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The Journal of Immunology, 2003, 170: 2923-2931.
Copyright © 2003 by The American Association of Immunologists

Acute Alcohol Inhibits TNF-{alpha} Processing in Human Monocytes by Inhibiting TNF/TNF-{alpha}-Converting Enzyme Interactions in the Cell Membrane 1

Xue-Jun Zhao2,*,{dagger}, Luis Marrero2,*, Kejing Song2,*,{dagger}, Peter Oliver2,*,{dagger}, So Yeon Chin*,{dagger}, Harriet Simon*, Jill R. Schurr*, Zili Zhang*,{dagger}, Deepu Thoppil*, Sharon Lee*, Steve Nelson*,{dagger} and Jay K. Kolls3,*,{dagger}

* Gene Therapy Program and {dagger} Alcohol Research Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alcohol abuse has long been known to adversely affect innate immune responses and predispose to infections. One cellular mechanism responsible for this effect is alcohol-induced suppression of TNF-{alpha} by mononuclear phagocytes. We undertook experiments to better understand the cellular mechanisms by which alcohol dose-dependently suppresses TNF elaboration by human monocytes. Here we show in human primary monocytes and cell lines that alcohol suppresses LPS-induced TNF secretion post-transcriptionally by inhibiting cellular processing by TNF-{alpha}-converting enzyme (TACE). Using fluorescent resonance energy transfer microscopy, physiological relevant levels of alcohol resulted in a reversible dose-dependent decrease in fluorescent resonance energy transfer efficiency between TNF and TACE. These data demonstrate that alcohol inhibits interactions between TNF and its converting enzyme, TACE, possibly by affecting membrane fluidity. These data in part explain the cellular mechanisms by which alcohol impairs monocyte function and may identify immunotherapeutic targets aimed at restoring immune function in this at-risk patient population.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alcohol is a potent immunosuppressive drug and has been widely recognized for many centuries as an important risk factor for the development of infections. As early as 1785, Dr. Benjamin Rush, the first Surgeon General of the United States, described in An Inquiry Into the Effects of Ardent Spirits Upon the Human Body and Mind that alcohol abusers are predisposed to a greater number of as well as more severe infections, particularly of the respiratory tract (1). In 1905, Sir William Osler wrote in his Principles and Practice of Medicine that alcoholism is "perhaps the most potent predisposing factor" to lobar pneumonia (2). It was not until 1923 that the first clinical study was performed to quantitatively assess the relationship between alcohol abuse and infection. Capps and Coleman at Cook County Hospital examined retrospectively the influence of alcohol abuse on the death rate in 3422 cases of lobar pneumonia occurring over an 8-year period (3). The mortality rate of patients with pneumonia who had no or light use of alcohol was 23%, while 35 and 50% of moderate and excessive drinkers died of pneumonia, respectively. Even with the advent of antibiotics, infection has continued to be a major problem among alcohol abusers. Nolan’s study of admissions to a university community hospital showed that 124 (13.8%) of the 900 patients admitted were alcoholics, and the most common presenting problem in these individuals (16.9%) was bacterial pneumonia, while only 6.5% of nonalcoholics presented with pneumonia (4). More recently, in 1995, a case-controlled study in Spain of 50 control subjects and 50 patients with community-acquired pneumonia identified high alcohol intake as a risk factor for pneumonia (5).

The cellular mechanisms by which alcohol results in immunosuppression have recently been reviewed (6). One proposed mechanism by which acute alcohol suppresses innate immunity is its dose-dependent suppression of TNF-{alpha} elaboration by mononuclear phagocytes (7, 8, 9, 10). The mechanism underlying this effect of acute ethanol (EtOH)4 has yet to be firmly established. Szabo and colleagues (7, 11) have reported decreased nuclear translocation of NF-{kappa}B and reduced steady-state levels of TNF mRNA in alcohol-exposed human monocytes. Studies in our laboratory have also shown a significant post-transcriptional and post-translational component of acute alcohol-mediated TNF suppression in both rodent and primate macrophages (12, 13, 14, 15). Based on these studies we hypothesized that acute EtOH results in decreased processing of TNF by TNF-{alpha}-converting enzyme (TACE), a member of the disintegrin and metalloproteinase (ADAM) family of proteins (16, 17).

To investigate whether acute alcohol specifically interferes with enzymatic processing of TNF, we transfected human TNF into murine fibroblasts that expressed wild-type TACE or an inactive form of TACE (TACE-/- fibroblasts) (18). To avoid issues of mRNA stability regulated by the TNF-{alpha} 3'-untranslated region (19, 20), we expressed TNF under control of the heterologous CMV promoter and a polyadenylation signal from SV40. Using this system, acute alcohol resulted in dose-dependent suppression of TACE-mediated processing of TNF. To further investigate whether acute alcohol interfered with human TACE/human TNF interactions on the cell surface, we used fluorescent resonance energy transfer to measure protein-protein interactions between TNF and TACE in three different systems: 1) Mono Mac 6 cells stimulated to produce TNF with LPS/PMA; 2) human A549 cells that express TACE constitutively, transfected with the human TNF cDNA; and finally, 3) human PBMC stimulated with LPS. In all three systems we observed a dose-dependent suppression of fluorescent resonance energy transfer (FRET) efficiency between TNF and TACE. Based on these results, we conclude that acute EtOH suppresses processing and secretion of TNF by altering membrane compartmentalization of the enzyme and substrate. This effect may be a critical mechanism by which acute alcohol suppresses innate immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of PBMCs

Human blood (20 cc) was collected from human volunteers, aged 18–65, using EDTA as an anticoagulant. The blood was mixed 1/1 with HBSS (Life Technologies, Invitrogen Corp., Carlsbad, CA) and then placed over Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) for centrifugation (900 x g, 30 min, 20°C). The PBMC layer was collected, rinsed in HBSS, and cultured as outlined below. All procedures were approved by the institutional review board of the Louisiana State University Health Sciences Center.

Cell culture

Human Mono Mac 6 cells or PBMCs were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 1x nonessential amino acids, and 1x penicillin-streptomycin (Life Technologies). The cells were incubated in 5% CO2 at 37°C and treated with 0, 50, and 100 mM EtOH 30 min before stimulation with 20 ng/ml Escherichia coli LPS 0111:B4 (List Biological Laboratories, Campbell, CA) and 60 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) for 1–3 h. Human PBMCs were treated with 0, 50, and 100 mM EtOH 30 min before stimulation with 100 ng/ml LPS. The EtOH was left in the culture during stimulation for both Mono Mac 6 cells and human PBMCs. The cell pellets and supernatant were collected for ELISA. Cell lysates were prepared in a lysis buffer containing PBS (pH 7.2), 1% Triton X-100, and proteinase inhibitor mixture (Roche, Indianapolis, IN) on ice for 30 min. The cell lysates were further centrifuged at 12,000 x g for 15 min.

A549 cells were maintained in DMEM/F-12 supplemented with 10% heat-inactivated FBS and 2 mM L-glutamine. Murine TACE-/- and TACE+/+ fibroblasts were gifts form Dr. R. Black and were cultured in DMEM/F-12 supplemented with 1% heat-inactivated FBS and 2 mM L-glutamine.

Construction of human TNF expression vector

The human full-length TNF-{alpha}-coding sequence was prepared by PCR from its cDNA in plasmid pcDV1 (ATCC 39894; American Type Culture Collection, Manassas, VA) with the COOH-terminal deletion mutant of the stop codon and then cloned in-frame into p3xFLAG-CMV-14 protein expression vector (Sigma-Aldrich). This vector was sequenced, and the expression of TNF was measured by transfecting the vector into A549 cells and measuring secreted TNF as well as cell surface TNF using an anti-TNF Ab (see below) as well as an anti-FLAG Ab (Sigma-Aldrich).

Transfection studies

A549 cells or murine TACE-/- and TACE+/+ fibroblasts were transfected with full-length TNF-3xFLAG plasmid vector using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer. A549 cells were incubated in the 37°C, 5% CO2 humidified incubator for ~24 h to obtain the greatest protein expression (data not shown). Murine TACE-/- and TACE+/+ fibroblasts were incubated in the 37°C, 5% CO2 humidified incubator for ~48–36 h to obtain the maximum protein expression (data not shown). Post-transfected A549 cells and murine TACE-/- and TACE+/+ fibroblasts were cultured for 1 h with fresh medium or medium with different concentrations of EtOH as indicated. Cell culture supernatants were collected, and cells were removed from the culture plate by incubation with 2 mM EDTA and lysed with 1% Nonidet P-40 in PBS containing 1 mM EDTA and protease inhibitor cocktail (Roche). Both culture media and cell lysates were spun by centrifugation for 15 min at 13,000 rpm in a microfuge. The levels of the cell-associated form of TNF in cell lysates and the secreted form of TNF in culture supernatant were measured by ELISA (R&D Systems, Minneapolis, MN) following the manufacturer’s recommendation. Total RNA from these cells was extracted using TRIzol reagent (Invitrogen), and TNF-{alpha} mRNA was measured by TaqMan RT-PCR using primers from Biosource (Camarillo, CA) and the following probe: 5'-FAM-CATCGCCGTCTCCTACCAGACCAAG-Black Hole Quencher 1-3'. Samples were run with and without reverse transcriptase to exclude DNA contamination.

RNase protection assays

Total RNA was isolated from Mono Mac 6 cells or PBMCs by lysing cells in TRIzol reagent (Life Technologies). An antisense mRNA probe for TNF, TACE, p75 TNF receptor II, and two housekeeping genes, L32 and GAPDH, were labeled with [32P]UTP (Amersham Pharmacia Biotech) based upon the manufacturer’s protocol. The remaining protected hybridized probes were fractionated on a 6% denaturing polyacrylamide gel. An aliquot of the probes was run to check the integrity and size of the probes. The gel was dried and exposed to a PhosphorImager screen, then analyzed using ImageQuant software (both from Molecular Dynamics, Mountain View, CA). The protected bands were quantified and normalized to the housekeeping gene L32.

Measurement of TNF-{alpha} and IL-8

TNF-{alpha} and IL-8 were measured in supernatants by ELISA (R&D Systems) or using Luminex beads specific for TNF-{alpha} and IL-8 (BioSource, Camarillo, CA) following the manufacturer’s recommendations. There was excellence concordance with ELISA and Luminex data (r > 0.97). Cell lysates for TNF-{alpha} were measured by ELISA as previously described (15) and normalized to total protein using a protein assay (Pierce, Rockford, IL).

Immunofluorescent labeling

Human monocytes, A549 cells, and Mono Mac 6 cells were stimulated as previously described and adhered to coverslips in culture. Immediately poststimulation, the coverslips were washed in cold PBS to remove excess medium and fixed in a 4% solution of 10% methanol (mEtOH)-free formaldehyde (Polysciences, Warrington, PA) in PBS for 15 min at room temperature. After thoroughly washing the fixative off with cold PBS, the cells were blocked with 5% rabbit serum in 1% BSA in PBS for 15 min. Subsequently, the blocker was replaced with the first primary Ab, mouse anti-human TACE clone M222 (Immunex, Seattle, WA), diluted in 1% BSA at 10 µg/ml. The coverslips were thoroughly washed in three PBS changes after the 45-min primary Ab incubation. The Ab was indirectly labeled with a rabbit anti-mouse, Cy-3-conjugated Fab (Jackson ImmunoResearch Laboratories, West Grove, PA) at 6.5 µg/ml in 1% BSA. Next, cells were washed and blocked with 5% mouse serum for 10 min to saturate any open Ag binding sites within the first primary Ab. The blocker was replaced with the second, primary Ab mouse anti-human, FITC-conjugated, TNF-{alpha} clone mAb11 (BD PharMingen, San Diego, CA) at a 10 µg/ml dilution in 1% BSA for 30 min. All Ab dilutions remained constant throughout the experiments and were titrated to be detected at their highest saturation point, hence maximizing to an optimal donor to acceptor ratio. Finally, the coverslips were washed, mounted cell side down in PBS on slides with spacers, and secured with nail polish.

FRET imaging and data collection

Human monocytes, Mono Mac 6 cells, and A549 cells mounted on coverslips were imaged using a digital fluorescence microscopy system. The system included a 12-bit, chilled, charged-coupled device (CCD) Sensicam QE with a 1376 x 1040 resolution and 65% quantum efficiency at 550 nm (The Cooke Corp., Auburn Hills, MI) on an automated Leica DMRXA microscope (Meyer Instruments, Houston, TX) using a 1.4 NA, x63 Plan-Apochromat objective. FITC and Cy3 were detected using appropriate filters (customized FITC filter cube: excitation, 480/40 nm; 505 long-pass dichroic; emission, 535/50; Cy3 filter cube: excitation, 545/30; 565 long-pass dichroic; emission 620/60; Chroma Technology, Brattleboro, VT). Fluorescence was excited with a 75-watt xenon arc lamp. The samples were photobleached with a 100-watt mercury source. All components were controlled using Slidebook software (Intelligent Imaging Innovations, Denver, CO), which was used for capture, nearest neighbor deconvolution, and FRET analysis. Image acquisition was adjusted in milliseconds to achieve the maximum CCD camera range. As part of detecting bleed-through, typical exposure times were used to confirm no visualization of FITC-labeled samples with the Cy3 filter cube and vice versa.

All experiments were based on FRET measured by acceptor photobleaching recovery using FITC as the donor and Cy3 as the acceptor. The Cy3 was determined to be sufficiently photolabile to undergo the premeasured 3-min photobleaching step for a subsequent <2% initial intensity measurement. The donor had a high enough quantum yield and stability to not fade significantly. All experiments included a donor-only and acceptor-only sample to verify minimal cross-over between them; hence, there was no significant energy transfer. Data were collected for 20 different fields from a single coverslip. Each fluorescent channel was collected at six consecutive z-planes measuring 0.3 µm each. The software allowed stack autoalignment between exposures to compensate for thermally induced shifts. The FRET experiment imaging began with an initial image stack of the FITC-labeled protein (in the presence of the Cy3-labeled Ab) obtained with the FITC filter set, immediately followed by an image stack captured with the Cy3 filter cube. Next, the sample was exposed to constant illumination for 3 min to photobleach. An image stack of the Cy3 fluorescence after photobleaching was then obtained, followed by another FITC fluorescence image stack using the FITC filter set. The exposure times were unchanged between pre- and postphotobleaching steps. Post-FRET calculation images were deconvolved using a nearest neighbor algorithm for presentation purposes only.

FRET data analysis

Images indicating FRET between the labeled Abs against TNF and TACE were calculated based in the increase in donor fluorescence after acceptor photobleaching using the following formula: E = FITCpostphotobleach - FITCprephotobleach/FITCpostphotobleach. Here, E represents FRET efficiency after performing a background subtraction caused by scattered light, autofluorescence, and dark current of the CCD camera. Fluorescence intensities were reported in arbitrary units and on a pixel-to-pixel basis. This was achieved by digitally performing the prephotobleaching channel subtraction from the postphotobleaching channel (see above formula) and thresholding the difference, labeled as mean FRET intensity.

Statistical methods

All data are presented as the mean ± SEM. Significance was estimated using ANOVA, followed by Tukey’s multiple comparison procedure, with p < 0.05 considered significant. Due to the variability in human donor TNF responses, data were normalized to the mean value of the prestimulation values as a percentage of the mean, and then ANOVA was performed on the percent change with EtOH.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Acute alcohol suppresses TNF secretion post-translationally

We examined the suppressive effects of alcohol on TNF secretion in both Mono Mac 6 cells (21) and human PBMCs. Acute EtOH added to Mono Mac 6 cells 30 min before stimulation with LPS/PMA resulted in a dose-dependent decrease in secretion of TNF at both 1 and 3 h after stimulation (Fig. 1A). There was no spontaneous release of TNF exposed to medium or EtOH in the absence of LPS/PMA stimulation (data not shown). The suppressive effect of EtOH was not associated with an inability of these cells to produce TNF. In fact, acute EtOH resulted in an increase in cell-associated TNF (Fig. 1B) and an increase in the ratio of cell-associated vs shed TNF 3 h after LPS/PMA stimulation, which is a measure of TNF processing by TACE (15, 18) (Fig. 1C). This was not due to a nonspecific inhibition of protein processing by the cell, as the secretion of IL-8 by Mono Mac 6 cells was not inhibited in this acute EtOH model (Fig. 1D). These data are in agreement with our prior published observations in Mono Mac 6 cells, where acute EtOH did not inhibit LPS/PMA-induced increases in steady-state TNF mRNA levels or mRNA half-life (15).



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  		FIGURE 1. Post-translational suppression of TNF by alcohol in Mono Mac 6 cells. A, Mono Mac cells were incubated with varying concentrations of EtOH 30 min before stimulation with (or without) 20 ng/ml LPS and 60 ng/ml PMA. TNF was measured in supernatants at 1 and 3 h (A) or in cell pellets at 3 h (B) by ELISA (n = 6–8; *, p < 0.05 compared with 0 mM control). There was no TNF detected in unstimulated cells with or without EtOH (data not shown). C, Ratio of cell associated to secreted TNF 3 h after LPS/PMA stimulation in Mono Mac 6 cells (n = 6–8; *, p < 0.05 compared with 0 mM control). D, Mono Mac cells were incubated with varying concentrations of EtOH 30 min before stimulation with or without LPS/PMA. IL-8 was measured in supernatants by ELISA 3 h after stimulation (n = 4–6).

 
We next extended these findings to human PBMCs. To avoid any activation of human monocytes by adherence, human PBMCs were incubated in polypropylene tubes with varying concentrations of alcohol, followed by stimulation with LPS. We observed a dose-dependent suppression of LPS-induced TNF production at 1 and 3 h after LPS-stimulation in five independent donors (Fig. 2, A and B, only 3 h shown). Since there was significant donor variability in their peak TNF response to LPS, we analyzed the effect of EtOH as a percentage of their control (Fig. 2B). This analysis verified a highly significant dose-dependent suppression of TNF. However, this suppression was not due to a decrease in cell-associated TNF, but was almost entirely due to a decease in the efficiency of shedding of TNF, as measured by the ratio of cell-associated vs shed TNF (Fig. 2, C and D). When plotted as a percentage of each donor’s control value (Fig. 2D), the percent reduction in secreted TNF (Fig. 2B) was accounted for solely by the percent increase in the ratio of cell-associated vs shed TNF (Fig. 2D). Moreover, we observed no effect of EtOH on steady transcripts for TNF mRNA as measured by RNase protection assay 2 h after stimulation with LPS (Fig. 2, E and F). These data further support our hypothesis that acute EtOH inhibits TNF post-translationally. Furthermore, acute EtOH-induced suppression of TNF secretion in human primary PBMCs could not be explained by a general defect in protein processing, since we did not observe a significant suppression of IL-8 from these same donors 3 h after LPS-stimulation (Fig. 2G).



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  		FIGURE 2. Post-translational suppression of TNF by alcohol in PBMCs. A, Human PBMCs from five different donors were incubated with 0, 50, or 100 mM EtOH 30 min before stimulation with or without LPS. TNF was measured in supernatants (A) or cell pellets by ELISA 3 h after stimulation. There was no TNF detected in unstimulated cells with or without EtOH (data not shown). B, Effect of EtOH on TNF secretion as a percentage of the donor’s baseline value (*, p < 0.01 compared with 0 mM control). C, Ratio of cell-associated vs shed TNF in PBMCs 3 h after stimulation with LPS. D, Effect of EtOH on the ratio of cell-associated vs shed TNF as a percentage of the donor’s baseline value 3 h after stimulation with LPS (*, p < 0.01 compared with 0 mM control). E, Representative RPA; D, mean increase in TNF mRNA in PBMCs 2 h after LPS stimulation normalized to baseline value and L32 transcripts for five human donors. G, Lack of an effect of EtOH added 30 min before LPS stimulation in PBMCs (n = 5). No IL-8 was detected in unstimulated PBMCs (data not shown).

 
Acute alcohol suppresses TACE-mediated TNF processing

To examine whether EtOH had a direct inhibitory effect on TACE, we incubated recombinant TACE (a gift from Dr. R. Black) (16) with a TNF-based TACE cleavage peptide that contains the alanine/valine cleavage site of TACE, as previously described (16). Analysis by HPLC showed efficient cleavage of the peptide by TACE, as previously described by our laboratory (22). However, we observed no inhibitory effect of EtOH in this in vitro assay at doses of 25, 50, and 100 mM EtOH. We concluded from this experiment that if EtOH acts on TACE-mediated TNF processing, this must occur in the context of the cell membrane.

To address this, we transfected the human TNF cDNA into TACE+/+ or TACE-/- fibroblasts (18). TACE+/+ fibroblasts efficiently processed TNF with a significant amount of TNF in the cell supernatant relative to the cell pellet (Fig. 3A). Addition of EtOH for 60 min resulted in a dose-dependent suppression of secreted TNF and an increase in the cell-associated TNF (Fig. 3A). Transfection and overexpression of human TNF into TACE-/- fibroblasts results in some processing of TNF, as measured by the ratio of secreted to cell-associated TNF (Fig. 3B). In fact, TACE-/- fibroblasts have a higher concentration of both cell-associated and secreted TNF due to a higher transfection efficiency than TACE+/+ fibroblasts (data not shown). However, when normalized to cell-associated TNF, as previously described by Black and colleagues (18), TACE-/- fibroblasts were not as efficient as TACE+/+ cells in processing TNF, similar to what we observed in our studies here (Fig. 3B). The addition of EtOH for 60 min to these transfected cells resulted in a dose-dependent suppression of TNF processing only in the TACE+/+ cells (Fig. 3B), but had no effect on the nonspecific processing in TACE-/- cells (Fig. 3B). Since these are murine fibroblast lines, we also performed transfection experiments in A549 cells, a human cell line that expresses human TACE (data not shown). Transfection of the human TNF cDNA in these cells resulted in processing of TNF, as measured by the ratio of secreted to cell-associated TNF, and this processing was blocked by the TACE inhibitor TAPI (Fig. 3C). Again, in this cell line EtOH resulted in a dose-dependent suppression of TNF processing. To investigate whether the EtOH effect was reversible, the EtOH-containing medium was removed by washing and replacing the medium with EtOH-free medium, followed by measuring secreted and cell-associated TNF 60 min later (Fig. 3D, Post-Wash). Compared with cells that had previously been exposed to 0 mM EtOH, cells previously exposed to 50 or 100 mM failed to show any suppression of TNF processing (data expressed as a percentage of the 0 mM control). These data strongly support the conclusion that acute EtOH inhibits TNF/TACE interactions in a cellular and reversible context.



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  		FIGURE 3. Alcohol-induced suppression of TNF-{alpha} processing by TACE. The human TACE cDNA was transfected into TACE+/+ or TACE-/- fibroblasts. Twenty-four hours later these cells were randomized to exposure with 0, 25, 50, 75, or 100 mM EtOH. TNF-{alpha} was measured in the cell supernatant or cell pellet 60 min later (n = 4; *, p < 0.05 compared with 0 mM control). TACE+/+ fibroblasts efficiently processed TNF-{alpha} by releasing TNF-{alpha} into the supernatant (A). B, Effect of EtOH on the ratio of shed vs cell-associated TNF in TACE+/+ or TACE-/- fibroblasts (n = 4; *, p < 0.05 compared with 0 mM control). C, Effect of EtOH and the TACE inhibitor TAPI on processing of TNF-{alpha} in transfected human A549 cells (n = 3; *, p < 0.05 compared with 0 mM control). No TNF-{alpha} was detectable in untransfected (un-ts) cells. D, Reversible effect of EtOH on TNF processing. TNF-transfected A549 cells were incubated with 0, 50, or 100 mM EtOH for 60 min (EtOH group) or were washed with fresh medium and incubated for another 60 min in EtOH-free medium (Post-Wash) before measuring shed and cell-associated TNF (*, p < 0.05 compared with 0 mM control).

 
Acute alcohol inhibits FRET efficiency between TNF-{alpha} and TACE

To further test this hypothesis we used FRET microscopy to image the cellular interactions of TNF with TACE in three systems: 1) LPS/PMA-stimulated Mono Mac 6 cells, 2) transfected A549 cells, and 3) LPS-stimulated human PBMCs. Unstimulated Mono Mac 6 cells or human PBMCs or untransfected A549 cells did not express TNF-{alpha} (data not shown), so all of the FRET data shown were obtained in stimulated or transfected cells. We also performed all FRET analysis on cells fixed with 4% mEtOH-free formaldehyde for 5 min, since we used Ab-based FRET, and TNF processing/secretion is a dynamic process. Fixation allowed us to examine a point in time in this highly dynamic process and did not affect FRET efficiency in transfected A549 cells where TNF production is more continuous than in LPS-stimulated monocytes (data not shown). Using deconvolution microscopy, LPS/PMA-stimulated Mono Mac 6 cells expressed TNF-{alpha} (Fig. 4a) and TACE (Fig. 4b), which upon stimulation showed significant colocalization (Fig. 4, c and d). We used these cells to establish FRET conditions using FITC (TNF) as the donor and Cy3 (TACE) as the acceptor. We next applied these conditions to transfected A549 cells and stimulated human PBMCs to investigate whether EtOH interfered with FRET efficiency.



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  		FIGURE 4. Representative Mono-Mac 6 cell expressing anti-TNF (FITC) and anti-TACE (Cy3) colocalization on its membrane. A stack of six deconvolved 0.3-µm images compose this compressed 2D projection and are comprised of the following channels: a) anti-TNF (FITC); b) anti-TACE (Cy3); and c) colocalization of both anti-TNF (FITC) and anti-TACE (Cy3). A pseudocolor rendering d, High (red pixels) colocalization areas to medium (yellow pixels) to low (gray and blue pixels; arrows) areas.

 
A549 cells, transfected with the human TNF cDNA, were incubated with 0, 50, or 100 mM EtOH for 60 min, followed by fixation in 4% mEtOH-free formaldehyde. Cells were stained for TNF and TACE, and FRET efficiency was measured and calculated from 20 representative fields/group. Both TNF and TACE were detectable on the cell surface (Fig. 5A, a and b) and demonstrated colocalization (Fig. 5Ae). The TACE (Cy3, acceptor) signal was photobleached (Fig. 5Ac), whereas the TNF (FITC) signal increased (Fig. 5Ad) consistent with FRET. FRET was quantified by using pixel-by-pixel subtraction of FITC fluorescent intensity post- and prephotobleaching. To depict FRET on the cell surface, the FRET intensity was superimposed over donor intensity as a pseudocolor image (Fig. 5A, panel f), where blue is little or no FRET efficiency, and red is peak FRET efficiency. Incubation with EtOH resulted in a dose-dependent decrease in FRET efficiency between TNF and TACE (Fig. 5B).



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  		FIGURE 5. A, Immunofluorescent detection, colocalization, and FRET intensity in a representative transfected A549 cell from a 0 mM EtOH group expressing anti-TACE (Cy3) and anti-TNF (FITC) as FRET acceptor and donor, respectively. The staining pattern is suggestive of both cell membrane and membrane vesicle localization: a) pre-photobleaching anti-TACE (Cy3; acceptor) specific to the cell surface; b) anti-TNF (FITC) (donor) prephotobleaching fluorescence; c) <2% initial anti-TACE (Cy3) intensity postphotobleaching; d) anti-TNF (FITC) donor intensity postphotobleaching; e) colocalization of membrane-bound TNF and TACE (prephotobleaching); and f) FRET intensity, calculated from the difference between donor pre- and postphotobleaching intensities shown in pseudocolor. B, Mean FRET intensity, calculated from the difference between donor (TNF) pre- and postphotobleaching intensity in 20 high power fields per group (*, p < 0.05 compared with 0 mM control; n = 4).

 
We next examined FRET efficiency between TNF and TACE in human PBMCs. Three individual donor’s PBMCs were incubated with 0, 50, or 100 mM EtOH, followed by stimulation with or without LPS for 1, 1.5, or 3 h. We analyzed the data at 1.5 h because this time point was associated with the highest amount of surface TNF and FRET intensity. Again TACE was labeled with Cy3 (Fig. 6, a, f, and k), and TNF was detected by FITC (Fig. 6, b, g, and l). Of note is that cells incubated with 100 mM EtOH (Fig. 5l) had significantly more surface TNF then those incubated with 0 mM EtOH (Fig. 6b). As seen with A549 cells, a significant amount of TNF was colocalized with TACE (Fig. 6, e, j, and o). As described above for A549 cells, photobleaching of the TACE (Cy3) signal is depicted in Fig. 6, c, h, and m, and FRET intensity is depicted in pseudocolor in Fig. 6, p, q, and r. Incubation with EtOH resulted in a dose-dependent suppression of FRET intensity (Fig. 6, p, q, r, and s), and this suppression was significant when the donors were corrected for their baseline FRET efficiency value (Fig. 6t). These data were also supported by a scatter plot of FRET intensity vs donor (TNF) signal, where there was significant FRET intensity in PBMCs stimulated with LPS and a reduction of pixels with FRET in the 50 and 100 mM EtOH groups (Fig. 7).



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  		FIGURE 6. Immunofluorescent detection, colocalization, and FRET intensity in human donor monocytes. Each row of images represents different experimental conditions: a–e, experimental control in the presence of LPS without EtOH; f–j, cells challenged with LPS and 50 mM EtOH; k–o, cells challenged with LPS and 100 mM EtOH. The monocytes were immunofluorescently labeled with anti-TACE (Cy3; acceptor) and anti-TNF (FITC; donor) to detect any membrane-bound interaction between TNF and its cleavage enzyme. Each image column illustrates all channels acquired during the FRET experiment, and the representative images were deconvolved using a Nearest Neighbor deconvolution algorithm after FRET calculations and for presentation purposes only. These include (from left to right): anti-TACE (conjugated to the acceptor Cy3) specific to the surface of the nonpermeabilized monocytes prephotobleaching (a, f, k); anti-TNF (conjugated to the FITC donor molecule) prephotobleaching localized to the membrane (b, g, l); <2% initial intensity anti-TACE (Cy3) postphotobleaching (c, h, m); donor postphotobleaching (d, i, n); donor and acceptor colocalization (e, j, o); and surface FRET intensity demonstrated by subtraction of the pre- from the postphotobleaching donor channels (p, q, r), emphasized with arrows. A donor and FRET channel pseudocolor was applied to the last series for better visualization and localization of the FRET intensity, which occurs at very low light levels. s, Energy transfer efficiencies for membrane colocalized TNF and TACE proteins in monocytes from human subjects (n = 3). Fluorescence intensities were obtained from 20 high power fields and identical planes of interest within each captured cell and all pre- and postphotobleaching channels. These efficiencies were calculated after performing a background subtraction. t, Effect of EtOH on Mean FRET efficiency compared with the donor’s baseline.

 


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  		FIGURE 7. Representative scatter plot of mean prephotobleaching donor intensity (x-axis) vs mean FRET intensity (y-axis) from donor 2. The colored lines represent lines of linear regression for 0 mM (black), 50 mM (blue), and 100 mM (red).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies show that acute EtOH blocks TNF secretion by human monocytes and PBMCs through a post-translational mechanism by inhibiting protein:protein interactions between TNF and TACE in the cell membrane. There have been many studies to better understand the mechanisms by which alcohol contributes to immunosuppression. Many types of infection, including bacterial pneumonia and, more recently, hepatitis C, have been shown to be adversely affected by concomitant alcohol abuse (23, 24, 25, 26). In addition, alcohol has been shown to be an independent risk factor for bacterial pneumonia and development of the adult respiratory distress syndrome (27).

One possible mechanism underlying the immunosuppressive effects of acute alcohol is its effects on innate immunity. In human volunteers and experimental animal models, alcohol dose-dependently suppresses neutrophil recruitment in response to chemotactic stimuli. One mechanism operative in the lung is suppression of the proinflammatory cytokine, TNF, which is required for induction of CXC chemokines critical for neutrophil recruitment (28, 29) as well as expression of adhesion molecules on vascular endothelium, which is required for neutrophil binding and diapedesis (28). Moreover, up-regulation of TNF by priming alveolar macrophages with IFN-{gamma} reverses EtOH-induced TNF suppression and improves pulmonary host defense against K. pneumoniae infection (30). In addition, up-regulation of TNF in the lung with gene delivery has been shown to augment pulmonary host defense against this same organism (31). These data highlight the critical role of TNF in pulmonary antibacterial host defense and underscore the importance of defining the mechanism by which alcohol suppresses this innate immune response.

Early studies in human PBMCs suggested that one potential mechanism was suppression of TNF mRNA (11, 32). However, in these studies, steady-state transcripts for TNF mRNA were suppressed between 20–30% in association with a >70% reduction in protein (32). Moreover, studies from our laboratory in human Mono Mac 6 cells and primary primate macrophages have shown that TNF transcription, steady-state mRNA transcripts, and TNF mRNA half-life are not altered by acute EtOH (14, 15). Thus, we investigated whether there exists a significant post-transcriptional/post-translational effect of acute EtOH on TNF production as well. Using both Mono Mac 6 cells and primary PBMCs and transfection studies, we show that acute EtOH suppresses TACE-mediated processing of TNF. Studies with FRET microscopy suggest that this occurs by interfering with enzyme-substrate interactions in the cell membrane, which results in an inefficient processing of TNF by EtOH. Although we observed a much greater effect of EtOH on TNF processing as opposed to FRET efficiency, it remains unclear what the relationship between FRET efficiency and enzyme function is. It is possible that a 25% reduction in FRET efficiency may result in a substantial decrease in TNF processing. Of interest, however, is that in our A549 system, the effect of EtOH is reversible, suggesting that EtOH has to be present in the system to observe the decrease in TNF processing. The mechanism of this reduction in interaction at present is unclear. Possibilities include changes in membrane fluidity (33) induced by EtOH (33), alterations in raft compartments, or perhaps abnormal ERK-mediated phosphorylation of TACE, which is critical for protein kinase C-regulated TrkA cleavage (34). Although there are no specific studies of yet examining TNF/TACE interactions in the context of membrane rafts, members of the TNF receptor superfamily, CD40 and CD120a (35, 36), as well as {alpha} secretases (37) have been localized to membrane rafts.

It is important to note that this study focused on the effects of acute EtOH, which has been shown to suppress TNF production in a variety of human and rodent models (9, 10, 15, 32). We did not investigate the effect of chronic EtOH. Data to date suggest that chronic EtOH significantly increases TNF production by both rodent and human macrophages (38, 39, 40) at least in part by increasing steady-state transcripts for TNF, possibly by increasing the TNF mRNA half-life (40), which may be mediated by increased ERK signaling. Preliminary studies from our laboratory suggest that this effect requires metabolism by EtOH through microsomal p450 enzymes (38). This effect of chronic EtOH on TNF production can be modeled in vitro in Mono Mac 6 (38) and Raw264.7 cells (39) and appears to be due in part to marked up-regulation of TNF transcription. In terms of alcohol’s impact on world health, this effect may be significant in terms of the development of liver disease (6). Of interest, however, is that acute EtOH ingestion along with chronic EtOH administration suppress TNF production in vivo (10).

The suppressive effects of EtOH do not require cellular metabolism of EtOH. Mono Mac 6 cells express only class V alcohol dehydrogenase and several isoforms of aldehyde dehydrogenase, such as aldehyde dehydrogenase 2 (determined by micorarray analysis on Affymetrix HG-U133A chips; data not shown). However, experiments with 4-methylpyrazole, an alcohol dehydrogenase inhibitor, and cynamide, an inhibitor of aldehyde dehydrogenase 2, did not alter effect of acute EtOH on TNF production or processing. Moreover, tertiary alcohols such as terbuatnol, which is not metabolized, also suppress TNF production by macrophages (12, 13). Taken together, these data suggest that the suppressive effects of EtOH on monocyte TNF production may be due to EtOH itself, whereas the up-regulation of TNF by monocytes probably requires metabolism and involves an alteration in the cellular redox state leading to increased TNF transcription (6).

Due to the critical role that TNF plays in the innate immune response, these mechanistic studies on both the suppressive and enhancing effects of EtOH on macrophage TNF production may lead to novel therapies to improve health in this high risk patient group.


   Acknowledgments
 
We thank Dr. Roy Black for providing M22, TACE+/+ and TACE-/- fibroblasts, and recombinant TACE.


   Footnotes
 
1 This work was supported by the National Institutes of Health through the following grants: AA010384 (to J.K.K.), AA009803 (to S.N.), and RR05096. Back

2 X.-J.Z., L.M., K.S., and P.O. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jay K. Kolls, CSRB, Room 601, 533 Bolivar Street, New Orleans, LA 70112. E-mail address: jkolls{at}lsuhsc.edu Back

4 Abbreviations used in this paper: EtOH, ethanol; CCD, charged-coupled device; FRET, fluorescent resonance energy transfer; TACE, TNF-{alpha}-converting enzyme; mEtOH, methanol. Back

Received for publication October 24, 2002. Accepted for publication January 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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