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(Journal of Leukocyte Biology. 2000;68:614-620.)
© 2000 by Society for Leukocyte Biology

Functional heterogeneity of rat hepatic and alveolar macrophages: effects of chronic ethanol administration

Lisa A. Morio, Hawjyh Chiu, Karen A. Sprowles and Debra L. Laskin

Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey

Correspondence: Dr. Debra Laskin, Rutgers University, Dept. of Pharmacology and Toxicology, 170 Frelinghuysen Road, Piscataway, NJ 08854-8020. E-mail: laskin{at}eohsi.rutgers.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic ethanol consumption is associated with increased incidence of hepatic and pulmonary infections. To determine if this is correlated with altered macrophage activity, we analyzed the functional properties of cells isolated sequentially from the liver and lung of rats fed a liquid diet containing ethanol (35% of calories) or malto-dextrin control for 9–12 weeks. Hepatic and alveolar macrophages from control animals were found to exhibit distinct morphologic and functional properties. Thus, hepatic macrophages were highly vacuolated and appeared larger and more irregular in shape than alveolar macrophages. These cells also displayed greater phagocytic activity and random migration. In contrast, lung macrophages produced more superoxide anion and nitric oxide, and exhibited enhanced chemotactic activity toward the complement fragment C5a. Whereas administration of ethanol to rats for 9–12 weeks resulted in decreased chemotaxis and superoxide anion production by alveolar macrophages, cell adhesion molecule expression was reduced in hepatic macrophages. Nitric oxide production and inducible nitric oxide synthase protein expression were decreased in both macrophage populations. These effects were not observed after 3–6 weeks of ethanol administration to rats. Our results suggest that changes in macrophage functioning may play a role in decreased host defense following chronic ethanol exposure.

Key Words: Kupffer cells • nitric oxide • lung • liver • ethyl alcohol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are relatively long-lived cells that play a central role in a variety of immunologic and inflammatory processes [1 ]. They are distributed widely in tissues where they acquire unique functional activities in response to their microenvironment. Alveolar macrophages are found primarily in the lower respiratory tract where they effectively remove inhaled contaminants such as microbial organisms and particulate matter from the lung [2 ]. These cells have been shown to exhibit increased oxidative metabolism when compared with other tissue macrophage subpopulations [3 ]. Hepatic macrophages (Kupffer cells) are localized in the liver sinusoids and constitute 80–90% of all tissue macrophages in the body [4 ]. They are highly phagocytic cells, primarily responsible for the clearance of endotoxin and other pathogens from the portal circulation [5 ]. Both alveolar and hepatic macrophages are responsive to xenobiotics, exhibiting altered biochemical and functional properties [6 , 7 ]. Changes in the activity of these cells may contribute to pathophysiologic responses of the lung and the liver to toxicants.

Repeated and excessive use of ethanol is known to increase opportunistic pulmonary and hepatic infections in humans [8 ]. It has been suggested that this is because of reduced macrophage functioning in these tissues [8 9 10 11 ]. In this regard, it has been shown that administration of ethanol to rats acutely or continuously for 2–3 weeks is associated with decreased production of nitric oxide (NO), superoxide anion, and tumor necrosis factor {alpha} by alveolar macrophages [12 13 14 15 16 ]. Furthermore, treatment of animals for 6 weeks resulted in impaired phagocytosis [17 18 19 ] and altered expression of Fc receptors by hepatic macrophages [19 ]. The comparative effects of ethanol administration to rats for more prolonged periods on lung and liver macrophages isolated sequentially from the same animal are unknown and represent the focus of the present studies. We found that treatment of rats with ethanol for 9–12 weeks affected hepatic and alveolar macrophages, however their responses were distinct. Thus, whereas decreases in cell adhesion molecule expression were observed in hepatic macrophages, reduced chemotactic responsiveness and oxidative metabolism were noted in alveolar macrophages. These changes may contribute to ethanol-induced susceptibility of the liver and lung to injury and/or infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Lipopolysaccharide (LPS) from Escherichia coli (serotype 0128:B12), ferricytochrome C (type iii), and superoxide dismutase (SOD; EC 1.15.1.1) were purchased from Sigma Chemical Co. (St. Louis, MO). Rat interferon-{gamma} (IFN-{gamma}) was from Gibco BRL (Gaithersburg, MD), and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was from LC Services (Woburn, MA). Anti-rat intercellular adhesion molecule-1 (ICAM-1, clone 1A29) and anti-rat-ß2-integrin (clone WT-3) antibodies were obtained from Seikagaku America (Rockville, MD). Anti-immunoglobulin G (IgG)1a antibody was from Coulter Immunotech (Miami, FL), and fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse IgG was from Jackson Immuno Research (West Grove, PA). Anti-mouse-inducible NO synthase (NOS II) antibody (clone N-39120) was purchased from Transduction Labs (Lexington, KY), and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was from Amersham Life Science (Arlington Heights, IL).

Animals
Male-specific pathogen-free Wistar rats (250–275 g) were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Animals were maintained on food and water ad libitum and housed individually in microisolator cages. Rats were fed a liquid diet composed of casein (20% of total calories), corn oil (35% of total calories), malto-dextrin (10% of total calories), ethanol (35% of total calories), and AIN-93 mineral salt and vitamin mixes. Pair-fed control animals were fed isocaloric malto-dextrin in place of ethanol. All diet components were purchased from Bioserve (Frenchtown, NJ). Blood-alcohol levels were determined using a diagnostic alcohol dehydrogenase kit (Sigma). Throughout the course of the experiments, alcohol levels were found to cycle between 20 mg/dl and 376 mg/dl, consistent with previous publications [20 21 22 ].

Macrophage isolation
Hepatic and alveolar macrophages were isolated sequentially from the liver and lung, as described previously, with some modifications [23 , 24 ]. Briefly, rats were anesthetized with 100 mg/kg ketamine HCl (Ketaset®; Fort Dodge Laboratories, Fort Dodge, IA) and 12 mg/kg xylazine HCl (Fermenta Animal Health Products, Kansas City, MO), and the livers and lungs perfused in situ via the portal vein with Leibowitz L-15 medium containing 100 U/ml collagenase Type IV. Livers were then excised, weighed, and combed, and the resulting cell suspension filtered through 60 µm mesh. Contaminating hepatocytes were removed by three successive centrifugations (50 g, 1 min). Nonparenchymal cells were recovered by centrifugation of the supernatants at 300 g for 5 min. Hepatic macrophages were purified from the nonparenchymal cell pellet on a Beckman JE-6 elutriator (Beckman Instruments Inc., Fullerton, CA), equipped with a centrifugal elutriator rotor set to a pump speed of 12 ml/min and a rotor speed of 2500 rpm. Macrophages were collected at a flow rate of 44 ml/min, enriched by differential centrifugation on a 18% metrizamide gradient, and then washed three times in L-15 medium. For isolation of alveolar macrophages, the lungs were exised en bloc and lavaged with 50 ml (5x10 ml) of Ca++/Mg++-free Hanks’ balanced saline solution (HBSS). The lavage fluid was centrifuged, and the pelleted cells were washed three times in HBSS containing 2% heat-inactivated fetal bovine serum (FBS). Cell viability was determined by trypan blue exclusion and for both macrophage types, was >95%. Alveolar macrophages were 95–99% pure, and hepatic macrophages, 80–85% pure, as determined morphologically and by peroxidase staining.

Analysis of cell adhesion molecule expression
Macrophages (1x106/ml) were incubated for 60 min in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.05% sodium azide, followed by incubation for 60 min with a 1:500 dilution of anti-rat-ICAM-1, anti-rat-ß2-integrin (CD18), or anti-IgG1a control antibody. Cells were then washed (300 g, 5 min) with PBS, incubated with FITC-conjugated goat anti-mouse IgG1 (1:500) for 30 min, washed, and then fixed in 4% buffered formalin. All incubations were carried out at 4°C. Cells were analyzed on a Coulter Epics Profile II flow cytometer (Coulter Instruments, Hialeah, FL). For each analysis, a minimum of 5000 events was collected in list-mode format. The instrument was standardized for fluorescence measurements using Immunobrite fluorospheres (Coulter Electronics, Hialeah, FL). The percentage binding was determined by subtracting the fluorescence distribution of cells stained with isotypic control antibody from the fluorescence distribution of cells stained with cell adhesion molecule-specific antibodies using Overton’s cumulative subtraction routine of the Coulter Cytologic software program.

Measurement of chemotaxis
Chemotactic responsiveness of macrophages was quantified using a Neuroprobe 48 microwell chemotaxis chamber [25 , 26 ]. Cells, suspended (2.5x106/ml) in serum-free RPMI medium 1640 containing 0.5% BSA, were incubated in the upper wells of the chamber at 37°C with TPA (50 nM), C5a (10%), or medium in the lower wells. Chemotaxis of the cells through a 5 µm pore polycarbonate filter was quantified 4 h later. Data are presented as the average number of cells migrating through 10 oil immersion fields.

Measurement of phagocytosis
Macrophage phagocytosis was quantified by uptake of propidium iodide (PI)-labeled Staphylococcus aureus (S. aureus) [27 ]. To prepare labeled bacteria, 1 ml of a 10% suspension of S. aureus (IgGsorb; The Enzyme Center, Malden, MA) was pelleted (13,000 g, 20 sec) and resuspended in 1 ml serum. After 30 min incubation at 37°C, the bacteria were washed twice (13,000 g, 20 sec) and incubated for 30 min at 25°C with 10 mM dimethyl pimelimidate (Pierce, Rockford, IL)/50 mM sodium tetraborate (pH 7.8). The bacteria were then washed with 10 mM glycine (pH 7.8) and incubated for 30 min at 25°C with papain (20 U; Sigma). This was followed by incubation for 30 min with 300 µg PI. To assess phagocytosis, macrophages (1x106/ml) were incubated with or without PI-labeled S. aureus (2x108 bacteria/ml) in a 37°C shaking water bath for 20 min. The cells were then fixed in 4% buffered formalin and red fluorescence associated with phagocytized S. aureus quantified by flow cytometry. For each analysis, a minimum of 5000 events was collected in list mode format. Phagocytosis is presented as the percentage increase in fluorescence, which was calculated by subtracting the background fluorescence distribution of cells from the fluorescence distribution of cells incubated with PI-labeled S. aureus using Overton’s cumulative subtraction routine as indicated above.

Measurement of superoxide anion and NO production
For superoxide anion determinations, macrophages (1.5x105 cells) were incubated in balanced salt solution (128 mM NaCl, 12 mM KCl, 1 mM CaCl2•2H2O, 2 mM MgCl2, 2 mM glucose, 3.43 mM anhydrous Na2HPO4, 0.57 mM anhydrous NaH2PO4) containing 44 µM ferricytochrome C, with or without 1 mM SOD and/or 170 nM TPA. Absorbance was determined spectrophotometrically 45 min later at 550 nm. The amount of superoxide anion released was calculated using a baseline value (E=21.1 nM-1cm-1 at 550 nm) obtained from samples containing SOD (26). For measurement of NO production, cells were inoculated into 24-well dishes (2.5x105 hepatic macrophages/well) or 96-well dishes (2x105 alveolar macrophages/well) in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 2 mM glutamine, penicillin (100 IU)-streptomycin (100 µg), and 0.05 IU/ml porcine pancreas insulin (complete DMEM). After 18 h in culture, the cells were washed twice and refed with DMEM, with and without LPS and/or IFN-{gamma}. NO production by the cells was quantified 48 h later by the accumulation of nitrite in the culture medium using the Griess reaction with sodium nitrite as the standard [28 ]. For nitrate determinations, samples were treated with nitrate reductase and NADPH for 30 min prior to analysis using a nitrate/nitrite assay kit (Boehringer-Mannheim, Indianapolis, IN). We found that in medium from cells culture-treated for 24 h with LPS and IFN-{gamma}, the ratio of nitrate:nitrite was 0.9:1.0 for alveolar macrophages and 1.3:1.0 for hepatic macrophages. These ratios did not change following ethanol administration.

Western blot analysis
Cells were incubated overnight in 24-well dishes (5x105 cells/well) in complete DMEM at 37°C. The cells were then washed twice and refed with DMEM containing LPS and IFN-{gamma} or medium control. After 24 h incubation, the cells were washed with PBS and lysed in buffer containing 10 mM Tris-HCl and 1% sodium dodecyl sulfate (SDS), pH 7.4. Protein concentrations were determined using the DC Protein Assay kit (Biorad, Hercules, CA). Cellular proteins (5 µg) were fractionated on 7.5% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were incubated with anti-NOS II antibody, followed by HRP-labeled goat anti-mouse IgG. Antibody binding was visualized by autoradiography with enhanced chemiluminescence Western blotting reagents (Amersham Life Science).

Statistics
All experiments used three to six animals per treatment group and were repeated at least three times. Data were analyzed using the Student’s t-test and analysis of variance (ANOVA). Results were considered statistically significant at P <= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Initially, we analyzed the effects of ethanol administration to rats on the physical and morphological properties of macrophages obtained from the lungs and livers. Flow cytometric analysis of alveolar macrophages from control animals revealed one large and relatively homogeneous population of cells with respect to size and density (Fig. 1 ). In contrast, three distinct subpopulations of hepatic macrophages were identified. These consisted of a population that was relatively small in size and density (subpopulation 1), a second larger and more dense population (subpopulation 2), and a third population (subpopulation 3) that was similar in size to subpopulation 1 and in density to subpopulation 2. To confirm the identity of the liver cells, the subpopulations were gated and sorted electronically. Each of the subpopulations stained >99% peroxidase-positive, demonstrating that they consisted entirely of macrophages [26 ]. Light microscopic examination of lung and liver macrophages cultured for 18 h showed marked differences in their morphology. Thus, alveolar macrophages appeared smaller, rounder, and more dense when compared with hepatic macrophages (Fig. 2A and C). Alveolar macrophages were also more homogeneous in appearance. These findings are consistent with our flow cytometric data. In contrast to alveolar macrophages, hepatic macrophages were characterized by an enlarged nucleus and highly vacuolated cytoplasm. Following treatment of the cells with the inflammatory mediators LPS and IFN-{gamma}, alveolar macrophages became enlarged, vacuolated and flattened, and spread on the culture dishes (Fig. 2B) . In contrast, no major changes were noted in hepatic macrophages (Fig. 2D) . Whereas ethanol administration to rats had no effect on the physical or morphological properties of the cells or on the number of alveolar macrophages recovered from the animals, hepatic macrophages decreased in number (Table 1 and unpublished results).



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  		Figure 1. Characterization of macrophages by flow cytometry. Alveolar macrophages (AM) and hepatic macrophages (HM) were analyzed by flow cytometry according to their laser light-scattering properties. Representative histograms are shown for each cell type. FALS, forward angle light scatter; SS, side scatter.

 


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  		Figure 2. Morphology of cultured macrophages. Alveolar macrophages (A and B) and hepatic macrophages (C and D) were cultured in medium control (A and C) or medium containing 10 ng/ml LPS and 100 U/ml IFN-{gamma} (B and D). After 18 h, the cells were stained with Giemsa. Original magnification, 400x.

 

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  		Table 1. Effects of Ethanol Administration on the Number of Hepatic and Alveolar Macrophages Recovered from the Rats

 
The effect of ethanol administration on expression of ICAM-1 and ß2-integrins (CD11/CD18) by alveolar and hepatic macrophages was next analyzed. Binding of antibodies against ICAM-1 and ß2-integrins to both macrophage types was found to be homogeneous. Thus, for each cell type, only one population was identified that expressed relatively large amounts of ICAM-1 and ß2-integrins. Although both macrophage populations expressed similar levels of ICAM-1, expression of ß2-integrins was significantly greater on alveolar macrophages when compared with hepatic macrophages (Fig. 3 ). Following chronic ethanol administration, expression of these cell adhesion molecules on hepatic macrophages was decreased with no effect on alveolar macrophages.



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  		Figure 3. Effects of ethanol administration on macrophage expression of ICAM-1 and ß2-integrins. Hepatic macrophages (HM) and alveolar macrophages (AM) from control rats or rats treated with ethanol for 9–12 weeks were stained with anti-ICAM antibody or anti-ß2-integrin antibody and analyzed by flow cytometry. Each bar represents the mean ± SEM of at least four experiments. aSignificantly different (P<=0.05) from macrophages from control animals.

 
In further studies, we analyzed the effects of chronic ethanol administration on chemotaxis in macrophages from the lung and the liver toward the complement cleavage product C5a and the phorbol ester TPA [25 , 26 ]. C5a was found to be a more effective chemoattractant than TPA in alveolar macrophages (Fig. 4 ). In contrast, the response of hepatic macrophages to these stimuli was similar. Hepatic macrophages also exhibited greater random migration in the presence of buffer control when compared with alveolar macrophages. Ethanol administration to rats for 9–12 weeks was found to depress chemotaxis of alveolar macrophages, but not hepatic macrophages, toward TPA and C5a (Fig. 4) . However, random migration in hepatic macrophages was decreased. The effects of ethanol on macrophage phagocytosis were evaluated also. Hepatic macrophages were found to phagocytize two to three times more PI-labeled S. aureus than alveolar macrophages (Fig. 5 , left panel). Treatment of rats with ethanol had no effect on phagocytosis by either macrophage population.



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  		Figure 4. Effects of ethanol administration on chemotaxis. Hepatic macrophages (HM) and alveolar macrophages (AM) were isolated from control rats or rats treated with ethanol for 9–12 weeks. Chemotaxis in response to TPA, C5a, or buffer control was measured using microwell chemotactic chambers. Each bar represents the mean ± SEM of three experiments. aSignificantly different (P<=0.05) from macrophages from control animals.

 


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  		Figure 5. Effects of ethanol administration on phagocytosis and superoxide anion production. Hepatic macrophages (HM) and alveolar macrophages (AM) were isolated from control rats or rats treated with ethanol for 9–12 weeks. Each bar is the mean ± SEM of at least three experiments. aSignificantly different (P<=0.05) from macrophages from control rats. Left panel, phagocytosis. Cells were incubated with PI-labeled S. aureus for 20 min, washed, and analyzed by flow cytometry. The data are presented as percentage increase in cell-associated fluorescence over background, which was calculated by subtracting background fluorescence of the cells from the cell-associated fluorescence following phagocytosis of PI-labeled S. aureus. Right panel, superoxide anion production. Cells were incubated with buffer or TPA for 45 min, supernatants collected, and superoxide anion production analyzed spectrophotometrically.

 
The effects of chronic ethanol administration on the production of reactive oxygen and reactive nitrogen intermediates by alveolar and hepatic macrophages were analyzed also. In the absence of stimulation, both macrophage populations produced relatively low levels of superoxide anion (Fig. 5 , right panel). Basal oxidative metabolism in alveolar macrophages was significantly greater than in hepatic macrophages. TPA, which is known to induce a respiratory burst in phagocytic cells [24 29 , 30 ], stimulated superoxide anion production by alveolar macrophages but had no effect on hepatic macrophages (Fig. 5 , right panel). Treatment of rats with ethanol was found to depress production of superoxide anion by alveolar macrophages with no significant effect on hepatic macrophages. Alveolar and hepatic macrophages were also found to produce NO following stimulation with LPS and/or IFN-{gamma}, a response that was dose- and time-dependent [24 , 29 ]. As observed with superoxide anion, alveolar macrophages produced significantly more NO than hepatic macrophages (Fig. 6 ). In hepatic and alveolar macrophages, NO production was correlated with expression of the inducible form of NOS (NOS II) (Fig. 7 ). It is interesting that low levels of NOS II protein were also observed in unstimulated hepatic macrophages. Ethanol administration to rats reduced NO production and NOS II protein expression in alveolar macrophages and hepatic macrophages (Figs. 6 and 7) .



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  		Figure 6. Effects of ethanol administration on NO production. Hepatic macrophages (HM) and alveolar macrophages (AM) isolated from control rats or rats treated with ethanol for 9–12 weeks were incubated with medium control, LPS (10 ng/ml), and/or IFN-{gamma} (100 U/ml). Culture supernatants were collected after 48 h and analyzed for nitrite content. Each value is the mean ± SEM from six to eight experiments. aSignificantly different (P<=0.05) from macrophages from control rats.

 


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  		Figure 7. Effects of ethanol administration on NOS II protein expression. Alveolar macrophages (AM; lanes 1–4) and hepatic macrophages (HM; lanes 5–8) from control rats (lanes 1, 2, 5, and 6) or from rats treated with ethanol for 12 weeks (lanes 3, 4, 7, and 8) were incubated for 24 h with (+) or without (-) LPS (10 ng/ml) plus IFN-{gamma} (100 U/ml) as indicated. NOS II expression was analyzed by Western blotting. The 130 kD NOS II protein is indicated. C, positive control.

 
For comparative purposes, we also evaluated the effects of ethanol administration to rats for 3–6 weeks on hepatic and alveolar macrophages. A significant decrease in the production of superoxide anion was observed in alveolar macrophages from rats treated with ethanol for 3–6 weeks (unpublished results). However, in contrast to chronic ethanol administration (9–12 weeks), other macrophage activities including chemotaxis, phagocytosis, cell adhesion molecule expression, NO production, and NOS II expression were unaltered.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies demonstrate that resident hepatic and alveolar macrophages are distinct cell populations functionally and structurally. Thus, differences were noted in the size and physical characteristics of the two populations, as well as in their functional capacity. Moreover, these cells responded distinctly to chronic ethanol administration. These data are consistent with previous studies on tissue macrophage heterogeneity [31 32 33 ] and provide additional evidence for the concept that macrophages acquire unique characteristics in response to their microenvironment [34 ] and exhibit distinct responses to toxins [6 7 35 ].

Macrophage expression of cell adhesion molecules is upregulated in response to tissue injury and various proinflammatory cytokines [36 , 37 ]. These proteins promote phagocyte adhesion to the endothelium and extravasation into the tissue [38 , 39 ]. We found that expression of ICAM-1 by hepatic and alveolar macrophages was similar. In contrast, alveolar macrophages expressed significantly more ß2-integrins than hepatic macrophages. This may reflect their greater migratory potential in the lung. Administration of ethanol to rats for 9–12 weeks resulted in a significant decrease in the number of macrophages recovered from the liver with no effect on alveolar macrophages. This was associated with reduced expression of ICAM-1 and ß2-integrins on the liver cells. ICAM-1 expression has been shown previously to be increased on hepatic macrophages, as well as endothelial cells and hepatocytes, after 16 weeks of ethanol administration to rats [40 ]. Differences between these findings and our work may reflect distinct methods used to quantify cell adhesion molecule expression on liver cells and/or in the length of exposure time to ethanol. Derangements in expression of ICAM-1 have been implicated in many pathological disorders, including endotoxemia and ischemia/reperfusion injury [41 42 43 ], and similar pathophysiologic processes may occur in ethanol-induced toxicity. The effect of chronic ethanol administration on ß2-integrin expression by hepatic and alveolar macrophages has not been evaluated previously. Decreases in expression of ß2-integrins and ICAM-1 on hepatic macrophages after prolonged ethanol administration may contribute to the reduced number of these cells in the liver.

Chemotaxis and phagocytosis are inflammatory functions essential for macrophage clearance of microorganisms as well as dead cells and debris from tissues. However, the extent to which macrophages exhibit these activities is dependent on their tissue origin and activating signals. Thus, although alveolar macrophages were found to be more responsive to C5a than hepatic macrophages, the liver cells exhibited greater random migration and chemotactic responsiveness to TPA. Increased random migration of hepatic macrophages may reflect a greater basal metabolic state of these cells because of endotoxin priming in vivo. Hepatic macrophages were also found to phagocytize two to three times more bacteria than alveolar macrophages, which is in accord with previous studies demonstrating that these cells express greater numbers of Fc receptors when compared with peritoneal macrophages [44 ]. Our studies also demonstrated that alveolar macrophages released greater quantities of reactive oxygen and reactive nitrogen intermediates than hepatic macrophages. This is consistent with their primary antimicrobial function in the lung [2 ]. In contrast to alveolar macrophages, hepatic macrophages constitutively expressed NOS II protein, which, most likely, reflects the fact that they are continually exposed to endotoxin in the portal circulation. Macrophage-derived mediators such as superoxide anion and NO facilitate microbial killing. Overproduction of these mediators by macrophages has also been implicated in cellular injury and tissue damage [6 7 ].

Prolonged ethanol exposure resulted in decreased chemotaxis, NO production, and oxidative metabolism by alveolar macrophages, and decreased NO production by hepatic macrophages. In contrast, this treatment had no effect on chemotaxis by hepatic macrophages or on phagocytosis by either cell type. Ethanol treatment of macrophages in culture has been shown to suppress adhesion, chemotaxis, phagocytosis, and oxidative metabolism [45 , 46 ]. Studies evaluating the effects of acute or chronic ethanol in vivo on these functions in liver and lung macrophages have led to contradictory conclusions [18 , 46 47 48 49 ], which may be a result of differences in the strains of animals used and/or in the doses or timing of ethanol administration. The present studies represent the first comprehensive analysis of lung and liver macrophages from the same animal after ethanol administration. The advantage of this approach is that individual differences between animals, which may confound a comparative study of liver and lung macrophages, have been eliminated. Our findings suggest that in general, alveolar macrophages are more sensitive than hepatic macrophages to the suppressive effects of ethanol on functional responsiveness. This may be important in the distinct responses of the liver and lung to this toxicant.

A significant decrease in superoxide anion production by alveolar macrophages was observed after 3–6 weeks and 9–12 weeks of ethanol treatment. Similar decreases in oxidative metabolism in alveolar macrophages have been described previously following 4 weeks and 12–14 weeks of ethanol administration to rats [12 , 50 ]. The mechanisms underlying the inhibitory effects of ethanol on macrophage superoxide anion release are unknown. It has been suggested that TPA-stimulated superoxide anion release is inhibited by acute ethanol-induced alterations in membrane fluidity, a process that can inactivate protein kinase C [50 ]. In this regard, ethanol feeding of rats for 9 weeks has been shown to downregulate membrane-associated protein kinase C [51 ].

NO production by alveolar macrophages and hepatic macrophages was decreased following 9–12 weeks of ethanol treatment of the animals. This was correlated with reduced expression of NOS II protein. Decreases in NO production by nonparenchymal liver cells and alveolar macrophages have also been shown following 8 weeks and 14 weeks, respectively, of ethanol administration [50 , 52 , 53 ]. These data demonstrate that ethanol administration inhibits macrophage priming for responsiveness to cytokines and bacterially derived products.

It is interesting that NOS II protein was not detected in alveolar macrophages from ethanol-treated animals, despite the fact that these cells released significant amounts of NO spontaneously and in response to stimulation with LPS + IFN-{gamma}. This may be a result of increased expression of NOS I or NOS III in these cells. It is also possible that ethanol exposure causes structural or antigenic changes in the NOS II protein, which decrease its ability to bind to antibodies. Our findings are in accord with previous studies that show NOS II protein was not detectable in alveolar macrophages from rats treated acutely with ethanol, which were attributed to ethanol-induced alterations in NOS II transcription and posttranscriptional modification [54 ]. It appears that chronic ethanol treatment decreases NO production, at least in part, by the blocking expression of the NOS II protein.

Chronic intake of ethanol causes immunosuppression, a process that has been correlated with a greater incidence of liver and lung infections in humans. Our data suggest that specific decreases in the functional capacities of hepatic and alveolar macrophages may contribute, in part, to the immunosuppressive effect of ethanol on the liver and lung.


   ACKNOWLEDGEMENTS
 
This work was supported by U.S. Public Health Service grants GM34310, ES04738, ES06897, and ES05022 from the National Institutes of Health and by a Career Development Award (AP #165) from the Burroughs Wellcome Fund. D. L. L. is a Burroughs Wellcome Scholar.

Received February 15, 2000; revised July 5, 2000; accepted July 6, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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