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. Author manuscript; available in PMC: 2013 Nov 1.
Published in final edited form as: Alcohol Clin Exp Res. 2012 May 2;36(11):1952–1962. doi: 10.1111/j.1530-0277.2012.01825.x

Ethanol induced TGF-β1 and ROS production are necessary for ethanol induced alveolar macrophage dysfunction and induction of alternative activation

Sheena D Brown 1,2, Lou Ann S Brown 1
PMCID: PMC3414680  NIHMSID: NIHMS365163  PMID: 22551312

Abstract

Rationale

Previous studies have shown that chronic ethanol ingestion results in impaired alveolar macrophage function, increased TGF-β1 production, and decreased antioxidant availability. Similarly, alternative activation (M2 activation) of alveolar macrophages also induces TGF-β1 production and impairs macrophage function. However, the potential links between ethanol-induced alveolar macrophage derangements, M2 activation, TGF-β1 production signaling, and oxidant stress has yet to be examined.

Objective

We hypothesized that ethanol-induced oxidant stress and induction of TGF-β1 signaling results in alternative activation which subsequently impairs the phagocytic capacity of alveolar macrophages.

Methods

Primary rat alveolar macrophages and the alveolar macrophages cell line NR8383 was treated with 0.08% ethanol ± the antioxidant glutathione (GSH) or a TGF-β1 neutralizing antibody for 5 days. Outcome measures included TGF-β1 production, reactive oxygen species (ROS) production, phagocytic capacity, and expression of markers of M2 activation.

Results

Chronic ethanol treatment greatly decreased alveolar macrophage phagocytic function, increased ROS production, increased TGF-β1, and increased expression of markers of M2 activation. Glutathione supplementation and inhibition of TGF-β1 signaling during ethanol treatment prevented these alterations.

Conclusions

Ethanol treatment increased oxidant stress, TGF-β1 production, and alternative activation in NR8383 cells. However, GSH supplementation and ablation of TGF-β1 signaling prevented these effects. This suggested the ethanol-induced switch to a M2 phenotype was a result of decreased antioxidant availability and increased TGF-β1 signaling. Preventing ethanol-induced induction of alternative activation may improve alveolar macrophage function in alcoholic subjects and decrease the risk of respiratory infections.

Keywords: macrophage, TGF-β1, oxidative stress, glutathione, alternative activation

INTRODUCTION

Abuse or excessive consumption of alcohol has numerous detrimental effects on physical and mental health, and causes extensive organ damage. Many of these effects contribute to increased mortality and morbidity and are irreversible (Boé et al., 2009; Cubero et al., 2009; Harper, 2009; Lands, 1998; Oscar-Berman and Marinkovic, 2003; Rehm et al., 2003; Room et al., 2005). Patients with alcohol use disorders (AUDs), including alcohol abuse and alcohol dependence, have a 2 fold increase in the incidence of ICU-related morbidity and mortality. This is commonly caused by acute respiratory distress syndrome (ARDS), which is characterized by lung inflammation that leads to impaired gas exchange and release of pro-inflammatory cytokines (Jurkovich et al., 1993; Moss et al., 1996; O Brien et al., 2007). The outcome of AUD patients with ARDS is worse than in non-alcoholics, as seen by significantly higher in-hospital mortality rates. The increased incidence of ICU admittance is primarily related to an increased risk of sepsis in AUD subjects. The increased risk of respiratory infections is partially due to an impaired immune response of the alveolar macrophages, the resident phagocyte in the airspace (Aytacoglu et al., 2006; Brown et al., 2004; Joshi and Guidot, 2007).

Depending on the different stimuli within the microenvironment within the alveolar space, alveolar macrophages are activated via distinct pathways that produce opposing effects on macrophage receptor expression, cytokine expression, and phagocytic function (Abramson and Gallin, 1990; Gordon, 2003; Gordon and Taylor, 2005; McKenzie et al., 1998; Muller et al., 2007; Taylor et al., 2005). Upon exposure to microbial stimuli such as lipopolysaccharide or TH1 cytokines, the alveolar macrophage is primed to become “classically activated” effector cells (Downing et al., 1999; Nathan and Hibbs, 1991; Noda and Amano, 1997). In response to TH2 cytokines, alveolar macrophages become alternatively activated and exhibit a phenotype that mediates allergic responses and promotes angiogenesis, tissue repair, and antiparasitic functions. Alternative activation is characterized by pronounced expression of arginase-1, a dampened immune response and promotion of tissue repair (Gordon, 2002, 2003; Gordon and Martinez; Gordon and Taylor, 2005; Martinez et al., 2009; Stein et al., 1992; Taylor et al., 2005). While alternative activation decreases inflammation, chronic impairment of the immune response decreases the capacity to remove microbial pathogens. Alternative activation has also been shown to induce lung fibrosis via production of profibrotic factors such as fibronectin, matrix metalloproteinases, and TGF-β1 (Isono et al., 2002; Leask and Abraham, 2004; Ruiz-Ortega et al., 2007; Wang et al., 2006). Alternative activation is distinguished by the upregulation of several markers including arginase-1, galectin-3, mannose receptor, Ym1/2, TGF-β1, and FIZZ-1 (Fichtner-Feigl et al., 2006; MacKinnon et al., 2008; Nair et al., 2003; Stein et al., 1992). Therefore, alternative activation is one mechanism by which the immune response or the pro-fibrotic response of alveolar macrophages is modulated.

In previous studies, chronic ethanol ingestion impaired alveolar macrophage binding and internalization of inactivated S. aureus. Over 50% of the macrophages isolated from ethanol-fed animals bound the pathogen but were unable to complete internalization (Brown et al., 2004). Additionally, chronic ethanol ingestion decreased antioxidant availability, leading to chronic ROS generation by alveolar macrophages (Bechara et al., 2004; Brown et al., 2004; Brown et al., 2007). In in vivo studies, ROS induces TGF-β1 expression(Koli et al., 2008) which is also a characteristic of alternatively activated macrophages (Martinez et al., 2009; Song et al., 2000; Stein et al., 1992). Once TGF-β1 is activated, it induces the depletion of the antioxidant glutathione (GSH) and increases intracellular ROS production. In contrast, in vitro treatments with GSH precursors induce proteolysis of the TGF-β1 type II receptor and disintegration of TGF-β1 (Koli et al., 2008). In other studies, TGF-β1 has been shown to directly induce arginase-1 activity and inhibit generation of nitric oxide by inducible NO synthase, a key step in the antimicrobial response during classical activation (Durante et al., 2001; Vodovotz et al., 1993). Lastly, Mitchell et al. demonstrated in conducting airways that chronic ethanol ingestion increased production of IL-13, a TH2 cytokine that induces alternative activation, as well as its receptor IL-13Rα1 (Mitchell et al., 2009).

Given the similarities in phenotypes, we hypothesized that ethanol-induced alveolar macrophage dysfunction is due to dysregulation of TGF-β1 signaling and increased ROS which induce alternative activation. Correspondingly, we examined whether antioxidant treatments and/or inhibition of TGF-β1 signaling prevent these ethanol-induced alterations. Our data suggests TGF-β1 signaling and GSH availability are an integral part of ethanol-induced alternative activation of alveolar macrophages, phagocytic dysfunction, and promotion of fibrogenesis.

METHODS

Cell Line Culture and Treatment

NR8383 cells, a rat alveolar macrophage–derived cell line (ATCC, Rockville, USA), were maintained in a humidified 5%CO2 incubator at 37°C with FK-12 media supplemented with 15% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (100 U/ml penicillin G sodium, 100 U/ml streptomycin, and 0.25 μg/ml amphotericin B). Cells were treated daily with fresh media containing 0.08% ethanol ± 500 μM GSH, ± 8 μg/ml TGF-β1 neutralizing antibody (AB-101-NA, R&D Systems, Minneapolis, USA), or ± 5 μg/ml IL-13 neutralizing antibody (MAB194, R&D Systems) for 5 consecutive days. As a control, some cells were treated with rat IgG for 5 consecutive days but no significant differences were observed when compared to ethanol alone (data not shown). Cells treated with ethanol were maintained in a closed chamber in order to maintain the ethanol concentration and media were changed daily. As a positive control for alternative activation, some cells were treated with 1.5 ng/ml IL-13 (1945-RL, R&D Systems) for 5 consecutive days.

Detection of Extracellular Hydrogen Peroxide (H2O2)

H2O2 release was determined using the oxidation of Amplex Red in the presence of horseradish peroxidase reagent according to the manufacturer’s instructions (Invitrogen; Carlsbad, CA). Briefly, cells were treated for 45 min with 100 μl of Amplex Red solution (50μM of Amplex Red reagent and 10 U/ml horseradish peroxidase in phosphate buffered saline). The H2O2 production was measured via a microplate reader by taking the absorbance at A560 and interpolating from a standard curve generated with known freshly prepared H2O2 concentrations.

Detection of Intracellular H2O2 Production

Intracellular H2O2 production was determined using the Amplite Green Peroxidase Sensor (Invitrogen; Carlsbad, CA), a cell permeable sensor that fluoresces green upon reaction with H2O2. Using an excitation wavelength of 480 nm and emission wavelength of 522 nm, fluorescence from Amplite Green was determined via quantitative digital analysis of microscopic images using FluoView (Olympus; Center Valley, PA). Background autofluorescence was determined and subtracted from the mean relative fluorescent units. Values are presented as the mean relative fluorescent units per cell (± SE) as tallied from at least 15 experimental fields per set.

Arginase Activity Measurements

Arginase activity was measured using the QuantiChrom Arginase Assay Kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s instructions. All values were normalized to protein concentration.

Measurement of TGF-β1 and IL-13 by ELISA

The TGF-β1 and IL-13 present in the cell lysates were quantitated by the commercially available ELISA kits TGF-β1 Emax immunoassay system (Promega; Madison, WI) and the Quantikine Rat IL-13 Immunoassay for IL-13 (Invitrogen; Carlsbad, CA), respectively. Cell lysates were loaded in a 1:10 dilution to measure production of IL-13 and TGF-β1. All values were normalized to the corresponding protein concentration.

Phagocytosis of pHrodo-labeled S. Aureus

NR8383 cells were plated onto 8 well chamber slides at a density of 106 cell/ml and allowed to adhere for 1 hr. After washing twice with sterile phosphate buffered saline, the cells were incubated with pHrodo-labeled S. aureus (2 hours; 1 mg/mL). The rhodamine-based pHrodo dye is pH-sensitive such that it is non-fluorescent at neutral pH and fluoresces red in acidic environments such as the phagolysosome. Thus, cells with red fluorescence indicate internalization, sequestration, and a respiratory burst within the phagolysosome. After incubation, the media was removed and the cells washed twice with phosphate buffered saline before fixation with 4% paraformaldehyde. Phagocytosis of pHrodo labeled S. aureus was determined via quantitative digital analysis of microscopic images using FluoView (Olympus; Melville, NY). Background autofluorescence was determined and subtracted from the mean relative fluorescent units. Values are presented as mean relative fluorescent units (RFU)/cell ± SEM, the mean percentage of cells fluorescently positive (per field) ± SEM, and phagocytic index ± SEM (RFU x % positive) as tallied from at least 15 experimental fields/set.

Western Blot Analysis

Cell lysates were prepared by adding radioimmunoprecipitation assay (RIPA) buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific; Rockford, IL). Protein concentration was determined using the Micro BCA Protein Assay Reagent Kit (Pierce). 30–50 μgs of total protein from each sample were electrophoresed on 4–15% gradient polyacrylamide gels at 200 V for 45 min and transferred to nitrocellulose membranes (Bio Rad Laboratories, Richmond, CA) at 100V for 1 hr. Membranes were blocked at room temperature for 1 hr in Tris buffered saline (TBS) with 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk. After washing (3 times; 10 min each), membranes were probed with primary antibodies to arginase-1 (1:100) (Santa Cruz Biotechnologies, Santa Cruz, CA) or GAPDH (1:500) (Santa Cruz Biotechnologies) diluted in 5% milk in TBS-T and kept at 4°C overnight. After 3 washes with T-TBS for 10 min, membranes were incubated at room temperature with secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnologies) for 1hr. After adding ECL chemiluminescence reagent (Denville Scientific, Metuchen, NJ) to the membranes, bands were detected and quantified via densitometry.

Capacity to Activate Fibroblasts

To determine if alternatively activated alveolar macrophages promoted fibroblast activation, NIH/3T3 fibroblasts stably transfected with a fibronectin promoter upstream of a luciferasereporter gene were used. These fibroblasts were seeded at a density of 106 cells/ml and allowed to adhere for 24 hrs. The 3T3 cells were then incubated for 24 hr with 300 μl of cell-free media from the treated NR8383 cells, the 3T3 cells were detached by scraping, washed with PBS containing 100 μl of reporter lysis buffer (Promega; Madison, WI), and then assayed for fibronectin transcription via luciferase activity using the Promega luciferase assay system (Promega; Madison, WI). Luciferase activity was normalized to total 3T3 cellular protein.

H2O2 Treatment

NR8383 cells were plated onto 8 well chamber slides (5X105 cells/ml) and allowed to adhere for 1 hr. After washing twice with sterile phosphate buffered saline, NR8383 cells were treated with 15 μM H2O2 for 30 minutes. Where appropriate, cells were pretreated with 8 ng/ml TGF-β1 neutralizing antibody for 1 hour prior to H2O2 treatment.

TGF-β1 Treatment

NR8383 cells were plated onto 8 well chamber slides at a density of 5X105 cells/ml and allowed to adhere for 1 hr. After washing twice with sterile phosphate buffered saline, NR8383 cells were treated with 10 ng/ml of TGF-β1 (T7039, Sigma-Aldrich) for 24 hours. Where appropriate, cells were co-treated with 500 μM GSH.

Cellular Viablity

To determine cellular viability following treatments, the trypan blue exclusion test was used. Cell were stained with equal volumes 0.4% trypan blue and counted using a Countess automated cell counter.

Quantitative PCR

RNA was isolated from NR8383 cells preserved in RNAlater according to the manufacturer’s instructions (RNeasy Mini; Qiagen, Valencia, Calif). Following isolation, 10 ng of total RNA per sample was reverse transcribed into cDNA using oligo (dT)-primers, according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). cDNA was then used to quantitate relative levels of arginase-1, CD32, and galection-3 gene expression, using the ABI Prism 7000 real time PCR system (Applied Biosystems). Data were normalized to glyceraldehydes 3-phosphate dehydrogenase (GAPDH). Net cycle threshold (CT) values for each gene or interest were used to calculate ΔCT values for each sample.

Alveolar Macrophage Isolation

Control male Sprague-Dawley rats (Harlan, St. Louis, MO) were used in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals) as described in protocols reviewed and approved by the Emory University Institutional Animal Care Committee. Briefly, after pentobarbital anesthesia, the trachea was cannulated and the rat lung underwent 5 bronchoalveolar lavages, consisting of 10 ml of sterile phosphate-buffered saline (37°C, pH 7.4). The lavage fluid was centrifuged at 500 g for 8 min, and the cell pellet was resuspended in Dulbecco’s modified Eagle’s medium supplemented with 2% fetal bovine serum plus penicillin and streptomycin (100 U/l each). Cell count and viability was determined on the Countess™ Automated Cell Counter (Invitrogen; Carlsbad, CA). Rat primary alveolar macrophages were plated at 0.25 million cells/ml and cultured at 37°C and 5% CO2 and allowed to adhere for 1 hr. Cells were then treated with ethanol (0.08%) ± 500 μM GSH or 8 ng/ml TGF-β1 neutralizing antibody for 5 consecutive days as described above for NR8383 cells.

Statistical Analysis

Statistical analysis was performed with Sigma Stat for Windows. The data is presented as means ± SE. Results were analyzed using the Students t-test or one-way analysis of variance where appropriate, followed by Student-Newman-Keuls test comparisons. A p-value of < 0.05 was considered significant.

RESULTS

Chronic Ethanol Treatment Induces Production of ROS and TGF-β1 in NR8383 Cells

Our previous studies demonstrated that chronic alcohol alters oxidant/antioxidant balance by decreasing GSH availability and increasing ROS (Brown et al., 2007; Moss et al., 2000). In the current study, we determined the effects of chronic ethanol treatment on intracellular and extracellular production of H2O2. As a positive control for alternative activation, some cells were treated with IL-13. After treatment of NR8383 cells (0.08% ethanol; 5 consecutive days), there was a 6 fold increase in extracellular H2O2 levels (Figure 1A; a p <0.05), and a 15-fold increase in intracellular H2O2 levels (Figure 1B; a p <0.05). Similarly, IL-13 led to a 6 fold increase in extracellular H2O2 and a ~19–fold increase in intracellular H2O2 levels (Figure 1-B). Representative images for intracellular H2O2 production are shown in Figure 1C. In NR8383 cells, these effects of ethanol on ROS generation were time-dependent (0.08% ethanol; 1–7 days) and dose-dependent (0.02% to 0.16%; 5 days) (Supplemental Figures 1–2). TGF-β1 was also increased ~2-fold in response to treatment with ethanol or IL-13 (Figure 1D; a p <0.05). To determine if ethanol-induced TGF-β1 production and ROS generation were dependant on one another, cells were treated with 0.08% ethanol plus GSH (500 μM), or TGF-β1 neutralizing antibody (8 ng/ml). After 5 days, GSH treatments or the TGF-β1 neutralizing antibody normalized extracellular and intracellular H2O2 (Figure 1A–B), as well TGF-β1 production (Figure 1D).

Figure 1. Chronic Ethanol Treatment Increases ROS andTGF-β1 Production.

Figure 1

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NR8383 cells were treated with 0.08% ethanol ± 500 μM GSH or 8 ng/ml TGF-β1 neutralizing antibody for 5 consecutive days. The extracellular level of H2O2 produced by NR8383 cells was determined using amplex red (A). Intracellular H2O2 production was quantified via amplite green fluorescence (B). Representative confocal images are shown (C). Intracelluar TGF-β1 was also measured via ELISA (D). Bars heights represent means ± SE. a p≤0.05 compared to controls, b p≤ 0.05 compared to the ethanol treatment. n=15 or more fields per group

Ethanol Exposure Induces IL-13 Expression in NR8383 Cells

Since the TH2 cytokine IL-13 induces alternative activation, we next sought to determine if ethanol induces expression of IL-13. In NR8383 cells, ethanol induced a 3.7 fold increase in IL-13 protein expression (Figure 2; a p <0.05). GSH treatments significantly reduced IL-13 protein expression but did not normalize its protein expression (Figure 2; b p <0.05). However, inhibition of TGF-β1 signaling with the TGF-β1 neutralizing antibody normalized IL-13 expression to control values (Figure 2; b p <0.05). It is also important to note that extracellular IL-13 upregulated intracellular IL-13 ~ 70%.

Figure 2. Chronic Ethanol Treatment Induces IL-13 Expression.

Figure 2

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The effects of chronic ethanol treatment on expression of IL-13 was examined in NR8383 treated with 0.08% ethanol ± 500 μM GSH or 8 ng/ml TGF-β1 neutralizing antibody for 5 consecutive days. Cells were also treated with IL-13 as a positive control. IL-13 protein expression was measured via ELISA (A). Bar heights represent mean ± SE. a p≤ 0.05 compared to controls, b p≤ 0.05 compared to ethanol, n=4 or more per group.

Ethanol-Induces M2 Activation of NR8383 Cells and Promotes a Pro-Fibrotic Phenotype

Since ethanol increased expression of IL-13, a cytokine that induces alternative activation, we next examined whether ethanol exposure upregulated markers of alternative activation. With ethanol treatment, NR8383 cells exhibited a ~2.8 fold increase in arginase-1 expression and a~50% increase in galectin-3 expression, similar to that observed with IL-13 alone (Figure 3A–C; a p <0.05). Additionally, ethanol treatments caused a 2.5 fold increase in arginase-1 activity (Figure 3D; a p <0.05). Treatment with GSH or the TGF-β1 neutralizing antibody prevented ethanol-induced increases in arginase-1 and galectin-3 protein expression, as well as arginase-1 activity (Figure 3A–D, bp <0.05). We next examined whether IL-13-induced alternative activation is associated with a macrophage phenotype that promotes fibroblast activation. When the cell-free supernatants from ethanol-treated NR8383 cells were overlaid onto the 3T3 fibroblasts, there was a 2.5-fold increase in fibronectin gene transcription by the 3T3 cells (Figure 3E; a p <0.05). When the NR8383 cells were treated concomitantly with ethanol plus the TGF-β1 neutralizing antibody, the increased fibroblast activation expected for ethanol treatment was blocked (Figure 3E; b p <0.05). Similar results were seen when the NR8383 cells were treated with GSH during the ethanol exposure.

Figure 3. Chronic Ethanol Treatment Induces Alternative Activation.

Figure 3

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The effects of chronic ethanol treatment on alternative activation was examined in NR8383 treated with 0.08% ethanol ± 500 μM GSH or 8 ng/ml TGF-β1 neutralizing antibody for 5 consecutive days. Cells were also treated with IL-13 as a positive control. Expression of arginase-1 (A) and galectin-3 (B) were determined via western blot analysis. Representative immunoblots are shown in C. Assessment of arginase-1 activity in was determined and normalized to protein concentrations (D). To measure fibroblast proliferation, transfected 3T3 cells were treated with the cell-free supernatants from all NR8383 cell treatment groups for 24 hrs. The 3T3 cells were then washed and lysed before fibronectin gene transcription was measured by luminescence (E). Data are presented as average luciferase activity ± SD. Bar heights represent mean ± SE. a p≤0.05 compared to controls, b p≤0.05 compared to ethanol, n=3 or more per group.

GSH Supplementation and Inhibition of TGF-β1 Prevented Ethanol–Induced Loss of NR8383 Cell Phagocytic Function

Chronic ethanol exposure significantly decreased the internalization of pHrodo red labeled S. aureus as determined by a ~55% decrease in the phagocytic function (Figure 4A, a p <0.05; Representative images in Figure 4B). In NR8383 cells, ethanol-induced suppression of phagocytosis was time- and dose-dependent (Supplemental Figures 1–2). For control cells, neither GSH supplementation nor inhibition of TGF-β1 signaling significantly altered the phagocytic capacity. However, daily supplementation with either GSH or the TGF-β1 neutralizing antibody prevented ethanol-induced decreases in NR8383 cell phagocytic function (Figure 4A, bp <0.05).

Figure 4. Chronic Ethanol Treatment Impaired Phagocytosis of pHrodo-Labeled S. aureus.

Figure 4

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Ethanol induced impairment of NR8383 cell phagocytosis was examined using the pH sensitive pHrodo labeled S. aureus. NR8383 cells were incubated with 1mg/ml pHrodo labeled S. aureus for 2 hr. Internalization was evaluated using quantitative digital analysis of fluorescence on the confocal microscopy. Representative photomicrographs at 20X magnification are shown in A. Bar heights represent the phagocytic index (B). Bars heights represent means ± SE. a p≤0.05 compared to controls, b p≤ 0.05 compared to ethanol, n=15 or more fields per group.

Ethanol-Induced M2 Activation and Decreased Phagocytic Function in NR8383 Cells is Dependent on IL-13

We next examined whether ethanol promotes alternative activation and decreases phagocytic function in an IL-13 dependant manner. Similar to our previous results, ethanol treatment resulted in a ~50% decrease in phagocytic capacity, a ~3 fold increase in arginase activity, and a ~2-fold increase in TGF-β1 (Figure 5A–C, ap <0.05). These effects of ethanol on alternative activation were time- and dose-dependent (Supplemental Figures 1–2). Treatment with the IL-13 antibody (5 μg/ml) during ethanol treatment normalized NR8383 cell phagocytic capacity and partially blocked ethanol-induced alternative activation, as determined by decreased arginase activity and TGF-β1 expression (Figure 5A–C, bp <0.05).

Figure 5. Ethanol Induced Alternative Activation and Loss of Phagocytic Function is Dependent on IL-13.

Figure 5

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The role of IL-13 in ethanol-induced NR8383 cell derangements was determined. Nr8383 cells were treated with 0.08% ethanol ± 5 μg/ml of IL-13 neutralizing antibody for 5 consecutive days. Phagocytic capacity (A), arginase activity (B), and TGF-β1 production (C) were determined. Bars heights represent means ± SE. a p≤0.05 compared to controls, b p≤0.05 compared to the ethanol treatment, n=3 or more per group.

H2O2 and TGF-β1–Induced NR8383 Cell Dysregulation

To delineate the relationship between ethanol-induced intracellular ROS generation, TGF-β1, and alternative activation, NR8383 cells were treated with H2O2 or TGF-β1 ± GSH. Where appropriate, cells were pretreated with TGF-β1 neutralizing antibody for 1 hour prior to treatment with H2O2 for 30 minutes. H2O2 treatment resulted in a ~65% decrease in phagocytic capacity, a 32% increase in IL-13 secretion, and a 73% increase in extracellular TGF-β1 (Figure 6; a,bp <0.05) Neutralization of TGF-β1 during H2O2 treatment partially prevented NR8383 cell phagocytic dysfunction and normalized IL-13 secretion. Similarly, exposure to TGF-β1 directly decreased phagocytic capacity by ~40%, increased intracellular ROS ~5.4 fold, and increased IL-13 production by ~35%. Co-treatment with 500 μM GSH partially blocked these effects (Figure 7; a,bp <0.05).

Figure 6. H2O2 Mediated Impairment of Phagocytic Function.

Figure 6

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N8383 cells were treated with H2O2 for 30 minutes ± pretreatment with 8 μg/ml TGF-β1 neutralizing antibody for 1 hour. H2O2 induced impairment of NR8383 cell phagocytosis was examined using the pH sensitive pHrodo labeled S. aureus. NR8383 cells were incubated with 1mg/ml pHrodo labeled S. aureus for 2 hr. Internalization was evaluated using quantitative digital analysis of fluorescence on the confocal microscope (A). Intracelluar TGF-β1 was also measured via ELISA (B). IL-13 protein expression was measured via ELISA (C). Bars heights represent means ± SE. a p≤0.05 compared to controls, b p≤0.05 compared to ethanol, n=15 or more fields per group.

Figure 7. TGF-β1-Induced Phagocytic Dysfunction and Increased ROS.

Figure 7

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N8383 cells were treated with TGF-β1 ± 500 μM GSH for 24 hr. Bar heights represent the phagocytic index (A). Intracellular H2O2 production was quantified via amplite green fluorescence (B). ExtracellularTGF-β1 was also measured via ELISA (C). IL-13 protein expression was measured via ELISA (D). Bars heights represent means ± SE. a p≤0.05 compared to controls, b p≤0.05 compared to the ethanol treatment. n=10 or more fields per group.

Chronic Ethanol-Induced Phagocytic Dysfunction, ROS Generation, and Upregulation of TGF-β1 and IL-13 in Rat Alveolar Macrophages

To determine physiological relevance, freshly isolated rat alveolar macrophages were treated with 0.08% ethanol for 5 consecutive days. Similar to that observed with NR8383 cells, ethanol treatment of freshly isolated rat alveolar macrophages resulted in a ~2.25 fold increases in intracellular ROS and ~41% decrease in phagocytic capacity (Figure 8A&B; a p <0.05). Treatment with either GSH or TGF-β1 neutralizing antibody prevented these effects (Figure 8A&B; b p <0.05). Importantly, none of the treatments resulted in altered cellular viability (data not shown). In response to ethanol treatment, alveolar macrophages displayed a 5-fold increase in TGF-β1 excretion and a 2-fold increase in extracellular IL-13 (Figure 8C&D; a p <0.05). Additionally, supplementation with either GSH or TGF-β1 neutralizing antibody partially blocked ethanol-induced IL-13 production (Figure 8D; b p <0.05).

Figure 8. Chronic Ethanol-Induced Phagocytic Dysfunction, ROS Generation, and Upregulation of TGF-β1 and IL-13 in Rat Alveolar Macrophages.

Figure 8

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Freshly isolated rat alveolar macrophages were treated with 0.08% ethanol ± 500 μM GSH or 8 ng/ml TGF-β1 neutralizing antibody for 5 consecutive days. Following treatment, alveolar macrophages were incubated with 1mg/ml pHrodo labeled S. aureus for 2 hr. Internalization was evaluated using quantitative digital analysisof fluorescence on the confocal microscopy. Bar heights represent the phagocytic index (A). Intracellular H2O2 production was quantified via amplite green fluorescence (B). ExtracellularTGF-β1 was also measured via ELISA (C). IL-13 protein expression was measured via ELISA (D). Bars heights represent means ± SE. a p≤0.05 compared to controls, b p≤0.05 compared to the ethanol treatment. n=10 or more fields per group

DISCUSSION

Numerous studies have demonstrated that patients with alcohol use disorders have an increased risk of respiratory infections, which is partially due to ethanol-induced impairment of the immune functions of alveolar macrophages. However, mechanisms by which alcohol impairs alveolar macrophage function are poorly understood (Boé et al., 2009; Esper A., 2006; Kane and Galanes, 2004; Moss et al., 1996; Moss and Burnham, 2003). On the other hand, previous studies suggest the majority of the negative effects of alcohol on alveolar macrophage dysfunctions are due to decreased GSH availability and subsequent oxidant stress within the alveolar space (Brown et al., 2004; Brown et al., 2007). Previous studies by this laboratory group demonstrated in rodent models of alcohol abuse that ethanol-induced phagocytic dysfunction could be reversed with ex-vivo GSH supplementation (Brown et al., 2007). Other studies showed that in vivo treatments with GSH precursors prevented ethanol-induced pulmonary macrophage dysfunction.

Previous studies have shown that chronic ethanol ingestion increases TGF-β1 production within alveolar macrophages. Our current studies demonstrated that chronic ethanol ingestion also leads to increased oxidant stress within the alveolar macrophage, as evidenced by increased intracellular and extracellular ROS. In other studies, ROS have been shown to induce TGF-β1 activation in vivo (Koli et al., 2008). Similarly, alternatively activated macrophages have robust increases in TGF-β1 production and secretion (Martinez et al., 2009; Song et al., 2000; Stein et al., 1992). Once activated, TGF-β1 has been shown to deplete intracellular glutathione stores, and increase intracellular ROS production, thereby, creating a vicious cycle of ROS generation. TGF-β1 also induces arginase-1 activity and inhibits generation of nitric oxide by inducible NO synthase, both markers of alternative activation. In the conducting airways, chronic ethanol ingestion increases production of IL-13, as well as its receptor IL-13Rα1 (Mitchell et al., 2009).

In the current study, chronic ethanol treatment increased expression of IL-13, a known inducer of alternative activation (Muller et al., 2007; Munder et al., 1998), as well as expression of established markers of alternative activation. Ethanol treatment also increased ROS generation and release, increased TGF-β1 production, and severely diminished phagocytic capacity. These effects were dependent on the ethanol concentration and the time of exposure. Ablation of TGF-β1 signaling through the use of a TGF-β1 neutralizing antibody blocked ethanol-induced ROS generation by NR8383 cells or control alveolar macrophages, expression of alternative activation markers, impaired phagocytosis and secretion of factors that upregulate fibroblast proliferation. This suggested that ethanol induced an autocrine loop between ROS generation and TGF-β1 expression, secretion, and signaling; thereby, making ethanol-induced increases in ROS and TGF-β1 central to alternative activation of alveolar macrophages and impaired immune responses. These results support other studies with TGF-β1 that demonstrate robust TGF-β1 production by alternatively activated macrophages (Gordon, 2003; Gordon and Martinez, 2010; Martinez et al., 2009) as well as the ability of TGF-β1 to upregulate arginase-1 activity and polyamine release, markers of alternative activation (Boutard et al., 1995; Lee et al., 2001).

In the current study, GSH treatments of NR8383 cells or primary alveolar macrophages also blocked ethanol-induced ROS generation, TGF-β1 expression and secretion, alternative activation, impaired phagocytic capacity, and the capacity of AM to secrete factors that promote fibroblast proliferation. This further supported the concept that ethanol-induced ROS generation was central to upregulation of TGF-β1 and subsequent switch of the alveolar macrophage to an alternatively activated phenotype with accompanying impaired immune responses. Ethanol-induced upregulation of IL-13 also plays a role in this feed forward loop as evidenced by the ability of the IL-13 neutralizing antibody to partially attenuate ethanol-induced expression of TGF-β1 and alveolar macrophage polarization. Additionally, our studies suggest the increased expression of IL-13 can be ablated by treatment with TGF-β1 inhibitors and attenuated with GSH. Therefore, we propose ethanol induced production of TGF-β1 is central to ethanol induced impairments of alveolar macrophage function and induction of alternative activation.

In conclusion, these studies with NR8383 cells and primary alveolar macrophages demonstrated that chronic ethanol exposure promotes a feed-forward loop between increased ROS generation and TGF-β1 expression and signaling. Ethanol-induced upregulation of IL-13 was also a component of this feed-forward loop. Increased signaling through IL-13 and TGF-β1 subsequently impaired the immune response of alveolar macrophages by inducing an alternatively activated phenotype. While the increases in galectin-3 were relatively modest compared to the other parameters, the increased activation of fibroblasts were most likely due to the combined effects of increased secretion of TGF-β1 and IL-13 by the alternatively activated alveolar macrophage. This phenotype switch may decrease the pro-inflammatory responses of alveolar macrophages, the chronic suppression of phagocytosis decreases the capacity of alveolar macrophages to clear microbes and may explain the increased risk of respiratory infections associated with chronic alcohol use disorders. Additional studies are needed to determine if in vivo treatments with GSH, TGF-β1 inhibitors or IL-13 inhibitors will reverse ethanol-induced suppression of the immune functions of alveolar macrophages and decrease the risk of respiratory infections.

Supplementary Material

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Acknowledgments

This study was funded by The National Institute of Alcohol Abuse and Alcoholism Grant R01 AA12197 and R01 HL096924 (LAB), the National Institute of Alcohol Abuse and Alcoholism Grant 1 P50 AA 135757 (LAB), and NIAAA Pre-doctoral Grant F31 AA017812 (SDB).

Abbreviations

ARDS

Acute respiratory syndrome

M2 activation

Alternative activation

AUD

Alcohol use disorders

GSH

Glutathione

ROS

Reactive oxygen species

RFU

Relative fluorescent units

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