Alveolar Macrophage Expression and Downstream Signaling in The

The Journal of Immunology

Chronic Ethanol Ingestion in Rats Decreases Granulocyte-Macrophage Colony-Stimulating Factor Receptor Expression and Downstream Signaling in the Alveolar Macrophage1

Pratibha C. Joshi,*† Lisa Applewhite,*† Jeffrey D. Ritzenthaler,† Jesse Roman,*† Alberto L. Fernandez,*† Douglas C. Eaton,‡ Lou Ann S. Brown,§ and David M. Guidot2*†

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-␣ 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. The Journal of Immunology, 2005, 175: 6837–6845.

or over a century, alcohol abuse has been well recognized tion, or poor oral hygiene. However, the precise mechanisms by as a significant risk factor for serious pulmonary infec- which chronic ethanol ingestion impairs alveolar macrophage F tions. For example, alcoholic patients are at increased risk function are poorly understood. for infection with necrotizing Gram-negative pathogens such as Within the alveolar space, relatively undifferentiated circulating Klebsiella pneumoniae (1) or to develop bacteremia and shock monocytes are recruited and undergo terminal maturation and dif- from typical pathogens, most notably Streptococcus pneumoniae ferentiation into alveolar macrophages in response to stimulation (2). The mechanisms by which alcohol abuse increases the risk of by GM-CSF. GM-CSF is a 23-kDa protein that was originally pneumonia are likely multiple and include increased risk of aspi- isolated from mouse lung extracts but was named because of its ration of oropharyngeal flora, decreased mucociliary clearance of potent effects on bone marrow development (fully reviewed in Ref. bacterial pathogens from the upper airway, and impaired pulmo- 11). However, when a GM-CSF knockout mouse was constructed nary host defenses. Perhaps the most prominent effects on host a little more than a decade ago, the phenotype was unexpected defense involve the alveolar macrophage, the first cellular line of (12). Specifically, the absence of GM-CSF expression had no dis- defense against pathogens within the lower airways. In experimen- cernible effect on hematopoiesis. However, the mice developed a tal models, chronic ethanol ingestion suppresses chemokine re- severe pulmonary phenotype that closely resembled pulmonary al- sponses and pathogen clearance from the airways (3–9) and im- veolar proteinosis (PAP)3 in humans. Insights from the mouse pairs alveolar macrophage innate immunity, including phagocytic studies ultimately led to the recognition that most patients with function and IL-12 secretion in response to endotoxin (10). Such PAP have acquired Abs to GM-CSF that neutralize the protein studies support the evolving recognition that alcohol abuse has within the alveolar space and prevent binding to its receptor on the specific effects on innate immune function within the lower air- alveolar macrophage membrane (13). Although PAP was first de- ways and that the increased risk of pneumonia in these patients scribed based on the accumulation of surfactant proteins and phos- cannot be ascribed solely to factors such as malnutrition, aspira- pholipids within the alveolar space, we now recognize that it is due to global defects in GM-CSF-dependent alveolar macrophage function that include impaired surfactant recycling, as well as de- *Atlanta Veterans Affairs Medical Center, †Department of Medicine, ‡Department of Physiology, and §Department of Pediatrics, Emory University School of Medicine, pressed innate immune functions (13). Therefore, patients with Atlanta, GA 30322 PAP have an acquired, functional deficiency in GM-CSF (as op- Received for publication May 11, 2005. Accepted for publication September 1, 2005. posed to a genetic mutation) that produces alveolar macrophage The costs of publication of this article were defrayed in part by the payment of page dysfunction. With this background, we hypothesized that alcohol- charges. This article must therefore be hereby marked advertisement in accordance mediated suppression of alveolar macrophage function could in- with 18 U.S.C. Section 1734 solely to indicate this fact. volve a functional defect in GM-CSF expression and/or signaling 1 This work is supported by National Institutes of Health, National Institute on Alcohol within the alveolar space. Abuse and Alcoholism P50 AA013757 and a Veterans Affairs Merit Review (to D.M.G.). 2 Address correspondence and reprint requests to Dr. David M. Guidot, Atlanta Vet- erans Affairs Medical Center (151-P), 1670 Clairmont Road, Decatur, GA 30033. 3 Abbreviations used in this paper: PAP, pulmonary alveolar proteinosis; GM- E-mail address: [email protected] CSFR␣, GM-CSF receptor ␣ subunit; GM-CSFR␤, GM-CSF receptor ␤ subunit.

GM-CSF is produced by the alveolar epithelium and binds to and GM-CSF, (sense) 5Ј-TCTGAGCCTCCTAAATGAC-3Ј, and (anti- specific GM-CSF receptors on the plasma membrane of the alve- sense) 5Ј-CATTTCTGGACCGGCTTC-3Ј. olar macrophage and thereby activates an intracellular signaling Rat GM-CSF primers were designed in our lab and were obtained from Sigma-Genosys. Rat G3PDH primers were purchased from Promega. Mo- pathway that ultimately leads to expression and nuclear binding of lecular mass marker HaeIII digest with fragment sizes 1358–72 bp was the transcription factor PU.1 (13). PU.1 is a member of the ETS purchased from Amersham Biosciences. family of transcription factors previously identified as a master Determination of GM-CSF protein levels in lung lavage fluid transcription factor in the proliferation and differentiation of my- eloid cells (14), and its expression is lost in alveolar macrophages In selected experiments, rat lungs were lavaged via a tracheostomy tube ϫ Ϯ both in patients with PAP and in GM-CSF knockout mice (11, 15). with saline (5 cc 3). The recovered lavage fluid (12 1 cc in all cases) was centrifuged at 1500 ϫ g for 10 min, and GM-CSF levels in the su- Lung-specific transgenic expression of GM-CSF in the type II cells pernatants were determined by a rat-specific ELISA (R&D Systems). The of these mice restores PU.1 expression and normalizes alveolar lower limit of detection was 10 pg/ml. Data are reported as total amount (in macrophage function (16). In fact, constitutive expression of PU.1 nanograms) of GM-CSF present in the lung lavage fluid. in alveolar macrophages of GM-CSF-deficient mice by transfec- Flow cytometric detection of membrane and intracellular tion with a PU.1-containing vector completely normalizes alveolar receptor expression macrophage function (17), confirming the critical role for PU.1 in GM-CSF signal transduction. Thus, GM-CSF-dependent expres- Membrane and intracellular expression of GM-CSF receptors on alveolar macrophages were measured by an established protocol (19). Briefly, cells sion of PU.1 appears to be absolutely required for terminal matu- were incubated for 30 min at room temperature with rabbit polyclonal Abs ration and function of the alveolar macrophage. However, to our (Santa Cruz Biotechnology) to either the rat GM-CSF receptor ␣ or ␤ knowledge, the effects of ethanol ingestion on GM-CSF expression subunit or to an isotype-matched control Ab. Cells were washed to remove and/or signaling to the alveolar macrophage within the alveolar unbound Ab followed by 30 min incubation at room temperature with space have not been examined. Therefore, we examined GM-CSF secondary anti-rabbit Ab conjugated to FITC. For intracellular staining of the receptors, cells were first permeabilized with 0.1% saponin in PBS, expression and key elements of its signaling, namely GM-CSF followed by staining with the Ab. Cells were washed with PBS-saponin receptor expression and PU.1 expression, in our rat model of before adding FITC-conjugated secondary Ab (Santa Cruz Biotechnology). chronic ethanol ingestion. We then determined the effects of rGM- Cells were washed with PBS and were kept in the dark at 4°C until ana- CSF treatment in vivo on restoring GM-CSF signal responsive- lyzed. The labeled cells were analyzed by FACScan flow cytometer (BD Biosciences). Data are expressed both as percentage of cells positive for the ness, as well as innate immune function, in the alveolar macro- ␣ subunit or the ␤ subunit, as well as the mean channel fluorescence for phages of ethanol-fed rats. positive cells in each group. Western blot analyses of PU.1 protein expression Materials and Methods Ethanol feeding Cell lysates were prepared by adding lysing reagent to isolated alveolar macrophages. Fifty micrograms of protein from each sample were loaded Adult Male Sprague-Dawley rats (initial weights, 150–200 g; Charles onto a 12% acrylamide gel and electrophoresed at 150 V for 75 min as River Laboratory) were fed the Lieber-DeCarli liquid diet (Research Diets) described previously (17). The separated proteins were transferred to a 0.45 containing either ethanol (36% of total calories) or an isocaloric substitu- ␮M polyvinylidene difluoride membrane at 15 V for 75 min. Membranes tion with maltin-dextrin ad lib for 6 wk as published previously (18). All were blocked at room temperature for1hinTBSwith 0.2% Tween 20 work was performed with the approval of the Institutional Care and Use of (TBS-T) containing 5% nonfat dry milk in TBS-T. Primary Ab for PU.1 Animals Committee at the Atlanta Veterans Affairs Medical Center. (Santa Cruz Biotechnology) at 1/50 in 5% milk in TBS-T was added to the membranes and kept at 4°C overnight. After several washing steps to re- rGM-CSF treatment via upper airway in vivo move unbound primary Ab, membrane was incubated at room temperature with HRP-labeled anti-rabbit IgG secondary Ab in 5% milk in TBS-T for In some experiments, control-fed and ethanol-fed rats were treated with 2 h. After adding ECL chemiluminescence reagent (Amersham Bio- recombinant rat GM-CSF (PeproTech) or PBS vehicle alone via intranasal sciences) to the membranes, bands were detected using a Bio-Rad Imaging instillation for 3 consecutive days as we published previously (18). Briefly, System. For those experiments involving prior treatment with GM-CSF, rats were anesthetized with 2% isofluorane before gently instilling 500 ng PU.1 expression was normalized to expression of the housekeeping protein of GM-CSF in 100 ␮l of PBS or 100 ␮l of PBS alone into one nostril with G3PDH to control for any potential proliferative effects of GM-CSF. a pipette, which is then delivered into the airway by reflex sniffing by the anesthetized rat. Rats were then sacrificed 24 h after the third treatment PU.1 electromobility shift assay with GM-CSF to obtain alveolar macrophages as described below. Cells were washed with cold PBS, and nuclear binding proteins were ex- Isolation of alveolar macrophages tracted. Protein concentration was determined by the Bradford method using Bio-Rad protein assay reagent. A double-stranded PU.1 consensus oli- Following pentobarbital anesthesia (100 mg/kg i.p.), a tracheostomy tube gonucleotide (5Ј-TGAAAGAGGAACTTGGT-3Ј) was radiolabeled with was placed and rat lungs were lavaged four times with 10 ml of sterile cold [32P]␥-ATP using T4 polynucleotide kinase enzyme. Nuclear protein (10 PBS (pH 7.4). The recovered lavage solution was centrifuged at 1500 rpm ␮g) was incubated with radiolabeled PU.1 for 30 min at room temperature. for 7 min, and the cell pellet resuspended in sterile medium for functional For competition reactions, nonradiolabeled consensus and mutated PU.1 studies. This procedure yielded Ͼ95% alveolar macrophages. double-stranded oligonucleotides (5Ј-TGAAAGAGCTACTTGGT-3Ј) were added to the reaction mixture at 50ϫ molar concentration as a control RNA extractions and RT-PCR for GM-CSF expression to confirm the identity of the PU.1-DNA complexes. DNA-protein com- Total RNA was extracted from lung tissue using Qiagen RNA extraction plexes were separated on 6% native polyacrylamide gel (20:1 acrylamide/ kit. RNA from each sample was reverse transcribed followed by PCR with bis ratio) for 2–3 h. Gels were fixed in a 10% acetic acid/10% methanol gene-specific primers. The number of cycles (35 for G3PDH and 40 for solution for 10 min, dried under vacuum, and exposed to phosphoscreen. GM-CSF) was chosen from our preliminary optimization experiments for Alveolar macrophage bacterial phagocytosis each gene product. PCR conditions were as follows: 5 min of denaturation at 94°C followed by 35–40 cycles of 45 s of denaturation at 94°C, 45 s In some experiments, alveolar macrophages were isolated from control-fed annealing at 60°C or 53°C, and 90-s extension at 72°C, followed by a final and ethanol-fed rats that had been treated with either GM-CSF or vehicle extension at 72°C for 7 min. PCR products were separated on a 2% agarose via the upper airway as described above. In those experiments, the mac- gel containing ethidium bromide. For quantitation, PCR bands were rophages were incubated for 4 h with FITC-labeled Staphylococcus aureus scanned using an imaging system linked to a computer with analysis soft- (Wood strain without protein A; Molecular Probes) in a 1:1 ratio; after ware. Relative amounts of G3PDH (983 bp) and GM-CSF (300 bp) were incubation, cells were washed several times with PBS and examined by quantitated and expressed as GM-CSF:G3PDH ratios. Specific primers confocal microscopy. Phagocytosis images were obtained by laser confocal were as follows: G3PDH, (sense) 5Ј-GAAGGTCGGTGTCAACGGATT microscopy with Fluoview analysis (Olympus). Representative photomi- GGC-3Ј, and (antisense) 5Ј-CATGTAGGCCATGAGGTCCACCAC-3Ј; crographs at ϫ60 magnification were obtained at a depth of 3–5 m in the The Journal of Immunology 6839 z-plane of the macrophage, and both fluorescent and Nomarski differential contrast images were obtained. The cell membranes in the differential contrast images were digitally outlined and then these digital outlines were superimposed on the corresponding fluorescent images. In other experiments, alveolar macrophages from control-fed and ethanol-fed rats were isolated and then incubated with the FITC-labeled S. aureus in a 1:1 ratio Ϯ rGM-CSF (10 ng/ml) in vitro for 4 h. TNF-␣ release from rat alveolar macrophages Freshly isolated alveolar macrophages (106 cells/ml) were incubated overnight Ϯ 100 ng/ml LPS (Escherichia coli 0111:B4). Supernatants were collected and frozen at Ϫ70°C. TNF-␣ in these supernatants was measured using a rat TNF-␣ ELISA kit from BioSource International. Statistics Data are presented as mean Ϯ SEM. Data analysis was done by ANOVA with Student-Newman-Keuls test for group comparison and were consid- ered statistically significant at a value of p Ͻ 0.05. Results Chronic ethanol ingestion had no apparent effect on pulmonary GM-CSF expression The first potential mechanism we examined was whether chronic ethanol ingestion dampened GM-CSF-dependent macrophage function by inhibiting expression of GM-CSF within the lung. We determined that ethanol ingestion in fact had no apparent effect on GM-CSF expression. As shown in Fig. 1, GM-CSF gene expression, as determined by RT-PCR (Fig. 1A shows a representative PCR gel and Fig. 1B shows the summary data from all experiments), was the same ( p Ͼ 0.05) in the lungs of control-fed and ethanol-fed rats. We next examined GM-CSF protein levels in the alveolar space where GM-CSF priming of alveolar macrophages occurs. As shown in Fig. 1C, chronic ethanol ingestion had no effect ( p Ͼ 0.05) on the levels of GM-CSF protein in the lung lavage fluid when compared with control-fed rats. Taken together, these initial studies indicate that chronic ethanol ingestion had no significant effect on GM-CSF expression within the lungs of ethanol-fed rats.

Chronic ethanol ingestion decreased membrane expression of the GM-CSF receptor in the alveolar macrophage As ethanol ingestion did not appear to affect GM-CSF protein availability within the alveolar space, we next examined whether ethanol ingestion could be interfering with GM-CSF signaling to the alveolar macrophage. As a first step in these experiments, we FIGURE 1. The effects of chronic ethanol ingestion on lung GM-CSF expression in rats. Shown in A is a representative RT-PCR gel showing examined membrane expression of the GM-CSF receptor in alve- mRNA levels for GM-CSF as well as the housekeeping gene G3PDH in the olar macrophages freshly isolated from control-fed and ethanol-fed lungs of a control-fed rat and an ethanol-fed rat. B, The summary data for rats. As shown in Fig. 2, chronic ethanol ingestion significantly the ratio of GM-CSF/G3PDH gene expression in control-fed and ethanol- ( p Ͻ 0.05) decreased membrane expression of both the GM-CSF fed rats. C, The levels of GM-CSF protein, as determined by a rat-specific receptor ␣ subunit (GM-CSFR␣) and the GM-CSF receptor ␤ sub- ELISA, in the lung lavage fluids of control-fed and ethanol-fed rats. B and unit (GM-CSFR␤). Fig. 2A shows the relative number of cells that C, Each value represents the mean Ϯ SEM of four rats in each group. were positive for the GM-CSFR ␣ and ␤ subunit, with cells from ethanol-fed rats expressed relative to cells from control-fed rats. atively specific, at least as reflected by our determination that Fig. 2B shows the relative mean channel fluorescence per cell for membrane expression for the IL-6R was the same ( p Ͼ 0.05) in those cells that were positive for the ␣ and ␤ subunit and again alveolar macrophages from ethanol-fed and control-fed rats (Fig. with the cells from ethanol-fed rats expressed relative to cells from 3). Therefore, chronic ethanol ingestion significantly decreased control-fed rats. Although ethanol ingestion did not significantly membrane expression for both subunits of the GM-CSF receptor in decrease the percentage of alveolar macrophages that were posi- alveolar macrophages, and this effect was more pronounced for the tive for GM-CSFR␣ membrane expression, the relative expression ␤ subunit, which is responsible for initiating intracellular signaling (mean channel fluorescence) for GM-CSFR␣ per positive cell was following GM-CSF binding. decreased by ϳ50% ( p Ͻ 0.05). By comparison, ethanol ingestion not only decreased the percentage of alveolar macrophages that In parallel, chronic ethanol ingestion decreased expression of were positive for GM-CSFR␤ membrane expression by ϳ50% the transcription factor PU.1 that is required for GM-CSF- ( p Ͻ 0.05), the relative expression for GM-CSFR␤ per positive dependent functions in alveolar macrophages cell was likewise decreased by ϳ50% ( p Ͻ 0.05). Importantly, We next compared the expression of PU.1, the master transcription decreased membrane expression of the GM-CSF receptor was rel- factor for GM-CSF-dependent functions, in alveolar macrophages 6840 ETHANOL AND GM-CSF SIGNALING

A t t n ns u t t u o co s u u c o o A cns cns

FL1-Height FL1-Height 40 FL1-Height FL1-Height 35 50 Membrane 30 Membrane GM-CSFR 25 IL-6R (% cells positive) (% cells positive) 20 25 15 * 10 5 0 0

B B 80 70 60 50 Membrane 50 * Membrane GM-CSFR * 40 IL-6R (Mean Channel (Mean Channel Fluorescence) 30 Fluorescence) 20 25 10 0 0 control ethanol control ethanol control ethanol FIGURE 3. Membrane expression of the IL-6R as determined by flow Alpha Beta cytometry in alveolar macrophages from control-fed and ethanol-fed rats. A FIGURE 2. The effects of chronic ethanol ingestion on membrane ex- , The relative number of cells that were positive for the membrane IL-6R, pression of the GM-CSF receptor in alveolar macrophages, as determined with cells from ethanol-fed rats expressed relative to cells from control-fed Insets by flow cytometry. A, The relative number of cells that were positive for rats. , Representative histograms of cell counts vs fluorescent inten- the GM-CSFR ␣ and ␤ subunit, with cells from ethanol-fed rats expressed sity for the expression of IL-6R (gray) in alveolar macrophages from con- left inset relative to cells from control-fed rats. Insets, Representative histograms of trol- and ethanol-fed animals. The histograms on the in each represent cells stained with an appropriate isotype-matched control Ab. B, cell counts vs fluorescent intensity for the expression of GM-CSFR ␣ and ␤ (gray line) in alveolar macrophages from control-fed animals. The his- The relative mean channel fluorescence per cell for those cells that were tograms on the left in each inset represent cells stained with an appropriate positive for IL-6R and again with the cells from ethanol-fed rats expressed Ϯ isotype-matched control Ab. B, The relative mean channel fluorescence per relative to cells from control-fed rats. Each value represents the mean p Ͻ ء cell for those cells that were positive for the ␣ and ␤ subunit and again with SEM of six determinations. , 0.05 compared with control. the cells from ethanol-fed rats expressed relative to cells from control-fed p Ͻ ethanol-fed rats. Therefore, we reasoned that similar treatment ,ء .rats. Each value represents the mean Ϯ SEM of six determinations 0.05 compared with control. could mitigate the dampening effects of chronic ethanol ingestion on GM-CSF-dependent functions of the alveolar macrophage. As from ethanol-fed and control-fed rats. Although ethanol ingestion a first step in these experiments, we examined the effects of rGM- significantly decreased GM-CSF receptor expression, these find- CSF treatment in vivo on membrane expression of the GM-CSF ings did not necessarily mean that GM-CSG signaling was suffi- receptor. As shown in Fig. 5, in these experiments ethanol inges- ciently impaired to explain the dampened macrophage function. tion again decreased membrane expression (as reflected by mean Therefore, we reasoned that the next target to examine was PU.1 channel fluorescence) by ϳ50% for both the ␣ and the ␤ subunits. expression because the loss of PU.1 expression in the alveolar However, rGM-CSF treatment significantly ( p Ͻ 0.05) increased macrophages of patients with alveolar proteinosis and in GM-CSF membrane expression for both the ␣ subunit (GM-CSFR␣; Fig. knockout mice is causally related to alveolar macrophage dysfunc- 5A) and the ␤ subunit (GM-CSFR␤; Fig. 5B). In fact, alveolar tion. As shown in Fig. 4, chronic ethanol ingestion significantly macrophage membrane expression for each subunit was increased ( p Ͻ 0.05) decreased PU.1 protein expression in alveolar macro- ϳ3-fold by GM-CSF treatment in ethanol-fed rats. In contrast, phages from ethanol-fed rats compared with control-fed rats. Shown GM-CSF treatment had no significant effect ( p Ͼ 0.05) on mem- in Fig. 4A are representative Western blots for PU.1 in macrophages brane expression of either the ␣ or the ␤ subunit in control-fed rats. from two control-fed and two ethanol-fed rats, while shown in Fig. 4B This GM-CSF-induced increase in membrane expression of the are the summary data for all of the experimental determinations. Im- GM-CSF receptor in alveolar macrophages from ethanol-fed rats portantly, decreased PU.1 expression by ethanol was associated with appeared to be mediated in significant part by increased translo- decreased nuclear binding of PU.1 as discussed later. cation of the receptor subunits from intracellular pools to the membrane. Specifically, rGM-CSF treatment significantly ( p Ͻ 0.05) Treatment with rGM-CSF in vivo restores alveolar macrophage increased the membrane to intracellular ratio by ϳ2-fold for both membrane expression of the GM-CSF receptor in ethanol-fed rats the ␣ subunit (Fig. 6A) and the ␤ subunit (Fig. 6B) in alveolar We had shown previously that treatment with rGM-CSF via the macrophages from ethanol-fed rats. In contrast, rGM-CSF treat- upper airway restores alveolar epithelial barrier function in chronic ment had no effect ( p Ͼ 0.05) on the relative cellular distribution The Journal of Immunology 6841

A -control- -ethanol- A t n u co s 4040kDa kDa → 160 140 FL1-Height 120 3 B Membrane 100 ** 2.5 GM-CSFRα (Mean Channel 80 Cellular 2 Fluorescence) 60 PU.1 protein 1.5 (relative 40 * densitometry) 1 * 20 0.5 0 0 control ethanol FIGURE 4. The effects of chronic ethanol ingestion on alveolar mac- B rophage protein expression of the GM-CSF-dependent transcription factor 160 t o PU.1. A, A representative Western blot of total cellular protein from alve- cuns 140 ** olar macrophages from two control-fed and two ethanol-fed rats probed 120 with an Ab against rat PU.1. B, The summary data of the relative densi- FL1-Height Membrane 100 tometry (in arbitrary units) of PU.1 protein in both experimental groups, GM-CSFRβ Mean Channel 80) ء Ϯ with each value representing the mean SEM of six determinations. , Fluorescence) p Ͻ 0.05 compared with control group. 60 * 40 20 of either subunit in alveolar macrophages from control-fed rats. 0 Fig. 7 shows representative fluorescent images for GM-CSFR␣ on control ethanol control ethanol the cell membranes of an alveolar macrophage from an ethanol-fed +GM-CSF rat (Fig. 7, left panel) and an alveolar macrophage from an ethanol- FIGURE 5. The effects of rGM-CSF treatment on GM-CSF receptor fed rat treated with rGM-CSF (Fig. 7, right panel). Consistent with expression in alveolar macrophages. Control-fed and ethanol-fed rats were the flow cytometry data in Fig. 6A, there is visual evidence of given either GM-CSF (500 ng in 100 ␮l of PBS) or PBS alone intranasally increased GM-CSFR␣ expression on the cell membrane following daily for 3 consecutive days. Twenty-four hours after the third treatment, alveolar macrophages were isolated and membrane expression of the GM- GM-CSF treatment. Taken together, the results in Figs. 5–7 sug- CSFR␣ (A) and the GM-CSFR␤ (B) determined by quantitating the mean gest that rGM-CSF restores membrane expression of the GM-CSF channel fluorescence (MCF) by flow cytometry and expressed as a per- receptor in alveolar macrophages from ethanol-fed rats, at least in centage of the MCF in macrophages from control-fed rats. Each value p Ͻ 0.05 compared ,ء .part, by mobilizing receptor subunits from the intracellular pool to represents the mean Ϯ SEM of six determinations ,p Ͻ 0.05 compared with untreated ,ءء .the plasma membrane. with untreated, control-fed group ethanol-fed group. Inset in each panel shows a representative histogram of Treatment with rGM-CSF in vivo restores PU.1 protein cell counts vs fluorescent intensity for membrane GM-CSF receptor ex- expression as well as PU.1 nuclear binding in alveolar pression in alveolar macrophages from ethanol-fed animals after GM-CSF macrophages in ethanol-fed rats treatment (gray peak on the right) as compared with no GM-CSF treatment (peak on the left). To determine whether restoration of GM-CSF receptor membrane expression translated to a restoration of PU.1 expression and therefore signaling capability, we next examined PU.1 expression as well as nuclear binding in alveolar macrophages from control-fed in Fig. 8 suggest that rGM-CSF treatment in vivo restores PU.1 and ethanol-fed rats with or without treatment with rGM-CSF in protein expression and nuclear binding in alveolar macrophages vivo. For these experiments, PU.1 protein expression was quanti- from ethanol-fed rats, and this increased PU.1 expression corre- tated and expressed relative to the housekeeping protein G3PDH. sponds to restoration of GM-CSF receptor expression (as shown in This was done to verify that any GM-CSF-mediated increases in Figs. 5–7). PU.1 protein expression were not due solely to generalized growth factor effects of GM-CSF on the alveolar macrophages. As shown Treatment with rGM-CSF in vivo restores alveolar macrophage in Fig. 8A, rGM-CSF treatment in vivo significantly ( p Ͻ 0.05) innate immune function in ethanol-fed rats increased cellular PU.1 protein expression in alveolar macro- Our final step in this study was to determine whether rGM-CSF phages from ethanol-fed rats by ϳ44%. By comparison, GM-CSF treatment in vivo could actually improve the functional status of treatment induced a much more modest, albeit significant ( p Ͻ the alveolar macrophage in ethanol-fed rats. Clearly, restoration of 0.05), increase in PU.1 protein expression in alveolar macrophages GM-CSF receptor and PU.1 protein expression in the alveolar from control-fed rats. In parallel, rGM-CSF treatment increased macrophage of ethanol-fed rats would be of limited significance if nuclear binding of PU.1 in alveolar macrophages from ethanol-fed this did not translate into improved immune function. We first rats. As shown in Fig. 8B, GM-CSF treatment in vivo increased examined endotoxin-induced secretion of TNF-␣ by freshly iso- PU.1 nuclear binding as determined by electromobility shift assay lated alveolar macrophages in vitro. As shown in Fig. 9, basal on nuclear extracts from freshly isolated alveolar macrophages in TNF-␣ secretion was the same ( p Ͼ 0.05) in alveolar macrophages each experimental group. Also evident in this representative gel is from control-fed and ethanol-fed rats, and prior GM-CSF treat- that chronic ethanol ingestion decreased PU.1 nuclear binding in ment had the same ( p Ͼ 0.05) modest effect on increasing basal parallel to the decrease in cellular PU.1 protein expression shown TNF-␣ secretion in each group. However, endotoxin-stimulated in Fig. 4. In contrast, GM-CSF treatment in vivo increased PU.1 TNF-␣ secretion was significantly decreased ( p Ͻ 0.05) in alve- nuclear binding in alveolar macrophages from both control-fed and olar macrophages from ethanol-fed rats (Fig. 9, third bar in each ethanol-fed rats, although in general, this effect was more dramatic group). However, alveolar macrophages from GM-CSF-treated, in macrophages from ethanol-fed rats. Taken together, the results ethanol-fed rats had the same ( p Ͼ 0.05) endotoxin-stimulated 6842 ETHANOL AND GM-CSF SIGNALING

1 t

A n u co s

0.75 * GM-CSFRα FL1-Height Ratio of Membrane to 0.5 Intracellular Mean Channel Fluorescence 0.25

0 s

1 t n

B u o c

0.75 FL1-Height GM-CSFRβ * Ratio of Membrane to 0.5 Intracellular FIGURE 7. Immunofluorescence labeling of membrane GM-CSFR␣ Mean Channel chain on rat alveolar macrophages. A, Representative images of intracel- Fluorescence 0.25 lular expression of GM-CSFR␣ in alveolar macrophages from control- and ethanol-fed animals. Cells were made permeable with saponin before staining with anti-GM-CSFR␣ Ab, followed by an appropriate FITC-conju- 0 gated secondary Ab. B, Representative images of membrane expression of control ethanol control ethanol GM-CSFR␣ in alveolar macrophages from control- and ethanol-fed rats +GM-CSF that were given either PBS or recombinant rat GM-CSF (500 ng/ml) intranasally for 3 days. Cells were stained with an anti-GM-CSFR␣ Ab, FIGURE 6. The effects of rGM-CSF treatment on the relative distribu- followed by an appropriate FITC-conjugated secondary Ab. Cells were tion of the GM-CSF receptor in the membrane vs the intracellular com- fixed in methanol, mounted on slides, and examined by fluorescent mi- partment of alveolar macrophages. Control-fed and ethanol-fed rats were croscopy. These qualitative images correlate with the quantitative analyses treated with either GM-CSF or PBS alone intranasally as detailed in Ma- of GM-CSFR␣ expression shown in Fig. 5. terials and Methods and in Fig. 5. Twenty-four hours after the third treatment, alveolar macrophages were isolated, and both membrane expression and intracellular expression of the GM-CSFR␣ (A) and the GM-CSFR␤ Taken together, the results shown in Figs. 9 and 10 indicate that (B) were determined by quantitating the mean channel fluorescence (MCF) GM-CSF treatment improved innate immune functions in the al- by flow cytometry. In each experimental determination, the ratio of the veolar macrophages of ethanol-fed rats. membrane MCF and the intracellular MCF was calculated and expressed as ء Ϯ a ratio. Each value represents the mean SEM of six determinations. , Treatment with rGM-CSF in vitro restores bacterial phagocytic p Ͻ 0.05 compared with untreated, ethanol-fed group. Inset in each panel capacity in alveolar macrophages from ethanol-fed rats shows representative histogram of cell counts vs fluorescent intensity for intracellular expression of GM-CSF receptor (gray) in alveolar macro- Although the results shown in Figs. 9 and 10 are striking, it is phages from control-fed animals. The histograms on the left in each inset possible that GM-CSF treatment in vivo recruited and primed a represent cells stained with an appropriate isotype-matched control Ab. new population of alveolar macrophages from peripheral monocyte pools and had no effect on the existing alveolar macrophage pool. To test whether or not GM-CSF treatment could secretion as alveolar macrophages from control-fed rats (hatched directly improve immune function in resident alveolar macro- gray line connects these two groups in Fig. 9). Notably, TNF-␣ phages in the alcoholic lung, we performed additional experi- secretion was greatest ( p Ͻ 0.05) in endotoxin-stimulated macro- ments in which macrophages were isolated and then stimulated phages from control-fed rats, indicating that even under “normal” with GM-CSF in vitro. In these conditions, alveolar macro- conditions, GM-CSF stimulation augments the endotoxin re- phages adhere tightly to plastic, and therefore, we could not sponse. We next examined the effects of GM-CSF treatment on the perform flow cytometry to assess GM-CSF receptor membrane ability of alveolar macrophages isolated from ethanol-fed rats to expression. However, adherent macrophages remain functional, phagocytose bacteria in vitro. We did not include macrophages and therefore, we assessed bacterial phagocytic capacity with from control-fed rats in these studies. The confocal microscopy and without GM-CSF treatment in vitro. We reasoned that if images in Fig. 10 illustrate that GM-CSF treatment augmented the GM-CSF treatment could directly increase a relevant macro- ability of macrophages from ethanol-fed rats to phagocytose the phage function in these conditions, this would provide further fluorescent bacteria. Fig. 10, A and B, shows the corresponding evidence that its effects in vivo could not be solely ascribed to fluorescent and differential contrast images for a macrophage from recruiting an entirely new alveolar macrophage population from an untreated, ethanol-fed rat, in which relatively few bacteria have an extrapulmonary monocyte pool. In these experiments, iso- been phagocytosed, and the majority of these remain in the pe- lated macrophages from control-fed and ethanol-fed rats were riphery of the cell. In contrast, as shown in Fig. 10, C and D, incubated with the fluorescent bacteria for 4 h, Ϯ recombinant alveolar macrophages from ethanol-fed rats treated with GM-CSF rat GM-CSF (10 ng/ml). The cells were examined by fluores- were able to ingest and internalize more of the fluorescent bacteria. cent microscopy and the percentage of macrophages that had The Journal of Immunology 6843

3000 *

A TNFα 2000 secretion (pg/ml) 0.3 ** 1000 ** Cellular * 0 PU.1 0.2 LPS --+ + --+ + protein GM-CSF -+ -+ -+ -+ expression control ethanol (relative to 0.1 FIGURE 9. The effects of rGM-CSF treatment on endotoxin-induced G3PDH) secretion of TNF-␣ by alveolar macrophages in vitro. Control-fed and ethanol-fed rats were treated with either GM-CSF or PBS alone intranasally. Twenty-four hours after the third treatment, alveolar macrophages were 0 6 Ϯ + GM-CSF +GM-CSF isolated, and 10 cells/ml were incubated overnight 100 ng/ml LPS (E. control ethanol coli 0111:B4). TNF-␣ concentrations in these supernatants were measured by ELISA as detailed in Materials and Methods. Each value represents the p Ͻ 0.05 increased compared ,ء .mean Ϯ SEM of three determinations -p Ͻ 0.05 de ,ءء ;with LPS-stimulated, untreated (no GM-CSF) control creased compared with LPS-stimulated, untreated (no GM-CSF) control. B GM-CSF PU.1 mPU.1 Gray hatched line, p Ͼ 0.05 same compared with LPS-stimulated, un- (10 ng/ml) 50x 50x treated (no GM-CSF) control. CE C E C E C E 01 2 34 56 7 8 bound vs 92 Ϯ 5%; p Ͻ 0.05) compared with macrophages from control-fed rats, and this was almost completely reversed with exogenous GM-CSF treatment (83 Ϯ 6% vs 56 Ϯ 8%; p Ͻ 0.05).

Discussion In this study, we determined that although chronic ethanol ingestion in rats did not affect GM-CSF expression within the alveolar space, it nevertheless dampened GM-CSF signaling capacity by decreasing expression of GM-CSF receptors on the surface of the free alveolar macrophage. This appeared to be relatively specific, as membrane expression of the IL-6R was not affected by chronic FIGURE 8. The effects of rGM-CSF treatment on PU.1 protein expres- ethanol ingestion. In parallel and likely as a consequence of de- sion (A) and nuclear binding (B) in alveolar macrophages. Control-fed and creased membrane receptors, the expression and nuclear binding of ethanol-fed rats were treated with either GM-CSF or PBS alone intrana- the GM-CSF-dependent transcription factor PU.1 was also de- sally as detailed in Materials and Methods and in Fig. 5. Twenty-four hours creased. Remarkably, treatment with rGM-CSF via the upper air- after the third treatment, alveolar macrophages were isolated. A, The cel- way restored membrane expression of the GM-CSF receptor as lular PU.1 protein expression relative to the housekeeping protein G3PDH well as the downstream expression and nuclear binding of PU.1 in in each experimental group, with each value representing the mean Ϯ SEM the alveolar macrophages of ethanol-fed rats. Even more impor- of six determinations. The inset shows a representative Western blot of cellular protein from two ethanol-fed animals treated with vehicle alone tantly in terms of potential clinical relevance, GM-CSF treatment and two ethanol-fed animals treated with GM-CSF that were probed with restored innate immune functions in alveolar macrophages of eth- a polyclonal Ab for PU.1. The band at 40 kDa is consistent with the known anol-fed rats, as reflected by endotoxin-induced secretion of size of PU.1, and as shown in the right side of the gel, this band is elim- TNF-␣ and bacterial phagocytosis. Taken together, these results -p Ͻ suggest that chronic ethanol ingestion inhibits alveolar macro ,ء .inated in the presence of a 20ϫ concentration of the control peptide -p Ͻ 0.05 compared phage immune function by dampening, but not completely block ,ءء .compared with untreated, control-fed group 0.05 with untreated, ethanol-fed group. B, A representative electromobility shift ing, macrophage responsiveness to the stimulatory effects of am- assay in which nuclear extracts from alveolar macrophages in each exper- bient GM-CSF within the alveolar microenvironment. ϭ ϭ imental group (C control diet and E ethanol diet). Lane 0 is free probe Furthermore, this study raises the possibility that in the appropriate without nuclear extract. Lanes 1–4 were probed with a 32P-labeled PU.1 clinical context impaired pulmonary host defenses in alcoholic pa- consensus oligonucleotide. Lanes 3 and 4, Rats were treated with rGM- CSF before macrophage isolation. Lanes 5–8, The results of probing nu- tients could be corrected with rGM-CSF treatment. clear extracts of macrophages from control-fed and ethanol-fed rats with For more than a century, it has been recognized that chronic the 32P-labeled PU.1 consensus oligonucleotide and either a 50ϫ concen- alcohol abuse is a major risk factor for the development of pneu- tration of unlabeled PU.1 consensus nucleotide (lanes 5 and 6)ora50ϫ monia. Although factors associated with alcoholism such as mal- concentration of a 32P-labeled mutated form of the PU.1 consensus nucle- nutrition, poor dentition, aspiration, smoking, and other drug likely otide (lanes 7 and 8). exacerbate the risk, experimental animal models have demon- strated that ethanol ingestion alone (in the absence of these other factors associated with chronic alcohol abuse in humans) impairs ingested one or more bacteria determined. As shown in Fig. 11 alveolar macrophage innate immune function (5, 7, 8, 10, 20). (and consistent with the results shown in Fig. 4 above), the However, the specific mechanisms by which ethanol ingestion percentage of macrophages from ethanol-fed rats that had any down-regulates macrophage function have not been identified. detectable phagocytic function in vitro was decreased (56 Ϯ 8% This study provides a plausible and specific mechanism by which 6844 ETHANOL AND GM-CSF SIGNALING

FIGURE 11. Bacterial phagocytosis in alveolar macrophages from control-fed and ethanol-fed rats Ϯ treatment with rGM-CSF in vitro. Macro- phages were incubated in a 1:1 ratio with FITC-labeled, inactivated S. aureus with or without recombinant rat GM-CSF (10 ng/ml) for 4 h. The percentage of macrophages that ingested ﬂuorescent bacteria was determined by direct observation under ﬂuorescent microscopy for each exper- iment. Shown are the means Ϯ SEM for four experiments in each condi- p Ͻ ,ءء ;p Ͻ 0.05 decreased compared with untreated control cells ,ء .tion 0.05 increased compared with untreated ethanol cells.

FIGURE 10. Confocal images of bacterial phagocytosis by alveolar macrophages from ethanol-fed rats Ϯ GM-CSF treatment in vivo. Shown are representative ϫ60 confocal images of bacterial phagocytosis in vitro activated initiates the intracellular signaling cascade (22). We did by alveolar macrophages isolated from ethanol-fed rats Ϯ prior treatment not examine this kinase or any of the other intracellular compo- with rGM-CSF in vivo. A and B, The corresponding fluorescent and dif- nents that transduce the GM-CSF signal from receptor binding to ferential contrast images for a macrophage from an untreated, ethanol-fed PU.1 expression and nuclear transcription activation, and one rat, in which relatively few bacteria have been phagocytosed, and the ma- would expect that these components would likewise be dampened. jority of these remains in the periphery of the cell. In contrast, as shown in Another complexity is that the GM-CSFR␤ is actually common to C and D, alveolar macrophages from ethanol-fed rats treated with GM-CSF the IL-3R and IL-5R (22, 23). However, IL-5R expression appears in vivo before macrophage isolation were able to phagocytose more bac- to be limited to eosinophils (23), whereas IL-3 expression is lim- teria, and most of the bacteria have been internalized. The cell membranes in the differential contrast images in B and C, respectively, were digitally ited to eosinophils, basophils, and mast cells (23). Therefore, eth- ␤ outlined, and these outlines were superimposed on the corresponding flu- anol-mediated changes in subunit expression in alveolar macro- orescent images in A and C, respectively, to better illustrate the cellular phages, as we observed, are likely specific to GM-CSF receptor localization of the bacteria relative to the plasma membranes. expression in this cell type, particularly as they parallel changes in the unique ␣ subunit. Whether alcohol abuse has any significant effects on IL-3 and/or IL-5 function in eosinophils and/or basophils alveolar macrophage maturation and function is dampened during is an open question. chronic ethanol ingestion. Specifically, ethanol ingestion interferes Our findings provide important new insights but also raise new with GM-CSF priming within the alveolar space that is absolutely questions. In particular, how does ethanol ingestion inhibit mobi- essential for the alveolar macrophage to acquire its full comple- lization and/or insertion of the GM-CSF receptor into the plasma ment of immune functions. membrane? In parallel, how does treatment with supraphysiologi- These findings are important because they provide novel in- cal levels of GM-CSF correct this defect when physiologic levels, sights into the fundamental mechanisms by which chronic alcohol which are not inhibited by ethanol ingestion, do not? At present we abuse impairs host defenses and renders patients susceptible to can only speculate. We know from previous studies that chronic pulmonary infections. They are also important because they raise ethanol ingestion causes oxidative stress and severe glutathione the provocative possibility that the alcoholic macrophage could be depletion within the alveolar space in experimental animals (24) as stimulated in vivo by exogenous GM-CSF treatment and thereby well as in humans (25), leading to diverse abnormalities in alveolar rapidly reacquire the innate immune function that protects the epithelial function that are prevented by supplementing the etha- lower airways from microbial invasion. As GM-CSF has already nol-containing diet with glutathione precursors (26–28). It is cer- been tested in a phase II clinical trial of sepsis and lung injury and tainly conceivable that ethanol-induced oxidative stress interferes was found to increase alveolar macrophage function (21), it is with GM-CSF trafficking to the plasma membrane. Acetaldehyde, reasonable to speculate that treating alcoholic patients with rGM- the first product of ethanol metabolism, produces endoplasmic re- CSF as adjunctive therapy for serious lung infections could aug- ticulum stress in hepatocytes, thereby inhibiting mitochondrial glu- ment their pulmonary host defense and improve outcome. tathione transport (29). If similar endoplasmic reticulum stresses Although we have identified significant defects in GM-CSF re- occurred within the alveolar macrophage, this could lead to mis- ceptor expression and parallel decreases in PU.1 expression in the folding of the GM-CSF receptor. However, this would have to be alcoholic macrophage, we recognize that GM-CSF signaling is a relatively specific inhibition as we determined that membrane complex and that alcohol abuse likely perturbs other components expression of the IL-6R was not affected. Although we did not find of the GM-CSF signal transduction pathway. For example, while evidence that ethanol ingestion decreased gene expression of GM- the GM-CSFR␤ initiates intracellular signaling following GM- CSF and/or its receptors, it is possible that supraphysiological lev- CSF binding, it contains no intrinsic catalytic activity. Rather, it is els of GM-CSF increased expression of one or more components constitutively associated with a tyrosine kinase, JAK2, that when of the pathway in addition to augmenting membrane expression of The Journal of Immunology 6845 the receptors. Regardless of the mechanism, it is intriguing that 2. Perlino, C. A., and D. Rimland. 1985. Alcoholism, leukopenia, and pneumococ- rGM-CSF treatment restored GM-CSF receptor membrane expres- cal sepsis. Am. Rev. Respir. Dis. 132: 757–760. 3. Arbabi, S., I. Garcia, G. J. Bauer, and R. V. Maier. 1999. Alcohol (ethanol) sion, PU.1 expression, and immune function in the alcoholic mac- inhibits IL-8 and TNF: role of the p38 pathway. J. Immunol. 162: 7441–7445. rophage. 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