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Our understanding of the intrinsic effects of cystic fibrosis (CF) transmembrane conductance regulator (cftr) deletion on resident neonatal alveolar macrophage (AM) remains limited. We previously demonstrated that diminished glutathione (GSH) or excessive AM transforming growth factor beta one (TGFβ1) contributes to AM dysfunction in a variety of disease states. In this study, using a gut-corrected cftr neonatal knockout (KO) mouse model and a siRNA-manipulated macrophage-like cell line (THP-1 cell), we hypothesized (1) that cftr mutation alone increases neonatal AM oxidant stress and cellular TGFβ1 signaling via altered GSH, thereby impairing cellular function, and (2) that exogenous GSH attenuates AM alterations and dysfunction in the KO AM. In neonatal KO mice, the baseline bronchoalveolar lavage fluid demonstrated a near doubling in mixed disulfides (P ≤ 0.05) and oxidized GSSG (P ≤ 0.05) without concurrent inflammation compared to WT littermates. KO AM demonstrated diminished AM thiols (P ≤ 0.05), increased AM mitochondrial ROS (P ≤ 0.05), increased AM TGFβ1 (P ≤ 0.05) with increased TGFβ1 signaling (P ≤ 0.05), and impaired phagocytosis (P ≤ 0.05). KO AM mitochondrial ROS was modulated by exogenous GSH (P ≤ 0.05). Conversely, TGFβ1 was reduced (P ≤ 0.05) and impaired phagocytosis was rescued (P ≤ 0.05) by exogenous GSH in the KO AM. These results suggest that an altered neonatal AM phenotype may contribute to the initiation of lung inflammation/infection in the CF lung. Modulation of the AM in the neonatal CF lung may potentially alter progression of disease. [ABSTRACT FROM AUTHOR]

Background Alcohol use disorders (AUDs) and cigarette smoking are associated with pulmonary oxidative stress, likely related to antioxidant depletion. Pulmonary oxidative stress may adversely affect innate immunity, leading to increased pneumonia susceptibility and severity, including development of the acute respiratory distress syndrome. In people with AUDs, most of whom smoke, antioxidant therapy can potentially restore immune cell function and attenuate pneumonia development. Challenges to human investigations of antioxidant therapies include an inability to identify pulmonary oxidative stress noninvasively and the optimal route to deliver pulmonary antioxidants. We sought to determine whether bronchoalveolar lavage (BAL) measures of thiol antioxidants from a 50-ml upper airway aliquot approximated those in the alveolar space and to determine whether AUDs and/or smoking affected these relationships. Methods Healthy human subjects with and without AUDs, including smokers and nonsmokers, underwent BAL. Samples obtained after the first 50-ml normal saline aliquot were analyzed as representing bronchial airways; subsequent 50-ml aliquots were analyzed as representative of the alveolar space. Reduced and oxidized (GSSG) glutathione, cysteine (Cys), and its oxidized species, cystine, along with mixed disulfides (MDs) were quantified using high-performance liquid chromatography. The percent of total thiols present in their oxidized forms, and thiol redox potentials, were calculated. Results Positive correlations between upper and lower BAL fluid thiol species were observed that were most robust for GSSG (ρ = 0.85), Cys (ρ = 0.83), and MDs (ρ = 0.69), but poor for thiol redox potential measures. In contrast to nonsmokers (either with or without AUDs), in subjects with AUDs who smoked, upper BAL fluid %GSSG, Cys, and MD measures were relatively increased compared to lower. Conclusions A small volume BAL procedure may be suitable to assess intrapulmonary oxidative stress related to thiol depletion. Factors including AUDs and smoking may disproportionately increase upper airways oxidative stress that could be relevant for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Background Chronic alcohol exposure alters the function of alveolar macrophages ( AM), impairing immune defenses in both adult and neonatal lungs. Fatty acid ethyl esters ( FAEEs) are biological markers of prenatal alcohol exposure in newborns. FAEEs contribute to alcohol-induced mitochondrial ( MT) damage in multiple organs. We hypothesized that in utero ethanol exposure would increase FAEEs in the neonatal lung and that direct exposure of neonatal AM to FAEEs would contribute to MT injury and cellular dysfunction. Methods FAEEs were measured in neonatal guinea pig lungs after ± in utero ethanol exposure via gas chromatography/mass spectrometry. The NR8383 cell line and freshly isolated neonatal guinea pig AM were exposed to ethyl oleate ( EO) in vitro. MT membrane potential, MT reactive oxygen species generation ( mROS), phagocytosis, and apoptosis were evaluated after exposure to EO ± the MT-specific antioxidant mito- TEMPO (mitoT) or ± the pan-caspase inhibitor Z- VAD- FMK. Whole lung FAEEs were compared using the Mann-Whitney U-test. Cellular results were analyzed using 1-way analysis of variance, followed by the Student-Newman-Keuls Method for post hoc comparisons. Results In utero ethanol significantly increased ethyl linoleate and the combinations of ethyl oleate + linoleate + linolenate ( OLL), and OLL + stearate in the neonatal lung. In vitro EO caused significant MT dysfunction in both NR8383 and primary neonatal AM, as indicated by increased mROS and loss of MT membrane potential. Impaired phagocytosis and apoptosis were significantly increased in both the cell line and primary AM after EO exposure. MitoT conferred significant but only partial protection against EO-induced MT injury, as did caspase inhibition with Z- VAD- FMK. Conclusions In utero ethanol exposure increased FAEEs in the neonatal guinea pig lung. Direct exposure to the FAEE EO significantly contributed to AM dysfunction, in part via oxidant injury to the MT and in part via secondary apoptosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Increased systemic oxidant stress contributes to a variety of maternal complications of pregnancy. Although the antioxidant glutathione (GSH) and its oxidized component glutathione disulfide (GSSG) have been demonstrated to be significantly altered in the adult alcoholic, the effects of maternal alcohol use during pregnancy on oxidant stress in the postpartum female remain under investigation. We hypothesized that maternal alcohol use would increase systemic oxidant stress in the pregnant female, evidenced by an oxidized systemic GSH redox potential. Methods: As a subset analysis of a larger maternal language study, we evaluated the effects of alcohol consumption during pregnancy on the systemic GSH redox status of the postpartum female. Using an extensive maternal questionnaire, postpartum women where queried regarding their alcohol consumption during pregnancy. Any drinking, the occurrence of drinking >3 drinks/occasion, and heavy drinking of >5 drinks/occasion during pregnancy were noted. Using HPLC, maternal plasma samples were analyzed for GSH, oxidized GSSG and the redox potential of the GSH/GSSG antioxidant pair calculated. Results: Maternal alcohol use occurred in 25% (83/321) of our study sample. Two in ten women reported consuming >3 drinks/occasion during pregnancy, while 1 in 10 women reported consuming alcohol at >5 drinks/occasion. Any alcohol use during pregnancy significantly decreased plasma GSH ( p < 0.05), while alcohol at >3 drinks/occasion or >5 drinks/occasion significantly decreased plasma GSH concentration ( p < 0.05), increased the percent of oxidized GSSG ( p < 0.05), and substantially oxidized the plasma GSH redox potential ( p < 0.05). Conclusions: Alcohol use during pregnancy, particularly at levels >3 drinks/occasion, caused significant oxidation of the systemic GSH system in the postpartum women. The clinical ramifications of the observed alcohol-induced oxidation of the GSH redox system on high risk pregnancies or on the exposed offspring require more accurate identification and further investigation. [ABSTRACT FROM AUTHOR]

Background: Alcoholic patients have an increased risk of respiratory infections, which is partially due to an impaired immune response of alveolar macrophages. The mechanisms by which alcohol impairs alveolar macrophage function are poorly understood. In this study, we demonstrated in a guinea pig model that chronic ethanol ingestion significantly impaired alveolar macrophage differentiation and function. Methods: Isolated alveolar macrophages were separated into 4 different subpopulations with varying densities and levels of maturation. Results:  Compared to control values, chronic ethanol ingestion decreased the percentage of alveolar macrophages in the mature fractions by ∼60%. Alveolar macrophage function in each subpopulation was determined by measuring phagocytosis of fluorescein isothiocyanate-labeled Staphylococcus aureus. Alveolar macrophages from ethanol-fed animals had ∼80% decrease in the phagocytic index. Western blot and immunohistochemical analysis of the differential markers granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor α (GM-CSFR-α), PU.1, CD11c, and CD11b verified that alcoholic macrophages displayed impaired terminal differentiation. While oral supplementation with the glutathione precursor S-adenosyl-methionine (SAM) did not alter the maturational status of control animals, SAM supplementation shifted the distribution of macrophages to more mature fractions, normalized the phagocytic index; as well as normalized expression of CD11c, CD11b, PU.1, and GM-CSFR-α. Chronic ethanol ingestion also impaired the differentiation status of interstitial macrophages which was normalized by SAM supplementation. Conclusion: This improvement in the maturational status suggested that ethanol-induced oxidant stress is a central feature in impaired terminal differentiation of macrophages in the interstitial and alveolar space. Therefore, strategies targeting pulmonary oxidant stress may restore macrophage differentiation and function even after chronic ethanol ingestion. [ABSTRACT FROM AUTHOR]

Background: Sepsis is the most common risk factor associated with acute respiratory distress syndrome (ARDS) and results in a 40–60% mortality rate due to respiratory failure. Furthermore, recent epidemiological studies have demonstrated that a history of alcohol abuse increases the risk of ARDS by 3.6-fold. More recently, group B streptococcus (GBS) infections in nonpregnant adults have been increasing, particularly in alcoholics where there is an increased risk of lobular invasion and mortality. We have shown in an established rat model that chronic ethanol ingestion impaired macrophage internalization of inactivated infectious particles in vitro and enhanced bidirectional protein flux across the alveolar epithelial-endothelial barriers, both of which were attenuated when glutathione precursors were added to the diet. We hypothesized that chronic ethanol ingestion would increase the risk of infection even though GBS is less pathogenic but that dietary N-acetylcysteine (NAC), a glutathione precursor, would improve in vivo clearance of infectious particles and reduce systemic infection. Methods: After 6 weeks of ethanol feeding, rats were given GBS intratracheally and sacrificed 24 hours later. GBS colony-forming units were counted in the lung, liver, spleen, and bronchoalveolar lavage fluid. Acute lung injury in response to GBS was also assessed. Results: Chronic ethanol exposure decreased GBS clearance from the lung indicating an active lung infection. In addition, increased colonies formed within the liver and spleen indicated that ethanol increased the risk of systemic infection. Ethanol also exacerbated the acute lung injury induced by GBS. NAC supplementation normalized GBS clearance by the lung, prevented the appearance of GBS systemically, and attenuated acute lung injury. Conclusions: These data suggested that chronic alcohol ingestion increased the susceptibility of the lung to bacterial infections from GBS as well as systemic infections. Furthermore, dietary NAC improved in vivo clearance of GBS particles, attenuated acute lung injury, and disseminated infection. [ABSTRACT FROM AUTHOR]

Background: The effects of fetal alcohol exposure on the risks of neonatal lung injury and infection remain under investigation. The resident alveolar macrophage (AM) is the first line of immune defense against pulmonary infections. In utero ethanol (ETOH) exposure deranges the function of both premature and term guinea pig AM. We hypothesized that fetal ETOH exposure would increase the risk of pulmonary infection in vivo. Methods: We developed a novel in vivo model of group B Streptococcus ( GBS) pneumonia using our established guinea pig model of fetal ETOH exposure. Timed-pregnant guinea pigs were pair fed ±ETOH and some were supplemented with the glutathione (GSH) precursor S-adenosyl-methionine (SAM-e). Term pups were given GBS intratracheally while some were pretreated with inhaled GSH prior to the experimental GBS. Neonatal lung and whole blood were evaluated for GBS while isolated AM were evaluated using fluorescent microscopy for GBS phagocytosis. Results: Ethanol-exposed pups demonstrated increased lung infection and sepsis while AM phagocytosis of GBS was deficient compared with control. When SAM-e was added to the maternal diet containing ETOH, neonatal lung and systemic infection from GBS was attenuated and AM phagocytosis was improved. Inhaled GSH therapy prior to GBS similarly protected the ETOH-exposed pup from lung and systemic infection. Conclusions: In utero ETOH exposure impaired the neonatal lung’s defense against experimental GBS, while maintaining GSH availability protected the ETOH-exposed lung. This study suggested that fetal alcohol exposure deranges the neonatal lung’s defense against bacterial infection, and support further investigations into the potential therapeutic role for exogenous GSH to augment neonatal AM function. [ABSTRACT FROM AUTHOR]

Alcohol-related chronic myopathy is characterized by severe biochemical and structural changes to skeletal muscle. Our goals were to: (1) identify early regulatory elements that precede the overt manifestation of plantaris atrophy; and (2) circumvent these derangements by supplementing alcohol-fed rats with the glutathione precursor, procysteine. After 6 weeks of daily ingestion, before the development of overt atrophy of the plantaris muscle, alcohol increased several markers of oxidative stress and increased gene expressions of atrogin-1 and transforming growth factor-beta1 (TGF-beta1) by approximately 60- and approximately 65-fold, respectively, which were attenuated by procysteine supplementation. Interestingly, after 28 weeks of alcohol ingestion, when overt plantaris atrophy had developed, atrogin-1 and TGF-beta1 gene expression had returned to baseline levels. Together, these findings suggest that alcohol-induced, redox-sensitive alterations drive pro-atrophy signaling pathways that precede muscle atrophy. Therefore, targeted anti-oxidant treatments such as procysteine supplementation may benefit individuals with chronic alcohol abuse, particularly if given prior to the development of clinically significant myopathy. [ABSTRACT FROM AUTHOR]

Background: The effects of in utero alcohol exposure on the immune function of the newborn remain under investigation. Fetal ethanol (ETOH) exposure increases oxidative stress in the developing lung, in part due to decreased availability of the antioxidant glutathione (GSH). We have previously shown that in utero ETOH impairs alveolar macrophage phagocytosis and viability in the premature pup, while maintaining GSH availability with maternal supplementation of S-adenosyl-methionine (SAM) during ETOH ingestion improves macrophage function and viability. We hypothesized that dysfunction of the neonatal alveolar macrophage exposed to ETOH in utero would persist at term gestation. Methods: Using a guinea-pig model of fetal ETOH exposure, timed-pregnant guinea-pigs were pair-fed ETOH±the GSH precursor SAM and the diet continued until spontaneous delivery. Term alveolar macrophages were evaluated using fluorescent microscopy for phagocytosis and apoptosis after in vitro incubation with Staphalococcus aureus. Using an in vivo model of intranasal Staph. aureus inoculation, the in vivo function of the term alveolar macrophage was also investigated using confocal fluorescent analysis. Results: In utero ETOH exposure increased oxidant stress in the alveolar macrophage and decreased phagocytosis and viability in vitro and in vivo. Confocal analysis of phagocytosis in vivo demonstrated a marked impairment of internalization of the bacteria by the ETOH-exposed alveolar macrophage. The addition of SAM during maternal ETOH ingestion prevented loss of alveolar macrophage function and viability in vitro and in vivo. Conclusions: In utero ETOH exposure impairs alveolar macrophage function and viability in vitro and in vivo even at term gestation. The ETOH-induced changes in macrophage function and viability can be ablated with maternal SAM supplementation. Further investigations are required to identify the mechanisms of ETOH-induced derangement of phagocytosis in the neonatal alveolar macrophage and the clinical ramifications of altered immune function after in utero alcohol exposure for the newborn. [ABSTRACT FROM AUTHOR]

This article represents the proceedings of the Alcohol and Immunology Research Interest Group (AIRIG) meeting, a satellite workshop held at the 37th Annual Meeting of the Society for Leukocyte Biology. The meeting was sponsored by the AIRIG and the National Institute on Alcohol Abuse and Alcoholism. The presentations were as follows: (1) Effects of Ethanol on Immune Response to Hepatitis C Virus by Jack R. Wands, (2) Alcohol and Alveolar Macrophage Dysfunction: The Role of Chronic Oxidant Stress by Lou Ann S. Brown, (3) T Cell Responses to Listeria monocytogenes in Mice on a Chronic Ethanol Exposure Protocol by Robert T. Cook, (4) Mechanisms of Acute and Chronic Alcohol Consumption on Severity of Viral Infections by the Liver and Pancreas by Thomas R. Jerrells, (5) Acute and Chronic Effects on Macrophage Ectodomain Shedding: Implications for Lung Host Defenses by Jay K. Kolls, (6) Increased Susceptibility to Pseudomonas Infection of Burn-Injured Mice Given Alcohol Before Injury by Elizabeth J. Kovacs, (7) Regulation of Tumor Necrosis Factor α Expression in Macrophages by Chronic Ethanol by Laura E. Nagy, and (8) Hepatitis C Virus Infection and Alcohol Use by Gyongyi Szabo. Meeting coorganizers were Elizabeth J. Kovacs, Lou Ann S. Brown, Thomas R. Jerrells, and Robert T. Cook. [ABSTRACT FROM AUTHOR]

Background The development of acute respiratory distress syndrome (ARDS) in patients with pre-existing cirrhosis of the liver is associated with very high mortality. One possible cause may be alteration of pulmonary antioxidant capacity as a result of chronic hepatic dysfunction. Glutathione (GSH) is the most substantial nonprotein thiol in living organisms and likely plays a key role in neutralizing the oxidants and reactive oxygen species that are increased in ARDS. The lung is unable to synthesize GSH and is dependent on the liver. During periods of oxidant stress, individuals may exhibit relative deficiencies of GSH. With cirrhosis, the end result of chronic alcohol ingestion, this deficiency is more profound. Methods Sixteen stable subjects with cirrhosis primarily due to alcohol consumption and 15 healthy controls underwent bronchoscopy and bronchoalveolar lavage with concurrent measurement of GSH in the plasma and the alveolar epithelial lining fluid (ELF). Results For standardizing for saline dilution of the epithelial lining fluid as a result of bronchoalveolar lavage, GSH values are expressed in relation to immunoglobulin A (IgA). GSH in the ELF was profoundly reduced in the cirrhotic group [12.5 μg of GSH per μg of IgA (5.3-16.9 μg)] compared with the control group [64.0 μg of GSH per μg of IgA (55.1-242.5 μg); p < 0.001]. The ratio of oxidized GSH to total GSH in the ELF was also significantly increased in the cirrhotic group [9.2% (5.1-16.4%) vs. 3.4% (1.7-5.7%); p < 0.003] Conclusions Despite a total reduction in GSH concentrations in the alveolar epithelial lining fluid of individuals with cirrhosis, the amount of oxidized GSH is increased. There is increased utilization of GSH despite the low supply in stable individuals with cirrhosis during steady state. These perturbations in GSH homeostasis in the alveolar epithelial lining fluid may be a factor in the poor outcomes seen in these individuals with ARDS. [ABSTRACT FROM AUTHOR]

Background Alcohol abuse increases the incidence of acute respiratory distress syndrome (ARDS). Previous evidence from our laboratory links ethanol-mediated glutathione depletion to impaired surfactant production by alveolar epithelial cells in vitro and to endotoxin-mediated edematous injury in isolated lungs ex vivo. ARDS patients have an imbalance between the inactive small aggregate (SA) and the bioactive large aggregate (LA) forms of surfactant phospholipid (as reflected by increased SA/LA ratios). Therefore, we hypothesized that ethanol ingestion, via glutathione depletion, could alter surfactant phospholipid distribution between LA and SA forms and thereby exacerbate sepsis-mediated lung dysfunction in vivo. Methods Rats fed an isocaloric diet with or without ethanol (36% total calories) for 6 weeks were made septic via cecal ligation and perforation. Some ethanol-fed rats had their diets supplemented with the glutathione precursor procysteine (l-2-oxothiaxolidine-4-carboxylate). Sepsis physiology was assessed by determining respiratory rates, arterial blood pressures, and plasma lactate levels, and lung dysfunction was assessed by determining lung lavage fluid protein levels (index of alveolar endothelial/epithelial barrier disruption), arterial hypoxemia (index of impaired gas exchange) and surfactant phospholipid SA and LA fractions (index of the alveolar epithelium's ability to maintain a functional surfactant pool during sepsis). Results Ethanol ingestion decreased ( p < 0.05) lung lavage fluid glutathione levels, and this defect was prevented by procysteine. Although ethanol ingestion had no effect ( p < 0.05) on any of the indices of sepsis, it increased ( p < 0.05) lung lavage fluid protein levels, worsened hypoxemia, and decreased the functional (LA) surfactant phospholipid pool after sepsis, all of which was prevented by procysteine. Conclusions Ethanol ingestion, via glutathione depletion, increased sepsis-mediated lung dysfunction, and these effects could contribute to the increased risk of ARDS seen in alcoholic patients. [ABSTRACT FROM AUTHOR]

Background : In septic patients, chronic alcohol abuse increases the incidence of the acute respiratory distress syndrome, a syndrome that requires alveolar type II cell proliferation and differentiation for repair of the damaged alveolar epithelium. We previously showed in a rat model that chronic ethanol ingestion decreased the antioxidant glutathione (GSH) in type II cells and exacerbated endotoxin-mediated acute lung injury. We hypothesized that this GSH depletion by ethanol, particularly mitochondrial GSH, predisposed type II cells to inflammatory mediator-induced apoptosis. Methods: Adult male rats were fed the Lieber-DeCarli diet for 2, 6, or 16 weeks. Alveolar type II cells were then isolated and treated with hydrogen peroxide or TNF-α. The effect on glutathione (cytosolic and mitochondrial), apoptotic events, and necrosis were determined. In other studies, rats were fed ethanol for 6 weeks and were treated with endotoxin and apoptosis of type II cells determined by the TUNEL method. Results: Chronic ethanol ingestion alone resulted in a progressive decrease in mitochondrial GSH and a progressive increase in the basal apoptosis and necrosis rate ( p≤ 0.05). Furthermore, there was a progressive increase in the sensitivity of the cells to H2O2 or TNF-α induced cytochrome c release, caspase 3 activation, apoptosis, and necrosis ( p≤ 0.05). Finally, there was a 2-fold increase in apoptotic type II cells in vivo when chronic ethanol ingestion was superimposed on endotoxemia. Conclusions: These results suggested that chronic ethanol ingestion resulted in a progressive depletion of mitochondrial GSH and sensitization of type II cells to inflammatory mediator-induced apoptosis and necrosis. These effects may be particularly relevant during acute stress when proliferation and differentiation of these cells are critical to repair of the damaged alveolar epithelium and may have important ramifications for the treatment of acute respiratory distress syndrome in patients with a history of alcohol abuse. [ABSTRACT FROM AUTHOR]

Background: Previous studies have shown that chronic ethanol (EtOH) ingestion results in impaired alveolar macrophage function, increased TGF-β(1) production, and decreased antioxidant availability. Similarly, alternative activation (M2 activation) of alveolar macrophages also induces TGF-β(1) production and impairs macrophage function. However, the potential links between EtOH-induced alveolar macrophage derangements, M2 activation, TGF-β(1) production signaling, and oxidant stress have yet to be examined. We hypothesized that EtOH-induced oxidant stress and induction of TGF-β(1) signaling result in alternative activation which subsequently impairs the phagocytic capacity of alveolar macrophages., Methods: Primary rat alveolar macrophages and the alveolar macrophages cell line NR8383 were treated with 0.08% EtOH ± the antioxidant glutathione (GSH) or a TGF-β(1) neutralizing antibody for 5 days. Outcome measures included TGF-β(1) production, reactive oxygen species (ROS) production, phagocytic capacity, and expression of markers of M2 activation., Results: Chronic EtOH treatment greatly decreased alveolar macrophage phagocytic function, increased ROS production, increased TGF-β(1) , and increased expression of markers of M2 activation. GSH supplementation and inhibition of TGF-β(1) signaling during EtOH treatment prevented these alterations., Conclusions: EtOH treatment increased oxidant stress, TGF-β(1) production, and alternative activation in NR8383 cells. However, GSH supplementation and ablation of TGF-β(1) signaling prevented these effects. This suggested that the EtOH-induced switch to an M2 phenotype was a result of decreased antioxidant availability and increased TGF-β(1) signaling. Preventing EtOH-induced induction of alternative activation may improve alveolar macrophage function in alcoholic subjects and decrease the risk of respiratory infections., (Copyright © 2012 by the Research Society on Alcoholism.)

Background: The effects of in utero alcohol exposure on the immune function of the newborn remain under investigation. Fetal ethanol (ETOH) exposure increases oxidative stress in the developing lung, in part due to decreased availability of the antioxidant glutathione (GSH). We have previously shown that in utero ETOH impairs alveolar macrophage phagocytosis and viability in the premature pup, while maintaining GSH availability with maternal supplementation of S-adenosyl-methionine (SAM) during ETOH ingestion improves macrophage function and viability. We hypothesized that dysfunction of the neonatal alveolar macrophage exposed to ETOH in utero would persist at term gestation., Methods: Using a guinea-pig model of fetal ETOH exposure, timed-pregnant guinea-pigs were pair-fed ETOH+/-the GSH precursor SAM and the diet continued until spontaneous delivery. Term alveolar macrophages were evaluated using fluorescent microscopy for phagocytosis and apoptosis after in vitro incubation with Staphalococcus aureus. Using an in vivo model of intranasal Staph. aureus inoculation, the in vivo function of the term alveolar macrophage was also investigated using confocal fluorescent analysis., Results: In utero ETOH exposure increased oxidant stress in the alveolar macrophage and decreased phagocytosis and viability in vitro and in vivo. Confocal analysis of phagocytosis in vivo demonstrated a marked impairment of internalization of the bacteria by the ETOH-exposed alveolar macrophage. The addition of SAM during maternal ETOH ingestion prevented loss of alveolar macrophage function and viability in vitro and in vivo., Conclusions: In utero ETOH exposure impairs alveolar macrophage function and viability in vitro and in vivo even at term gestation. The ETOH-induced changes in macrophage function and viability can be ablated with maternal SAM supplementation. Further investigations are required to identify the mechanisms of ETOH-induced derangement of phagocytosis in the neonatal alveolar macrophage and the clinical ramifications of altered immune function after in utero alcohol exposure for the newborn.

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.

Background: Since chronic alcohol use suppresses the adult immune system, we tested the hypothesis that maternal alcohol ingestion increases the risk of infection in term newborns., Methods: Analysis of a large case-control study of birth weight for gestational age was performed focusing on maternal alcohol ingestion and the development of infection in term newborns > or =36 weeks gestation. After delivery, mothers were asked about alcohol and tobacco use in the 3 months prior to conception, the 1st, 2nd, and 3rd trimester of pregnancy., Results: Eight hundred and seventy-two singleton newborns (872) > or = 36 weeks gestation were identified for analysis. A total of 51 (5.8%) had newborn infections. Gestational age, sex, and small for gestational age (SGA) were similar in the newborns with and without infection (p = NS). Infants whose mothers reported alcohol use, excessive drinking or smoking in pregnancy were more likely to have a newborn diagnosed with an infection than were mothers who reported abstaining from alcohol or cigarettes (p < 0.05). When controlling for race and smoking, SGA infants whose mothers used any alcohol had a 2.5-fold increase risk of infection, while excessive alcohol use increased the risk 3-4-fold. In a multivariable logistic regression analysis controlling for low maternal income, smoking, and SGA, excessive alcohol use during the 2 trimester increased the risk of newborn infection (OR 3.7 [1.1,12.8], p < 0.05)., Conclusions: Excessive maternal alcohol use is associated with an increased risk of newborn infection in this patient sample. Increased awareness and further clinical investigations are warranted to address the detrimental effects of fetal alcohol exposure on the developing immune system.

Background: An association between the development and severity of the acute respiratory distress syndrome has been described in individuals who abuse alcohol chronically, possibly through a mechanism involving the deficiency of pulmonary glutathione. In a rodent model of chronic alcohol abuse, this antioxidant contributes to the maintenance of alveolar-capillary membrane integrity. We postulated that humans who chronically abuse alcohol will have similar alterations in alveolar-capillary barrier function., Methods: Bronchoalveolar lavage was performed in 18 healthy chronic alcoholics and 18 control subjects; total protein and glutathione concentrations were measured within the epithelial lining fluid. To examine possible protracted effects of alcohol abuse, a subset of 11 chronic alcoholic subjects underwent a second bronchoalveolar lavage after a week of abstinence., Results: Chronic alcoholic subjects had significantly elevated protein concentrations compared with controls (8.64 microg protein/ng immunoglobulin A vs. 5.91 microg protein/ng immunoglobulin A, p = 0.01). After a week of abstinence, no significant increase in either the glutathione levels or normalization of the protein concentrations in the epithelial lining fluid was demonstrable., Conclusions: Increased protein levels in the epithelial lining fluid of individuals who abuse alcohol chronically may signify abnormal alveolar epithelial barrier function that does not appear to readily reverse after a period of abstinence.

Background: Alcohol abuse increases the risk of acute lung injury in critically ill patients. We have shown that alveolar epithelial cell (AEC) apoptosis in response to inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha), parallels endotoxin-mediated acute lung injury in ethanol-fed rats. Although angiotensin II mediates TNF-alpha-induced apoptosis of AECs in vitro, its role in ethanol-mediated susceptibility to AEC apoptosis is unknown., Methods: Adult male rats were fed the Lieber-DeCarli diet for 6 weeks. AECs were isolated, and TNF-alpha- and angiotensin II-induced cytotoxicity (by terminal transferase-mediated dUTP nick end labeling staining) was determined with or without the addition of the angiotensin-converting enzyme inhibitor (lisinopril) or a selective blocker of the angiotensin II type 1 receptor (AT(1)) or type 2 receptor (AT(2)). Finally, the relative expression of the AT(1) and AT(2) receptors in AECs was determined by Western blot analysis., Results: TNF-alpha-induced cytotoxicity, but not angiotensin II-induced cytotoxicity, was prevented by lisinopril, indicating that de novo angiotensin II synthesis is required for TNF-alpha-induced apoptosis in these cells. Both TNF-alpha- and angiotensin II-induced cytotoxicity in AECs from control-fed and ethanol-fed rats were inhibited by the selective AT(2) blocker, PD123319, but not by the selective AT(1) blocker, losartan. In parallel, ethanol ingestion doubled AT(2) expression in AECs (by Western blot) but had no significant effect on AT(1) receptor expression., Conclusions: Chronic ethanol ingestion increases AT(2) expression in the alveolar epithelium and enhances TNF-alpha- and angiotensin II-induced cytotoxicity, both of which act via AT(2). Together, these findings suggest that selective AT(2) receptor inhibition could limit the development of acute lung injury in alcoholic patients.