We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF- ) production by whole blood and ...
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, July 1996, p. 392–398 1071-412X/96/$04.0010 Copyright q 1996, American Society for Microbiology
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Alcohol Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Gene Expression by Peripheral Blood Mononuclear Cells as Measured by Reverse Transcriptase PCR In Situ Hybridization M. P. N. NAIR,1* N. M. KUMAR,2 Z. A. KRONFOL,3 J. F. GREDEN,3 J. S. LWEBUGA-MUKASA,2 1 AND S. A. SCHWARTZ Departments of Medicine and Microbiology1 and Pulmonary Medicine and Critical Care Division, Lung Biology Research, Department of Medicine,2 State University of New York at Buffalo, Buffalo General Hospital, Buffalo, New York 14203, and Department of Psychiatry, The University of Michigan, and Alcohol Research Center, Ann Arbor, Michigan 481093 Received 29 September 1995/Returned for modification 2 November 1995/Accepted 1 April 1996
We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-a) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-a gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 mg/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-a gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-a. These studies confirm our earlier finding that alcohol suppressed the production of TNF-a by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction. alcohol on TNF-a mRNA expression in vitro by using a semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization method.
The body’s ability to mount an antigen-specific response is directly dependent on the production of multiple immunoregulatory proteins, cytokines (15, 17). Tumor necrosis factor (TNF) is an important cytokine which is involved in host defense mechanisms against tumors and infectious and inflammatory diseases (1). Previous studies showed that TNF modulates the functions of polymorphonuclear neutrophils (41), T cells (24, 29), B cells (9, 19, 22, 23), monocytes/macrophages (36), natural killer cells (35), and lymphokine-activated killer cells (37). TNF also interacts with other cytokines, particularly gamma interferon, granulocyte-macrophage colony-stimulating factor, and interleukin 1 (IL-1), IL-2, and IL-4 (2, 12, 31, 38). Thus, TNF plays an important role in host defense. Previous studies have shown that chronic alcohol consumption is associated with dysfunctions of the immune system (11, 20, 21, 26–28, 43, 47). It was demonstrated that acute alcohol intoxication in rats markedly suppressed levels of TNF in both serum and lungs elicited in response to lipopolysaccharide (LPS) (32) or intratracheal challenge with Staphylococcus aureus or Klebsiella pneumoniae (33). These studies suggest that altered TNF release may have a role in the reduced immune response to infections observed in alcoholics. We recently showed that alcohol significantly suppressed LPS-induced TNF alpha (TNF-a) production by whole blood and peripheral blood mononuclear cells (PBMC) from healthy adult subjects as measured by a bioassay using the TNF-sensitive WEHI 164 subclone 13 cell line (30). The present investigation examined the effect of
MATERIALS AND METHODS Blood donors. Blood donors were apprised of this study, and consents were obtained consistently with the policies of the appropriate institutions and the National Institutes of Health. Peripheral blood from healthy, nonalcoholic individuals was drawn into a syringe containing heparin (20 U/ml). Subjects were free of medical or psychiatric illness and were not taking medications known to affect immune functions, including nonsteroidal anti-inflammatory agents and substances of abuse. Experimental design. (i) Separation of PBMC. Mononuclear cells were isolated from heparinized venous blood by a modification of the method of Boyum (4). The blood was diluted with an equal volume of normal saline and was centrifuged at 400 3 g for 30 min at 188C. The mononuclear cell band was harvested, washed three times with saline, and resuspended in RPMI 1640 medium containing 25 mM HEPES (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid) buffer supplemented with 10% heat-inactivated fetal calf serum (Gibco/BRL, Gaithersburg, Md.), 80 mg of gentamicin (Schering, Kenilworth, N.J.) per ml, and 300 mg of fresh glutamine per ml (complete medium). PBMC were cultured in complete medium. Triplicate PBMC cultures (106/ml) received ethanol at final concentrations of 0.1, 0.2, and 0.3%. Triplicate cultures also received ethanol plus LPS (Escherichia coli L-2630; Sigma Chemical Co., St. Louis, Mo.) at a final concentration of 10 mg/ml. As controls, triplicate samples also received LPS or medium alone. Control and treated PBMC were incubated at 378C for 4 to 8 h in a 5% CO2 and 95% air incubator. This was based on our previous studies (30) as well as studies by other investigators (10) which showed that 4- to 8-h incubation of lymphocytes with LPS was necessary to induce maximum TNF message. The viability of leukocytes was examined at the end of the 4- to 8-h incubation by a trypan blue dye exclusion assay. The viability of alcohol-treated cultures was not affected and was found to be similar to that of control cultures (;95% viability). (ii) Solution RT-PCR analysis. The treated and control samples were centrifuged at 900 3 g for 10 min, and total cellular RNA was isolated according to the procedure of Chomczynski and Sacchi (7). First-strand cDNA was synthesized from total RNA derived from treated and untreated cultures. Two micrograms
* Corresponding author. Mailing address: Buffalo General Hospital, 100 High St., Buffalo, NY 14203. Phone: (716) 859-2985. Fax: (716) 859-2999. 392
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FIG. 1. Detection of TNF-a gene expression in human PBMC by conventional in situ hybridization. PBMC were incubated with and without 10 mg of LPS per ml in the presence or absence of alcohol (0.3%, vol/vol). TNF-a message was barely detectable in cells treated with medium alone (A); cells treated with LPS showed moderate levels of TNF-a message (B); and cells treated with alcohol produced no appreciable change in the LPS-induced TNF message (C). This experiment was repeated three times using PBMC from three different donors, with similar results.
of total RNA was reverse transcribed with Moloney murine leukemia virus (MMLV) RT (Bethesda Research Laboratories, Gaithersburg, Md.). The reaction tubes were incubated at 378C for 60 min, further denatured at 958C for 10 min, and quick-chilled on ice, and 2 ml was run on an agarose gel to examine cDNA. One-tenth the volume of cDNA obtained from different RNA samples was amplified in 2 mM MgCl2–13 PCR buffer (Promega)–0.2 mM deoxynucleoside triphosphates (dNTPs)–50 pmol of 59-upstream and 39-downstream specific primer pairs–1 to 2 U of Taq DNA polymerase (Promega). For the semiquantitative PCR, the newly synthesized DNA sample was mixed with the housekeeping glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primer pairs plus the TNF-a primer pairs in the same tube. All PCRs were carried out with a hot-start program (608C for 10 min). The reaction tubes were heat denatured at 958C for 60 s, annealed at 50 to 608C for 60 s, and primer extended at 728C for 90 s. The reaction parameters were programmed in a Programmable Thermal Controller (Thermolyne-Temp-Tronic, Dubuque, Iowa) and repeated for 30 cycles. The final step was set at 728C for 10 min and then 48C indefinitely. One-tenth of the PCR mixtures was electrophoresed on 4% agarose gels (3% Nusieve and 1% SeaKem; FMC Corp.), stained with ethidium bromide, and photographed with Polaroid 557 negative-positive films. Differences in loading were corrected by
comparing equal amounts of G3PDH or b-actin in all the PCRs. For semiquantitation, the photographic negatives were scanned in a densitometer (Molecular Dynamics, Sunnyville, Calif.). The densitometric values were normalized to values for control housekeeping genes. The following primer sequences were used in the RT-PCR experiments: human TNF-a, 59-CAGAGGGAATTCCCCAG-39 (upstream) and 59-CCTTGGTCTGGTAGGAGACG-39 (downstream) (325 bp; GeneAmplimer, Perkin-Elmer Cetus); human G3PDH, 59-TGAAGGTCGGTG TCAACG-39 (upstream) and 59-CATGTAGGCCATGAGGTC-3 (downstream) (983 bp; GeneAmplimer, Perkin-Elmer Cetus); and rat b-actin, 59-TTGTAACC AACTGGGACGATATGG-39 (upstream) and 59-GATCTTGATCTTCATGG TGCTAGG-39 (downstream) (764 bp) (34). The amplified products yielded expected sizes for TNF-a (325 bp), G3PDH (983 bp), and b-actin (764 bp), as previously described (34). (iii) Fixation of cells. Alcohol-treated and -untreated cells were centrifuged at 400 3 g for 30 min at 48C, and the pellet was resuspended, fixed in freshly made 4% paraformaldehyde in 13 phosphate-buffered saline (PBS) for 15 to 20 min at 48C, and then kept in 70% ethanol in diethylpyrocarbonate (DEPC)-H2O at 48C until used. The fixed cells were microcentrifuged for 2 min, and the alcohol was carefully vacuum removed. The cell pellet was thoroughly resuspended in 13 PBS and divided into two sets. Cells from set 1 were cytocentrifuged onto glass slides for conventional in situ hybridization and stored in 70% ethanol at 48C until used. Cells from set 2 were thoroughly resuspended and rehydrated with 5 mM MgCl2 in 13 PBS containing DEPC-H2O. The cell suspension was microcentrifuged at 48C, and the buffer was carefully removed by vacuum. The cell pellet was resuspended with RNase-free proteinase K (10 mg/ml) and incubated at 378C for 15 to 30 min. Before and after addition of each reagent, the cells were thoroughly washed twice with 13 PBS in DEPC-H2O and microcentrifuged, and all traces of buffer were carefully vacuum removed. The treated samples were then placed on ice. (iv) RT-PCR in situ. Proteinase K-treated cells were resuspended in 0.1 M glycine–0.2 M Tris-HCl (pH 7.4) for 10 min at room temperature, and all the cells were washed thoroughly in 13 PBS in DEPC-H2O and centrifuged. The cell pellet was resuspended in DNase buffer containing RNase-free DNase (10 mg/ ml) and incubated for 30 min at 378C. The cell suspension was heat inactivated at 708C for 5 to 10 min and washed twice with 13 PBS. The pellet was resuspended in MMLV RT buffer containing RNasin (2 U/ml), 5 mM MgCl2, 1 mM dNTPs, and 2.5 mM random hexamers. The mixture was reverse transcribed with 200 to 400 U of MMLV RT enzyme and incubated at 378C for 1.5 h. The products were denatured at 908C for 5 to 10 min and quick-chilled on ice. All reactions were carried out in 0.5-ml Eppendorf tubes. The cells were washed twice with 13 PBS made in DEPC-H2O and centrifuged. PBS was carefully
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FIG. 2. Suppression of TNF-a gene expression in human PBMC by alcohol as detected by RT-PCR in situ hybridization. PBMC were incubated with and without 10 mg of LPS per ml in the presence or absence of alcohol (0.3%, vol/vol). (A) Enhancement of TNF-a gene expression compared with Fig. 1A; (B) marked enhancement of TNF-a gene expression with LPS; and (C) significant suppression of LPS-induced TNF-a gene expression by alcohol. The presence of dark silver granules indicates in situ RT-PCR-amplified gene. This experiment was repeated three times using PBMC from three different donors, with similar results.
aspirated and the pellets were resuspended in 100 ml of a PCR mixture consisting of 13 PCR buffer (Promega), 0.2 mM dNTPs, 2 mM MgCl2, 200 to 500 pmol of 59 sense and 39 antisense TNF-a primers, and Taq DNA polymerase (5 U). The reaction tubes were heat denatured at 958C for 5 min in step 1 and 948C for 2 min in step 2, annealed at 558C for 2 min in step 3, and primer extended at 728C for 5 min in step 4. Steps 2 to 4 were repeated for 30 cycles, and further extension was done at 728C for 15 min in step 5. The samples were left at 48C overnight in step 6 (18). An additional 5.0 U of enzyme was added, and the samples were subjected to an additional 30 cycles of amplification (for a total of 60 cycles) to ensure availability of enzyme within the cells and further amplification (18). The tubes containing the cells and PCR mixture were microcentrifuged for 5 min, and the mineral oil and reaction mixture were removed by aspiration. The cell pellet was resuspended in 100 ml of 13 PBS made in DEPC-H2O, mixed thoroughly, and transferred to 1.5-ml Eppendorf tubes, and the cells were then deposited on glass microscope slides (superfrost plus; Fisher) by cytocentrifugation (450 to 500 rpm for 5 min; Shandon Cytocentrifuge). Cellular RNA was further digested with 100 mg of DNase-free RNase A (Boehringer Mannheim, Indianapolis, Ind.) per ml in 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate; pH 7.2) buffer for 1 h at 378C. The cells were then postfixed with 4% paraformaldehyde for 15 to 30 min to improve retention of the amplified products during subsequent denaturation, hybridization, and washing (18). (v) In situ hybridization. Cells on glass slides were denatured in 95% formamide–0.13 SSC for 15 min at 658C. The slides were cooled to 48C in 0.13 SSC, dipped in DEPC-H2O, and dehydrated in 70% ethanol. From this step onwards, conventional in situ hybridization and RT-PCR in situ hybridization slides were treated identically, as described previously for in situ hybridization procedures
(25). Prior to in situ hybridization, cells from set 1 (stored in 70% ethanol at 48C) were rehydrated with 5 mM MgCl2 in PBS and treated with proteinase K (1 mg/ml) and then with 0.1 M glycine–0.2 M Tris-HCl (pH 7.4) as described above for set 2 cells. The TNF-a cDNA probe used for both conventional and RT-PCR in situ hybridization studies was a 1.38-kb HindIII-BamHI fragment from pFC54 (American Type Culture Collection catalog no. 39918). The TNF-a probe was random labeled with [32P]dATP, and unincorporated label was removed by using Sephadex G-50 quick columns (Boehringer Mannheim) and standard methods. The 32P-cDNA probes were heat denatured at 958C for 10 min and quick-chilled prior to in situ hybridization. The cells were washed in 13 PBS in DEPC-H2O and then overlaid with a few drops of prehybridization solution (50% deionized formamide in 23 SSC) for 1.5 h at room temperature in a moist chamber. Each slide was then placed over 50- to 100-ml hybridization mixture containing 32Plabeled cDNA probes (50,000 to 100,000 cpm) and incubated overnight in a moist chamber at room temperature. After hybridization, the cells were immersed in 0.3 M NaCl–20 mM Tris-HCl (pH 7.4)–5 mM EDTA–25% formamide for 2 h at room temperature. They were then washed in 50% formamide in 23 SSC and in 50% formamide in 13 SSC for 30 min each at 378C and in 13 SSC for 30 min at room temperature. The last washes were done with 0.53 SSC at 428C followed by 0.53 SSC at room temperature for 30 min each. Afterwards, the slides were air dried, dipped in NTB-2 emulsion (Kodak, Rochester, N.Y.), and exposed at 48C for 5 to 7 days. The slides were developed in D-19-3 developer (Kodak) for 5 min, in 2% acetic acid for 3 min, and then in rapid fixer for 5 min. Finally, they were rinsed in tap water and air dried. The cells were subsequently stained with filtered (0.2-mm pore size) 0.1% toluene blue and photographed under a microscope (3400).
RESULTS Data presented in Fig. 1 show TNF-a gene expression in PBMC cultured in medium alone or with LPS and LPS plus 0.3% (vol/vol) alcohol as examined by conventional in situ hybridization. TNF-a mRNA was barely detectable in the cells cultured in medium alone (Fig. 1A). Fifty-six percent of cells treated with LPS showed TNF-a message expression, as evidenced by low to moderate deposition of dark silver grains (Fig. 1B). The addition of alcohol to LPS produced a slight decrease in the number of cells (48% of cells) showing the deposition of dark silver grains by conventional in situ hybridization (Fig. 1C). In contrast, RT-PCR in situ showed marked enhancement of TNF-a gene expression and permitted recog-
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FIG. 3. Alcohol suppresses LPS-induced TNF-a gene expression in human PBMC as detected by semiquantitative RT-PCR in solution. RNAs from LPS-treated control PBMC and PBMC treated with LPS plus different concentrations of alcohol were reverse transcribed and amplified (RT-PCR) with rat b-actin and human TNF-a primers and electrophoresed. (A) b-Actin gene expression (764 bp). (B) TNF-a gene expression (325 bp). Lanes A, control; lanes B, LPS-treated control; lanes C and D, cultures treated with LPS plus 0.2 and 0.3% (vol/vol) alcohol, respectively; lanes M, molecular size markers (fX174; Bethesda Research Laboratories). Sizes are indicated in base pairs. Alcohol demonstrated dose-response inhibitory effects on LPS-induced TNF-a gene expression. EtOH, ethanol.
nition of low levels of TNF-a gene expression even in control cells (Fig. 2A). Cultures treated with LPS showed greater enhancement of TNF-a gene expression, as manifested by the deposition of dark silver grains over most of the cells (Fig. 2B). Cells treated with LPS plus alcohol showed significant suppression of LPS-induced TNF-a message (Fig. 2C), as evidenced by the reduction in silver grains in most of the cells compared with the amount in LPS-treated cultures alone (Fig. 2B). To confirm these observations, we further evaluated the expression of the TNF-a gene using a semiquantitative RTPCR method in solution. RNA extracted from cultures treated with alcohol at various concentrations was reverse transcribed
and amplified with specific primers for TNF-a and b-actin or G3PDH. The densitometry readings (optical density units) of the TNF-a bands normalized to the b-actin or G3PDH values were expressed as percent increases or decreases over control values. Figure 3A shows the b-actin gene (764 bp) expression in control cultures (lane A) and cultures treated with LPS (lane B) and LPS plus 0.2% (vol/vol) (lane C) or 0.3% (vol/vol) (lane D) alcohol. LPS or LPS plus alcohol did not affect the b-actinspecific RNA gene expression. Data presented in Fig. 3B showed that cultures treated with LPS alone demonstrated upregulation of the TNF-a message (lane B; 23% increase) compared with the control culture (lane A). Lymphocytes
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FIG. 4. Alcohol suppresses endogenous TNF-a message. RNAs from control and alcohol-treated PBMC were reverse transcribed and amplified with human G3PDH and TNF-a primer pairs and electrophoresed. G3PDH and TNF-a messages amplified at around 983 and 325 bp, respectively. Alcohol at a 0.2% (vol/vol) concentration demonstrated a significant suppression of endogenous TNF-a message (lane C, 57% suppression) compared with that in the control culture (lane A). Results are shown also for a culture treated with 0.1% (vol/vol) alcohol and a positive reagent control (lanes B and D, respectively). Molecular markers were used to determine the sizes (in base pairs) of the resultant fragments (lane M). These results are from a single experiment which was repeated three times using PBMC from three different donors, with similar results.
treated with LPS plus 0.2 and 0.3% (vol/vol) alcohol produced 31% (Fig. 3B, lane C) and 44% (lane D) suppression of TNF-a message compared with the expression in cultures treated with LPS alone (lane B). The suppression was most pronounced at the 0.3% (vol/vol) alcohol concentration (lane D). We further studied the effect of alcohol on endogenous TNF-a message of PBMC cultured in medium alone (without stimulation with LPS). In this system, reverse-transcribed RNA products were amplified simultaneously with both internal control gene primers (G3PDH) and TNF-a primers in the same tube. Data presented in Fig. 4 demonstrate that both amplified human G3PDH gene (983-bp) and TNF-a gene (325-bp) products amplified at the expected regions. Cells cultured with 0.1% alcohol did not produce any significant effect on endogenous TNF-a message (Fig. 4, lane B; 2% inhibition). However, alcohol at a 0.2% concentration significantly suppressed TNF-a message (Fig. 4, lane C; 57% suppression) compared with expression in the control culture (lane A). Lane D in Fig. 4 represents a positive reagent template control (308 bp). Thus, the semiquantitative in vitro RT-PCR analysis confirmed our RT-PCR in situ hybridization which demonstrated that alcohol suppresses LPS-induced TNF-a gene activation. DISCUSSION Previous studies have shown that alcohol consumption is associated with abnormalities of humoral (11, 12, 28) and cellular (5, 21) immunity, including dysfunctions of suppressor (47), helper (26), and cytotoxic (27, 39, 43) lymphocyte activities and production of various soluble immune mediators (39). Rats fed a diet including ethanol also demonstrated a loss of cells from the thymus and lymph nodes and showed reduced proliferative responses to concanavalin A (20), although their ability to produce IL-2 and the numbers of IL-2 receptors per cell were not affected (21). Ethanol also modulated the production of IL-6 and susceptibility to Legionella pneumonia in mice (48). TNF-a is a multipotential cytokine that appears to have an
important role in the pathogenesis of various diseases (45). Although TNF-a is produced mainly by macrophages, this cytokine is also produced by a variety of other cells, including NK cells, T cells, and fibroblasts (40). A recent study demonstrated the expression of TNF-a mRNA in hepatocytes and Kupffer cells from healthy individuals and patients with alcoholic liver diseases as measured by in situ hybridization (16). Previous studies demonstrated that acute ethanol intoxication in rats markedly suppressed levels of TNF in serum and lungs elicited in response to LPS and also suppressed the recruitment of neutrophils to the alveoli of rats (33). Recently, it was shown that acute alcohol administration markedly reduced S. aureus- or K. pneumoniae-induced TNF activity in lung lavage fluids of rats (32). D’Souza et al. (13) showed that acute ethanol administration decreased circulating serum TNF levels in rats. Administration of recombinant TNF enhanced the bactericidal capacity of the lungs against S. aureus (3), suggesting that TNF plays an important role in protecting the host against infection. Acute alcohol intoxication of rats also decreased TNF-a receptors on neutrophils (8). In a murine model, dietary alcohol did not significantly affect in vitro TNF-a production, while splenocytes from mice fed either ethanol or a control diet manifested significantly lower levels of mitogeninduced TNF production when cultured with ethanol (6). Acute ethanol treatment of peripheral blood monocytes in vitro resulted in the suppression of TNF activity (46). A recent study also showed decreased TNF-a production by activated monocytes of trauma patients during very early postinjury periods, while elevated levels of TNF-a were observed during the late phase of the injury (44). Furthermore, Staphylococcus enterotoxin B- and LPS-induced TNF-a mRNA also was inhibited by acute treatment of normal monocytes with ethanol in vitro (42). We recently reported that when added directly to normal whole blood or separated total mononuclear cells, alcohol at intoxicating levels in vivo significantly suppressed LPS-induced TNF production in vitro as assessed by bioassay using the TNF-sensitive WEHI 164 subclone 13 cell line (30).
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The results presented here show significant inhibition of LPS-induced TNF-a message expression by alcohol as demonstrated by semiquantitative RT-PCR of isolated RNA and RTPCR in situ hybridization using whole cells. This study confirms our earlier finding (30) of the effects of alcohol on the reduction of TNF production as measured by bioassay. The results demonstrate that RT-PCR in situ markedly improves the levels of detection of TNF-a gene expression compared with those of the conventional in situ hybridization technique. Further studies comparing amplification using 30 and 60 cycles of PCR demonstrated similar results for the untreated control cultures, suggesting that 60 cycles of PCR do not cause amplification of nonspecific messages (data not presented). These studies confirm an earlier study (18) which showed that two rounds of amplification (60 cycles total) by the addition of another 5 U of Taq DNA polymerase may be necessary to ensure the availability of enzymes within the cells. The exact mechanisms of ethanol-induced suppression of LPS-activated TNF-a gene expression are not clearly understood. It is possible that ethanol may interfere with the number, affinity, or avidity of TNF receptors on the cells (14), may block the LPS-binding protein that is required for the maximum expression of TNF message, or may induce the production of toxic oxygen metabolites (42). These possibilities are being investigated. The results of these studies are consistent with other in vivo findings (13, 32, 33) and present direct evidence that one of the mechanisms of alcohol-mediated immunosuppression may be a decrease in the production of TNF. Our findings may explain, in part, the increased susceptibility to infections observed in alcoholic patients. ACKNOWLEDGMENTS We express our sincere appreciation to Carol Sperry and Gerry Sobkowiak for their excellent secretarial assistance. This work was supported in part by NIH grants 1 RO1 MH 47225, 1 P50 AA07378, and RO1 CA 35922 and by the Margaret Duffy and Robert Cameron Troup Memorial Fund for Cancer Research of the Buffalo General Hospital and the Buffalo General Foundation. REFERENCES 1. Beutler, B., and A. Cerami. 1986. Cachectin and tumor necrosis factors as two sides of the same biological coin. Nature (London) 320:584–588. 2. Bevilacqua, M. P., J. S. Pober, G. R. Majeau, R. S. Cotran, and M. A. Gimbrone, Jr. 1984. Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells. J. Exp. Med. 160:618–623. 3. Blanchard, D. K., J. Y. Djeu, T. W. Klein, H. Friedman, and W. E. Stewart II. 1988. Effect of acute alcohol intoxication on granulocyte mobilization and kinetics. J. Leukocyte Biol. 43:429–435. 4. Boyum, A. 1990. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21:77–89. 5. Chang, M. P., D. C. Norman, and T. Makinodan. 1990. Immunotoxicity of alcohol activities of and B cells of aging mice. Alcohol. Clin. Exp. Res. 14: 210–215. 6. Chen, G. J., D. S. Huang, B. Watzl, and R. R. Watson. 1993. Ethanol modulation of tumor necrosis factor and gamma interferon production by murine splenocytes and macrophages. Life Sci. 52:1319–1326. 7. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159. 8. Deaciue, I. V., N. B. D’Souza, G. J. Bagby, C. H. Lang, and J. J. Spitzer. 1992. Effect of acute alcohol administration on TNF-alpha binding to neutrophils and isolated liver plasma membranes. Alcohol. Clin. Exp. Res. 16:533–538. 9. Decker, T., M. I. Lohmann-Matthes, and G. E. Gifford. 1987. Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages. J. Immunol. 138:957–962. 10. DeForge, L. E., and D. G. Remick. 1991. Kinetics of TNF, IL-6, and IL-8 gene expression in LPS-stimulated human whole blood. Biochem. Biophys. Res. Commun. 174:18–24. 11. Delacroix, D. L., K. B. Elkon, A. R. Genbel, H. F. Hedgson, C. Dive, and J. F. Verman. 1982. Changes in size, subclass and metabolic properties of serum immunoglobulin A in liver diseases and in other diseases with high serum
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