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. 1996 Jul;3(4):392–398. doi: 10.1128/cdli.3.4.392-398.1996

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 Nair 1, N M Kumar 1, Z A Kronfol 1, J F Greden 1, J S Lwebuga-Mukasa 1, S A Schwartz 1
PMCID: PMC170356  PMID: 8807202

Abstract

We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) 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-alpha 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 micrograms/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-alpha 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-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha 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.

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Selected References

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  1. Beutler B., Cerami A. Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature. 1986 Apr 17;320(6063):584–588. doi: 10.1038/320584a0. [DOI] [PubMed] [Google Scholar]
  2. Bevilacqua M. P., Pober J. S., Majeau G. R., Cotran R. S., Gimbrone M. A., Jr Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells. J Exp Med. 1984 Aug 1;160(2):618–623. doi: 10.1084/jem.160.2.618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blanchard D. K., Djeu J. Y., Klein T. W., Friedman H., Stewart W. E., 2nd Protective effects of tumor necrosis factor in experimental Legionella pneumophila infections of mice via activation of PMN function. J Leukoc Biol. 1988 May;43(5):429–435. doi: 10.1002/jlb.43.5.429. [DOI] [PubMed] [Google Scholar]
  4. Chang M. P., Norman D. C., Makinodan T. Immunotoxicity of alcohol in young and old mice. I. In vitro suppressive effects of ethanol on the activities of T and B immune cells of aging mice. Alcohol Clin Exp Res. 1990 Apr;14(2):210–215. doi: 10.1111/j.1530-0277.1990.tb00474.x. [DOI] [PubMed] [Google Scholar]
  5. Chen G. J., Huang D. S., Watzl B., Watson R. R. Ethanol modulation of tumor necrosis factor and gamma interferon production by murine splenocytes and macrophages. Life Sci. 1993;52(15):1319–1326. [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. D'Souza N. B., Bagby G. J., Nelson S., Lang C. H., Spitzer J. J. Acute alcohol infusion suppresses endotoxin-induced serum tumor necrosis factor. Alcohol Clin Exp Res. 1989 Apr;13(2):295–298. doi: 10.1111/j.1530-0277.1989.tb00329.x. [DOI] [PubMed] [Google Scholar]
  8. D'Souza N. B., Nelson S., Summer W. R., Deaciuc I. V. Expression of tumor necrosis factor-alpha and interleukin-6 cell-surface receptors of the alveolar macrophage in alcohol-treated rats. Alcohol Clin Exp Res. 1994 Dec;18(6):1430–1435. doi: 10.1111/j.1530-0277.1994.tb01446.x. [DOI] [PubMed] [Google Scholar]
  9. DeForge L. E., Remick D. G. Kinetics of TNF, IL-6, and IL-8 gene expression in LPS-stimulated human whole blood. Biochem Biophys Res Commun. 1991 Jan 15;174(1):18–24. doi: 10.1016/0006-291x(91)90478-p. [DOI] [PubMed] [Google Scholar]
  10. Deaciuc I. V., D'Souza N. B., Bagby G. J., Lang C. H., Spitzer J. J. Effect of acute alcohol administration on TNF-alpha binding to neutrophils and isolated liver plasma membranes. Alcohol Clin Exp Res. 1992 Jun;16(3):533–538. doi: 10.1111/j.1530-0277.1992.tb01412.x. [DOI] [PubMed] [Google Scholar]
  11. Decker T., Lohmann-Matthes M. L., Gifford G. E. Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages. J Immunol. 1987 Feb 1;138(3):957–962. [PubMed] [Google Scholar]
  12. Delacroix D. L., Elkom K. B., Geubel A. P., Hodgson H. F., Dive C., Vaerman J. P. Changes in size, subclass, and metabolic properties of serum immunoglobulin A in liver diseases and in other diseases with high serum immunoglobulin A. J Clin Invest. 1983 Feb;71(2):358–367. doi: 10.1172/JCI110777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drew P. A., Clifton P. M., LaBrooy J. T., Shearman D. J. Polyclonal B cell activation in alcoholic patients with no evidence of liver dysfunction. Clin Exp Immunol. 1984 Aug;57(2):479–486. [PMC free article] [PubMed] [Google Scholar]
  14. Gemlo B. T., Palladino M. A., Jr, Jaffe H. S., Espevik T. P., Rayner A. A. Circulating cytokines in patients with metastatic cancer treated with recombinant interleukin 2 and lymphokine-activated killer cells. Cancer Res. 1988 Oct 15;48(20):5864–5867. [PubMed] [Google Scholar]
  15. González-Amaro R., García-Monzón C., García-Buey L., Moreno-Otero R., Alonso J. L., Yagüe E., Pivel J. P., López-Cabrera M., Fernández-Ruiz E., Sánchez-Madrid F. Induction of tumor necrosis factor alpha production by human hepatocytes in chronic viral hepatitis. J Exp Med. 1994 Mar 1;179(3):841–848. doi: 10.1084/jem.179.3.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grunfeld C., Palladino M. A., Jr Tumor necrosis factor: immunologic, antitumor, metabolic, and cardiovascular activities. Adv Intern Med. 1990;35:45–71. [PubMed] [Google Scholar]
  17. Haase A. T., Retzel E. F., Staskus K. A. Amplification and detection of lentiviral DNA inside cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4971–4975. doi: 10.1073/pnas.87.13.4971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jelinek D. F., Lipsky P. E. Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1. J Immunol. 1987 Nov 1;139(9):2970–2976. [PubMed] [Google Scholar]
  19. Jerrells T. R., Peritt D., Marietta C., Eckardt M. J. Mechanisms of suppression of cellular immunity induced by ethanol. Alcohol Clin Exp Res. 1989 Aug;13(4):490–493. doi: 10.1111/j.1530-0277.1989.tb00363.x. [DOI] [PubMed] [Google Scholar]
  20. Jerrells T. R., Smith W., Eckardt M. J. Murine model of ethanol-induced immunosuppression. Alcohol Clin Exp Res. 1990 Aug;14(4):546–550. doi: 10.1111/j.1530-0277.1990.tb01197.x. [DOI] [PubMed] [Google Scholar]
  21. Kashiwa H., Wright S. C., Bonavida B. Regulation of B cell maturation and differentiation. I. Suppression of pokeweed mitogen-induced B cell differentiation by tumor necrosis factor (TNF). J Immunol. 1987 Mar 1;138(5):1383–1390. [PubMed] [Google Scholar]
  22. Kehrl J. H., Miller A., Fauci A. S. Effect of tumor necrosis factor alpha on mitogen-activated human B cells. J Exp Med. 1987 Sep 1;166(3):786–791. doi: 10.1084/jem.166.3.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kelso A., Gough N. M. Coexpression of granulocyte-macrophage colony-stimulating factor, gamma interferon, and interleukins 3 and 4 is random in murine alloreactive T-lymphocyte clones. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9189–9193. doi: 10.1073/pnas.85.23.9189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Meadows G. G., Blank S. E., Duncan D. D. Influence of ethanol consumption on natural killer cell activity in mice. Alcohol Clin Exp Res. 1989 Aug;13(4):476–479. doi: 10.1111/j.1530-0277.1989.tb00359.x. [DOI] [PubMed] [Google Scholar]
  25. Morgan M. Y., Ross M. G., Ng C. M., Adams D. M., Thomas H. C., Sherlock S. HLA-B8, immunoglobulins, and antibody responses in alcohol-related liver disease. J Clin Pathol. 1980 May;33(5):488–492. doi: 10.1136/jcp.33.5.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nair M. P., Schwartz S. A., Kronfol Z. A., Hill E. M., Sweet A. M., Greden J. F. Suppression of tumor necrosis factor production by alcohol in lipopolysaccharide-stimulated culture. Alcohol Clin Exp Res. 1994 Jun;18(3):602–607. doi: 10.1111/j.1530-0277.1994.tb00917.x. [DOI] [PubMed] [Google Scholar]
  27. Nawroth P. P., Bank I., Handley D., Cassimeris J., Chess L., Stern D. Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1. J Exp Med. 1986 Jun 1;163(6):1363–1375. doi: 10.1084/jem.163.6.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nelson S., Bagby G. J., Summer W. R. Alcohol-induced suppression of tumor necrosis factor--a potential risk factor for secondary infection in the acquired immunodeficiency syndrome. Prog Clin Biol Res. 1990;325:211–220. [PubMed] [Google Scholar]
  29. Nelson S., Bagby G., Summer W. R. Alcohol suppresses lipopolysaccharide-induced tumor necrosis factor activity in serum and lung. Life Sci. 1989;44(10):673–676. doi: 10.1016/0024-3205(89)90472-4. [DOI] [PubMed] [Google Scholar]
  30. Nudel U., Zakut R., Shani M., Neuman S., Levy Z., Yaffe D. The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res. 1983 Mar 25;11(6):1759–1771. doi: 10.1093/nar/11.6.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ortaldo J. R., Mason L. H., Mathieson B. J., Liang S. M., Flick D. A., Herberman R. B. Mediation of mouse natural cytotoxic activity by tumour necrosis factor. Nature. 1986 Jun 12;321(6071):700–702. doi: 10.1038/321700a0. [DOI] [PubMed] [Google Scholar]
  32. Oster W., Lindemann A., Horn S., Mertelsmann R., Herrmann F. Tumor necrosis factor (TNF)-alpha but not TNF-beta induces secretion of colony stimulating factor for macrophages (CSF-1) by human monocytes. Blood. 1987 Nov;70(5):1700–1703. [PubMed] [Google Scholar]
  33. Owen-Schaub L. B., Gutterman J. U., Grimm E. A. Synergy of tumor necrosis factor and interleukin 2 in the activation of human cytotoxic lymphocytes: effect of tumor necrosis factor alpha and interleukin 2 in the generation of human lymphokine-activated killer cell cytotoxicity. Cancer Res. 1988 Feb 15;48(4):788–792. [PubMed] [Google Scholar]
  34. Pober J. S., Gimbrone M. A., Jr, Lapierre L. A., Mendrick D. L., Fiers W., Rothlein R., Springer T. A. Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon. J Immunol. 1986 Sep 15;137(6):1893–1896. [PubMed] [Google Scholar]
  35. Roselle G. A., Mendenhall C. L., Grossman C. J. Ethanol and soluble mediators of host response. Alcohol Clin Exp Res. 1989 Aug;13(4):494–498. doi: 10.1111/j.1530-0277.1989.tb00364.x. [DOI] [PubMed] [Google Scholar]
  36. Rubin B. Y., Anderson S. L., Sullivan S. A., Williamson B. D., Carswell E. A., Old L. J. Nonhematopoietic cells selected for resistance to tumor necrosis factor produce tumor necrosis factor. J Exp Med. 1986 Oct 1;164(4):1350–1355. doi: 10.1084/jem.164.4.1350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shalaby M. R., Aggarwal B. B., Rinderknecht E., Svedersky L. P., Finkle B. S., Palladino M. A., Jr Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors. J Immunol. 1985 Sep;135(3):2069–2073. [PubMed] [Google Scholar]
  38. Spitzer J. J., Bautista A. P. Alcohol, cytokines and immunodeficiency. Adv Exp Med Biol. 1993;335:159–164. doi: 10.1007/978-1-4615-2980-4_22. [DOI] [PubMed] [Google Scholar]
  39. Stacey N. H. Inhibition of antibody-dependent cell-mediated cytotoxicity by ethanol. Immunopharmacology. 1984 Dec;8(3-4):155–161. doi: 10.1016/0162-3109(84)90020-1. [DOI] [PubMed] [Google Scholar]
  40. Szabo G., Mandrekar P., Verma B., Isaac A., Catalano D. Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor alpha production late postinjury. J Clin Immunol. 1994 Nov;14(6):340–352. doi: 10.1007/BF01546318. [DOI] [PubMed] [Google Scholar]
  41. Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol. 1992;10:411–452. doi: 10.1146/annurev.iy.10.040192.002211. [DOI] [PubMed] [Google Scholar]
  42. Verma B. K., Fogarasi M., Szabo G. Down-regulation of tumor necrosis factor alpha activity by acute ethanol treatment in human peripheral blood monocytes. J Clin Immunol. 1993 Jan;13(1):8–22. doi: 10.1007/BF00920631. [DOI] [PubMed] [Google Scholar]
  43. Watson R. R., Eskelson C., Hartmann B. R. Severe alcohol abuse and cellular immune functions. Ariz Med. 1984 Oct;41(10):665–668. [PubMed] [Google Scholar]
  44. Yamamoto Y., Klein T. W., Friedman H. Ethanol affects macrophage production of IL-6 and susceptibility to infection by Legionella pneumophila. Adv Exp Med Biol. 1993;335:169–173. doi: 10.1007/978-1-4615-2980-4_24. [DOI] [PubMed] [Google Scholar]

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