelial cells and macrophages contribute to the alcohol-mediated disruption of epithelial barrier function via the expression and activation of TGFß1 at points of cell-cell contact.

Chronic alcohol ingestion in rats decreases Krüppel-like factor 4 expression and intracellular zinc in the lung.

BACKGROUND: Chronic alcohol ingestion alters the dynamic balance between granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta1 (TGFß1) signaling within the alveolar space and, in parallel, impairs alveolar macrophage and epithelial cell function by inhibiting expression of the zinc importer ZIP4 and decreasing zinc bioavailability in the alveolar compartment. As the transcription factor Krüppel-like factor 4 (KLF4 ) binds to ZIP4 , we hypothesized that alcohol exposure and consequent perturbations in GM-CSF and TGFß1 signaling could decrease cellular KLF4 expression and/or binding as a mechanism by which it inhibits ZIP4 expression and decreases cellular zinc levels. METHODS AND RESULTS: Alcohol exposure in vitro or chronic ingestion in vivo decreased KLF4 expression in alveolar macrophages and epithelial cells. Treatment with GM-CSF or TGFß1 showed an enhancing or dampening effect on KLF4 expression and binding, respectively. Further, treatment of a rat alveolar macrophage cell line with alcohol in vitro for 4 weeks decreased the expression of the zinc transporters ZIP4 and ZNT1, and of the zinc storage protein metallothionein 1. In parallel, treating these macrophages with KLF4 siRNA decreased ZIP4 expression and decreased cellular zinc and phagocytic capacity to levels equivalent to those following alcohol exposure. In epithelial monolayers, transepithelial electrical resistance (TER) was significantly decreased by alcohol ingestion as compared with control diets, and it was restored by in vitro GM-CSF treatment. In contrast, in vitro TGFß1 treatment of the epithelial monolayers from control-fed rats significantly decreased TER as compared with untreated control monolayers. CONCLUSIONS: Taken together, these results suggest that within the alveolar space, chronic alcohol exposure decreases KLF4 and ZIP4 expression and consequently decreases zinc transport into cells, which, in turn, impairs their function. Furthermore, the dynamic decrease in the relative influence of GM-CSF versus TGFß1 could mediate the zinc deficiency and consequent cellular dysfunction that characterize the "alcoholic lung" phenotype.

HIV-1 transgene expression in rats induces differential expression of tumor necrosis factor alpha and zinc transporters in the liver and the lung.

BACKGROUND: Highly effective antiviral treatment can suppress HIV-1 infection, but the chronic effects of HIV-1-related viral proteins, including gp120 and Tat, on organs such as the lungs can be damaging. HIV-1 transgenic rodent models are useful for studying the systemic effects of these proteins independently of viral infection. We have previously shown that HIV-1 transgene expression (and therefore, HIV-1-related protein expression) in rats decreases alveolar macrophage zinc levels and phagocytic capacity by unknown mechanisms. We hypothesized that HIV-1 transgene expression induces chronic inflammation and zinc sequestration within the liver and thereby decreases zinc bioavailability in the lung. We examined the expression of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα), the zinc storage protein, metallothionein (MT1), and the zinc exporter, ZNT1 in the livers and the lungs of wild type and HIV-1 transgenic rats ± dietary zinc supplementation. In addition, we measured zinc levels, the zinc importing protein ZIP1, and the phagocytic capacity in the alveolar macrophages. RESULTS: HIV-1 transgene expression increased the liver-specific expression of TNFα, suggesting a chronic inflammatory response within the liver in response to HIV-1-related protein expression. In parallel, HIV-1 transgene expression significantly increased MT1 and ZNT1 expression in the liver as compared to the lung, a pattern that is consistent with zinc sequestration in the liver as occurs during systemic inflammation. Further, HIV-1 transgene expression decreased intracellular zinc levels and increased expression of ZIP1 in the alveolar macrophages, a pattern consistent with zinc deficiency, and decreased their bacterial phagocytic capacity. Interestingly, dietary zinc supplementation in HIV-1 transgenic rats decreased gene expression of TNFα, MT1, and ZNT1 in the liver while simultaneously increasing their expression in the lung. In parallel, zinc supplementation increased alveolar macrophage intracellular zinc levels and bacterial phagocytic capacity in HIV-1 transgenic rats. CONCLUSION: Taken together, these findings suggest that chronic HIV-1-related protein expression causes liver inflammation and zinc sequestration, which in turn limits zinc bioavailability in the lung and thereby impairs alveolar macrophage phagocytic function. Importantly, dietary zinc supplementation decreases liver inflammation and zinc sequestration and restores alveolar macrophage phagocytic function in HIV-1 transgenic rats, a result with potential clinical implications for improving lung health in HIV-1-infected individuals.

Chronic alcohol ingestion exacerbates lung epithelial barrier dysfunction in HIV-1 transgenic rats.

BACKGROUND: Alcohol abuse and HIV-1 infection frequently coexist, and these individuals are at high risk for serious lung infections and respiratory failure. Although alcohol ingestion and HIV-1 transgene expression have been shown to independently cause oxidative stress and disrupt alveolar epithelial barrier function in experimental models, their interactive effects have not been examined. METHODS AND RESULTS: In this study, we determined that chronic alcohol ingestion (12 weeks) exacerbated the already significant defects in alveolar epithelial paracellular permeability and lung liquid clearance in HIV-1 transgenic rats. Further, immunocytochemical analyses of tight junction protein expression in primary alveolar epithelial cells showed that occludin and zonula occludens-1 localization within the plasma membrane was more disrupted than in either condition alone, consistent with the observed defects in epithelial barrier function. Interestingly, expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), the transcription factor required to activate the antioxidant-response element, was decreased in primary alveolar epithelial cells isolated from HIV-1 transgenic rats. In parallel, exposing lung epithelial cells in vitro to either alcohol or the HIV-related protein gp120 also decreased Nrf2 expression. Importantly, treatment with procysteine, which increases thiol antioxidants including glutathione, improved tight junction protein localization in the plasma membrane and restored alveolar epithelial barrier function in alcohol-fed HIV-1 transgenic rats. CONCLUSIONS: These results provide novel evidence that HIV-related proteins and alcohol together causes more barrier dysfunction in the lung epithelium than either stress alone. However, these significant effects on the alveolar barrier can be mitigated by augmenting the thiol antioxidant pool, a strategy with potential clinical applications in subjects who are highly vulnerable to lung disease because of coexistent alcohol abuse and HIV infection.

Zinc supplementation restores PU.1 and Nrf2 nuclear binding in alveolar macrophages and improves redox balance and bacterial clearance in the lungs of alcohol-fed rats.

BACKGROUND: Chronic alcohol abuse causes oxidative stress, impairs alveolar macrophage immune function, and increases the risk of pneumonia and acute lung injury. Recently we determined that chronic alcohol ingestion in rats decreases zinc levels and macrophage function in the alveolar space; provocative findings in that zinc is essential for normal immune and antioxidant defenses. Alveolar macrophage immune function depends on stimulation by granulocyte/monocyte colony-stimulating factor, which signals via the transcription factor PU.1. In parallel, the antioxidant response element signals via the transcription factor Nrf2. However, the role of zinc bioavailability on these signaling pathways within the alveolar space is unknown. METHODS: To determine the efficacy of dietary zinc supplementation on lung bacterial clearance and oxidative stress, we tested 3 different groups of rats: control-fed, alcohol-fed, and alcohol-fed with zinc supplementation. Rats were then inoculated with intratracheal Klebsiella pneumoniae, and lung bacterial clearance was determined 24 hours later. Isolated alveolar macrophages were isolated from uninfected animals and evaluated for oxidative stress and signaling through PU.1 and Nrf2. RESULTS: Alcohol-fed rats had a 5-fold decrease in lung bacterial clearance compared to control-fed rats. Dietary zinc supplementation of alcohol-fed rats normalized bacterial clearance and mitigated oxidative stress in the alveolar space, as reflected by the relative balance of the thiol redox pair cysteine and cystine, and increased nuclear binding of both PU.1 and Nrf2 in alveolar macrophages from alcohol-fed rats. CONCLUSIONS: Dietary zinc supplementation prevents alcohol-induced alveolar macrophage immune dysfunction and oxidative stress in a relevant experimental model, suggesting that such a strategy could decrease the risk of pneumonia and lung injury in individuals with alcohol use disorders.

HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction.

BACKGROUND: HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined. RESULTS: HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization. CONCLUSION: Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

Zinc deficiency mediates alcohol-induced alveolar epithelial and macrophage dysfunction in rats.

Chronic alcohol abuse impairs both alveolar epithelial and macrophage function, and renders individuals susceptible to acute lung injury, pneumonia, and other serious lung diseases. Zinc deficiency, which is known to impact both epithelial and immune cell functions, is also associated with alcohol abuse. In this study, chronic alcohol ingestion (6 wk) in rats altered expression of key zinc transporters and storage proteins in the small intestine and the lung, and decreased zinc levels in the alveolar compartment. Zinc supplementation of alveolar epithelial monolayers derived from alcohol-fed rats in vitro, or of the diets of alcohol-fed rats in vivo, restored alveolar epithelial barrier function, and these improvements were associated with salutary changes in tight junction protein expression and membrane localization. In parallel, dietary zinc supplementation increased intracellular zinc levels, GM-CSF receptor expression, and bacterial phagocytic capacity in the alveolar macrophages of alcohol-fed rats. Together, these studies implicate zinc deficiency as a novel mechanism mediating alcohol-induced alveolar epithelial and macrophage dysfunction. Importantly, these findings argue that dietary supplementation can overcome alcohol-induced zinc deficiency and restore alveolar epithelial and macrophage function, and therefore could be an effective treatment for the susceptible alcoholic lung phenotype.

HIV-1-transgene expression in rats decreases alveolar macrophage zinc levels and phagocytosis.

HIV-1 infection impairs alveolar macrophage immune function and renders patients susceptible to pneumonia by poorly understood mechanisms. Alveolar macrophage maturation and function depends on granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced and secreted by the alveolar epithelium. Macrophages respond to GM-CSF through the GM-CSF receptor (GM-CSFR), which has a binding subunit (GM-CSFRalpha) and a signaling subunit (GM-CSFRbeta). In this study, we measured GM-CSFR expression and alveolar macrophage function in a transgene HIV-1 rat model (NL4-3Delta gag/pol); this construct bears a pro-virus with gag and pol deleted, but other HIV-1-related proteins, such as gp120 and Tat, are expressed, and the rats develop an AIDS-like phenotype as they age. We first determined that HIV-1-transgenic expression selectively decreased alveolar macrophage expression of GM-CSFRbeta and impaired bacterial phagocytosis in vitro. Next, we examined the role of zinc (Zn) deficiency as a potential mechanism underlying these effects, and determined that HIV-1-transgenic rats have significantly lower levels of Zn in the alveolar space and macrophages. To test the direct effect of Zn deficiency on macrophage dysfunction, we treated rat alveolar macrophage cell line with a Zn chelator, N,N,N',N'-tetrakis-(2-pyridyl-methyl) ethylenediamine, and this decreased GM-CSFRbeta expression and phagocytosis. In parallel, treatment with Zn acetate in vitro for 48 hours restored intracellular Zn levels and phagocytic function in alveolar macrophages from HIV-1-transgenic rats. Taken together, these data suggest that pulmonary Zn deficiency could be one of the mechanisms by which chronic HIV-1 infection impairs alveolar macrophage immune function and renders these individuals susceptible to serious lung infections.

Na,K-ATPase expression is increased in the lungs of alcohol-fed rats.

BACKGROUND: Alcohol abuse independently increases the risk of developing the acute respiratory distress syndrome (ARDS), a disease characterized by diffuse alveolar epithelial damage, lung edema, and consequent severe hypoxemia. Chronic alcohol abuse increases alveolar epithelial permeability both in vitro and in vivo, in part due to altered tight junction formation. However, both alcohol-fed animals and otherwise healthy alcoholic humans do not have pulmonary edema at baseline, even though their lungs are highly susceptible to acute edematous injury in response to inflammatory stresses. This suggests that active fluid transport by the alveolar epithelium is preserved or even augmented in the alcoholic lung. Chronic alcohol ingestion increases expression of apical sodium channels in the alveolar epithelium; however, its effects on the Na,K-ATPase complex that drives sodium and fluid transport out of the alveolar space have not been examined. METHODS: Age- and gender-matched Sprague-Dawley rats were fed the Lieber-DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 6 weeks. Gene and protein expression of lung Na,K-ATPase alpha1, alpha2, and beta1 subunits were quantified via real-time PCR and immunobiological analyses, respectively. Alcohol-induced, Na,K-ATPase-dependent epithelial barrier dysfunction was determined by calculating lung tissue wet:dry ratios following an ex vivo buffer-perfused challenge for 2 hours in the presence of ouabain (10(-4) M), a Na,K-ATPase inhibitor. RESULTS: Chronic alcohol ingestion significantly increased gene and protein expression of each Na,K-ATPase subunit in rat lungs. Immunohistochemical analyses of the alcoholic lung also revealed that protein expression of the Na,K-ATPase alpha1 subunit was increased throughout the alveolar epithelium. Additionally, lungs isolated from alcohol-fed rats developed more edema than comparably treated lungs from control-fed rats, as reflected by increased lung tissue wet:dry ratios. CONCLUSIONS: These findings indicate that chronic alcohol ingestion, which is known to increase alveolar epithelial paracellular permeability, actually increases the expression of Na,K-ATPase in the lung as a compensatory mechanism. This provides a potential explanation as to why the otherwise healthy alcoholic does not have evidence of pulmonary edema at baseline.

The alcoholic lung: epidemiology, pathophysiology, and potential therapies.

Epidemiological evidence gathered only in the past decade reveals that alcohol abuse independently increases the risk of developing the acute respiratory distress syndrome by as much as three- to fourfold. Experimental models and clinical studies are beginning to elucidate the mechanisms underlying this previously unrecognized association and are revealing for the first time that chronic alcohol abuse causes discrete changes, particularly within the alveolar epithelium, that render the lung susceptible to acute edematous injury in response to sepsis, trauma, and other inflammatory insults. Recent studies in relevant animal models as well as in human subjects are identifying common mechanisms by which alcohol abuse targets both the alveolar epithelium and the alveolar macrophage, such that the risks for acute lung injury and pulmonary infections are inextricably linked. Specifically, chronic alcohol ingestion decreases the levels of the antioxidant glutathione within the alveolar space by as much as 80-90%, and, as a consequence, impairs alveolar epithelial surfactant production and barrier integrity, decreases alveolar macrophage function, and renders the lung susceptible to oxidant-mediated injury. These changes are often subclinical and may not manifest as detectable lung impairment until challenged by an acute insult such as sepsis or trauma. However, even otherwise healthy alcoholics have evidence of severe oxidant stress in the alveolar space that correlates with alveolar epithelial and macrophage dysfunction. This review focuses on the epidemiology and the pathophysiology of alcohol-induced lung dysfunction and discusses potential new treatments suggested by recent experimental findings.

GM-CSF receptor expression and signaling is decreased in lungs of ethanol-fed rats.

Alcohol abuse dramatically increases the risk of acute lung injury. In an experimental rat model of ethanol-mediated susceptibility to lung injury, recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) restored alveolar epithelial barrier function both in vitro and in vivo, even during acute endotoxemia. These findings suggested that the alveolar epithelium, which secretes GM-CSF into the airway where it is required for alveolar macrophage maturation, likewise responds to GM-CSF priming in a receptor-mediated manner. In this study we determined that both the GM-CSF receptor alpha- and beta-subunits (GM-CSFRalpha and GM-CSFRbeta) are expressed throughout the rat airway epithelium and that this expression was significantly decreased in the alveolar epithelium following chronic ethanol ingestion (6 wk). In parallel, PU.1, the master transcription factor for GM-CSF signaling in hematopoietic cells, is also expressed in alveolar epithelial cells, and ethanol ingestion likewise decreased PU.1 protein expression and nuclear binding in the alveolar epithelium. Finally, GM-CSF signaling as reflected by PU.1 expression and nuclear binding was restored with recombinant GM-CSF treatment in vitro. We conclude that chronic ethanol ingestion decreases GM-CSF receptor expression and signaling in the lung epithelium. Consequently, we speculate that dampening of GM-CSF stimulation of the alveolar epithelium is responsible at least in part for the diverse functional defects that characterize the alcoholic lung and could be a therapeutic target in acute lung injury.

Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-stimulating factor receptor expression and downstream signaling in the alveolar macrophage.

Although it is well recognized that alcohol abuse impairs alveolar macrophage immune function and renders patients susceptible to pneumonia, the mechanisms are incompletely understood. Alveolar macrophage maturation and function requires priming by GM-CSF, which is produced and secreted into the alveolar space by the alveolar epithelium. In this study, we determined that although chronic ethanol ingestion (6 wk) in rats had no effect on GM-CSF expression within the alveolar space, it significantly decreased membrane expression of the GM-CSF receptor in alveolar macrophages. In parallel, ethanol ingestion decreased cellular expression and nuclear binding of PU.1, the master transcription factor that activates GM-CSF-dependent macrophage functions. Furthermore, treatment of ethanol-fed rats in vivo with rGM-CSF via the upper airway restored GM-CSF receptor membrane expression as well as PU.1 protein expression and nuclear binding in alveolar macrophages. Importantly, GM-CSF treatment also restored alveolar macrophage function in ethanol-fed rats, as reflected by endotoxin-stimulated release of TNF-alpha and bacterial phagocytosis. We conclude that ethanol ingestion dampens alveolar macrophage immune function by decreasing GM-CSF receptor expression and downstream PU.1 nuclear binding and that these chronic defects can be reversed relatively quickly with rGM-CSF treatment in vivo.

Mechanical ventilation exacerbates alveolar macrophage dysfunction in the lungs of ethanol-fed rats.

BACKGROUND: Patients with alcohol abuse have a two- to three-fold increased risk of acute lung injury and respiratory failure after sepsis or trauma but are also at increased risk of nosocomial pneumonia. Mechanical ventilation exacerbates lung injury during critical illnesses. In this study we tested whether mechanical ventilation of the alcoholic lung promotes on balance a proinflammatory phenotype favoring ventilator-induced lung injury or an immunosuppressive phenotype favoring ventilator-associated pneumonia. METHODS: Lungs from rats fed an isocaloric diet with or without ethanol (six weeks) were isolated and ventilated ex vivo with a low-volume (protective) or high-volume (injurious) strategy for two hours with or without prior endotoxemia (two hours). In other experiments, rats were subjected to high-volume ventilation in vivo. Airway levels of the proinflammatory cytokines tumor necrosis factor-alpha, macrophage inflammatory protein-2, and interleukin-1beta were determined after mechanical ventilation ex vivo and compared with edematous lung injury after high-volume ventilation in vivo. In parallel, alveolar macrophage phagocytosis of bacteria and secretion of interleukin-12 during ventilation ex vivo and endotoxin-stimulated alveolar macrophage phagocytosis and tumor necrosis factor-alpha secretion in vitro were determined. RESULTS: Ethanol ingestion suppressed the proinflammatory response to injurious mechanical ventilation and did not increase experimental ventilator-induced lung injury. In parallel, ethanol ingestion blunted the innate immune response of alveolar macrophages during injurious ventilation ex vivo and after endotoxin stimulation in vitro. CONCLUSIONS: Ethanol ingestion dampens ventilator-induced inflammation but exacerbates macrophage immune dysfunction. These findings could explain at least in part why alcoholic patients are at increased risk of ventilator-associated pneumonia.

Angiotensin II mediates glutathione depletion, transforming growth factor-beta1 expression, and epithelial barrier dysfunction in the alcoholic rat lung.

Alcohol abuse markedly increases the risk of sepsis-mediated acute lung injury. In a rat model, ethanol ingestion alone (in the absence of any other stress) causes pulmonary glutathione depletion, increased expression of transforming growth factor-beta1 (TGF-beta1), and alveolar epithelial barrier dysfunction, even though the lung appears grossly normal. However, during endotoxemia, ethanol-fed rats release more activated TGF-beta1 into the alveolar space where it can exacerbate epithelial barrier dysfunction and lung edema. Ethanol ingestion activates the renin-angiotensin system, and angiotensin II is capable of inducing oxidative stress and TGF-beta1 expression. We determined that lisinopril, an angiotensin-converting enzyme inhibitor that decreases angiotensin II formation, limited lung glutathione depletion, and treatment with either lisinopril or losartan, a selective angiotensin II type 1 receptor blocker, normalized TGF-beta1 expression. The glutathione precursor procysteine also prevented TGF-beta1 expression, suggesting that TGF-beta1 may be induced indirectly by angiotensin II-mediated oxidative stress and glutathione depletion. Importantly, lisinopril treatment normalized barrier function in alveolar epithelial cell monolayers from ethanol-fed rats, and treatment with either lisinopril or losartan normalized alveolar epithelial barrier function in ethanol-fed rats in vivo, as reflected by lung liquid clearance of an intratracheal saline challenge, even during endotoxemia. In parallel, lisinopril treatment limited TGF-beta1 protein release into the alveolar space during endotoxemia. Together, these results suggest that angiotensin II mediates oxidative stress and the consequent TGF-beta1 expression and alveolar epithelial barrier dysfunction that characterize the alcoholic lung.

Transforming growth factor beta1 expression and activation is increased in the alcoholic rat lung.

Alcohol abuse increases the incidence of acute respiratory distress syndrome more than threefold in patients with septic shock. We have shown that chronic ethanol ingestion in a rat model impairs alveolar epithelial barrier function and enhances lung injury during sepsis. We speculated that transforming growth factor beta(1) (TGFbeta(1)), a pluripotent cytokine implicated in models of epithelial barrier disruption and lung injury, could mediate alveolar epithelial injury in the alcoholic lung. We report that chronic ethanol ingestion (6 weeks) in rats increased both TGFbeta(1) mRNA and protein tissue expression (p < 0.05), but alone did not induce the release of TGFbeta(1) into the alveolar space. However, during endotoxemia, ethanol-fed rats released fivefold more TGFbeta(1) protein (by ELISA, p < 0.05) into the alveolar space than control-fed rats. Furthermore, lung lavage fluid from endotoxemic, ethanol-fed rats had more biologically active TGFbeta(1) protein than control-fed rats (p < 0.05), as reflected by anti-TGFbeta(1) antibody-inhibitable induction of permeability in rat alveolar epithelial monolayers in vitro. We conclude that chronic ethanol ingestion increases lung expression of TGFbeta(1,) which, during endotoxemia, is released and activated in the alveolar space in which it can disrupt the normally tight epithelial barrier. We speculate that this mechanism could contribute to the increased risk of acute respiratory distress syndrome in alcoholic patients.

Granulocyte/macrophage colony-stimulating factor treatment improves alveolar epithelial barrier function in alcoholic rat lung.

Chronic alcohol abuse increases the risk of developing acute lung injury approximately threefold in septic patients, and ethanol ingestion for 6 wk in rats impairs alveolar epithelial barrier function both in vitro and in vivo. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a trophic factor for the alveolar epithelium, and a recent phase II clinical study suggests that GM-CSF therapy decreases sepsis-mediated lung injury. Therefore, we hypothesized that GM-CSF treatment could improve ethanol-mediated defects in the alveolar epithelium during acute stresses such as endotoxemia. In this study, we determined that recombinant rat GM-CSF improved lung liquid clearance (as reflected by lung tissue wet:dry ratios) in ethanol-fed rats anesthetized and then challenged with 2 ml of saline via a tracheostomy tube. Furthermore, GM-CSF treatment improved lung liquid clearance and decreased epithelial protein leak in both control-fed and ethanol-fed rats after 6 h of endotoxemia induced by Salmonella typhimurium lipopolysaccharide given intraperitoneally, but with the greater net effect seen in the ethanol-fed rats. Our previous studies indicate that chronic ethanol ingestion decreases lung liquid clearance by increasing intercellular permeability. Consistent with this, GM-CSF treatment in vitro decreased permeability of alveolar epithelial monolayers derived from both control-fed and ethanol-fed rats. As in the endotoxemia model in vivo, the effect of GM-CSF was most dramatic in the ethanol group. Together, these results indicate that GM-CSF treatment has previously unrecognized effects in promoting alveolar epithelial barrier integrity and that these salutary effects may be particularly relevant in the setting of chronic alcohol abuse.