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. 1992 Jun 1;89(11):4864–4868. doi: 10.1073/pnas.89.11.4864

Constitutive synthesis of tumor necrosis factor in the thymus.

B P Giroir 1, T Brown 1, B Beutler 1
PMCID: PMC49188  PMID: 1594585

Abstract

Although tumor necrosis factor (TNF) is a major mediator of endotoxic shock, the normal function of TNF that has preserved this protein throughout mammalian evolution remains unknown. If the protein serves a role in normal development or homeostasis, it must be produced under physiologic conditions. To determine whether TNF secretion occurs in normal animals, and to define the tissue sources of the protein, we prepared a reporter construct in which the TNF coding sequence and introns are replaced by the chloramphenicol acetyltransferase (CAT) coding sequence. This construct was inserted into the murine genome, yielding 13 transgenic founders. Macrophages harvested from 4 of the transgenic lines expressed CAT activity after stimulation with Escherichia coli lipopolysaccharide in vitro. Each of these 4 transgenic lines also constitutively expressed CAT activity in the thymus but in no other tissue examined. Cultured thymocytes secrete TNF, as demonstrated both by cytotoxicity assays and by immunoprecipitation of radiolabeled thymic culture medium. CAT activity was associated with the thymic lymphocyte population and not with thymic macrophages or dendritic cells. CAT activity was present in thymic lymphocytes irrespective of CD4 or CD8 expression; T cells from the spleen, however, had no detectable CAT activity. The biosynthesis of TNF in the thymus of normal animals implies a role for this protein in the development or regulation of the immune response.

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Selected References

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  1. BILLINGHAM R. E., BRENT L. A simple method for inducing tolerance of skin homografts in mice. Transplant Bull. 1957 Apr;4(2):67–71. [PubMed] [Google Scholar]
  2. Beller D. I., Unanue E. R. Thymic maturation in vitro by a secretory product from macrophages. J Immunol. 1977 May;118(5):1780–1787. [PubMed] [Google Scholar]
  3. Beutler B. A., Milsark I. W., Cerami A. Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. J Immunol. 1985 Dec;135(6):3972–3977. [PubMed] [Google Scholar]
  4. Beutler B., Brown T. A CAT reporter construct allows ultrasensitive estimation of TNF synthesis, and suggests that the TNF gene has been silenced in non-macrophage cell lines. J Clin Invest. 1991 Apr;87(4):1336–1344. doi: 10.1172/JCI115137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beutler B., Milsark I. W., Cerami A. C. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science. 1985 Aug 30;229(4716):869–871. doi: 10.1126/science.3895437. [DOI] [PubMed] [Google Scholar]
  6. Boehmer H., Sprent J., Nabholz M. Tolerance to histocompatibility determinants in tetraparental bone marrow chimeras. J Exp Med. 1975 Feb 1;141(2):322–334. doi: 10.1084/jem.141.2.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brent L., Brooks C., Lubling N., Thomas A. V. Attempts to demonstrate an in vivo role for serum blocking factors in tolerant mice. Transplantation. 1972 Sep;14(3):382–387. doi: 10.1097/00007890-197209000-00016. [DOI] [PubMed] [Google Scholar]
  8. Cerami A., Ikeda Y., Le Trang N., Hotez P. J., Beutler B. Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: the role of cachectin (tumor necrosis factor). Immunol Lett. 1985;11(3-4):173–177. doi: 10.1016/0165-2478(85)90165-8. [DOI] [PubMed] [Google Scholar]
  9. Chung I. Y., Benveniste E. N. Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta. J Immunol. 1990 Apr 15;144(8):2999–3007. [PubMed] [Google Scholar]
  10. Cseh K., Beutler B. Alternative cleavage of the cachectin/tumor necrosis factor propeptide results in a larger, inactive form of secreted protein. J Biol Chem. 1989 Sep 25;264(27):16256–16260. [PubMed] [Google Scholar]
  11. Cuturi M. C., Murphy M., Costa-Giomi M. P., Weinmann R., Perussia B., Trinchieri G. Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes. J Exp Med. 1987 Jun 1;165(6):1581–1594. doi: 10.1084/jem.165.6.1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dayer J. M., Beutler B., Cerami A. Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J Exp Med. 1985 Dec 1;162(6):2163–2168. doi: 10.1084/jem.162.6.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Engelmann H., Holtmann H., Brakebusch C., Avni Y. S., Sarov I., Nophar Y., Hadas E., Leitner O., Wallach D. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity. J Biol Chem. 1990 Aug 25;265(24):14497–14504. [PubMed] [Google Scholar]
  14. Goeddel D. V., Aggarwal B. B., Gray P. W., Leung D. W., Nedwin G. E., Palladino M. A., Patton J. S., Pennica D., Shepard H. M., Sugarman B. J. Tumor necrosis factors: gene structure and biological activities. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):597–609. doi: 10.1101/sqb.1986.051.01.072. [DOI] [PubMed] [Google Scholar]
  15. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grau G. E., Fajardo L. F., Piguet P. F., Allet B., Lambert P. H., Vassalli P. Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science. 1987 Sep 4;237(4819):1210–1212. doi: 10.1126/science.3306918. [DOI] [PubMed] [Google Scholar]
  17. Han J., Brown T., Beutler B. Endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level. J Exp Med. 1990 Feb 1;171(2):465–475. doi: 10.1084/jem.171.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacob C. O., McDevitt H. O. Tumour necrosis factor-alpha in murine autoimmune 'lupus' nephritis. Nature. 1988 Jan 28;331(6154):356–358. doi: 10.1038/331356a0. [DOI] [PubMed] [Google Scholar]
  19. Jue D. M., Sherry B., Luedke C., Manogue K. R., Cerami A. Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages. Biochemistry. 1990 Sep 11;29(36):8371–8377. doi: 10.1021/bi00488a025. [DOI] [PubMed] [Google Scholar]
  20. Kawakami M., Cerami A. Studies of endotoxin-induced decrease in lipoprotein lipase activity. J Exp Med. 1981 Sep 1;154(3):631–639. doi: 10.1084/jem.154.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keshav S., Lawson L., Chung L. P., Stein M., Perry V. H., Gordon S. Tumor necrosis factor mRNA localized to Paneth cells of normal murine intestinal epithelium by in situ hybridization. J Exp Med. 1990 Jan 1;171(1):327–332. doi: 10.1084/jem.171.1.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kinkhabwala M., Sehajpal P., Skolnik E., Smith D., Sharma V. K., Vlassara H., Cerami A., Suthanthiran M. A novel addition to the T cell repertory. Cell surface expression of tumor necrosis factor/cachectin by activated normal human T cells. J Exp Med. 1990 Mar 1;171(3):941–946. doi: 10.1084/jem.171.3.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kojima A., Tanaka-Kojima Y., Sakakura T., Nishizuka Y. Spontaneous development of autoimmune thyroiditis in neonatally thymectomized mice. Lab Invest. 1976 Jun;34(6):550–557. [PubMed] [Google Scholar]
  24. Kruys V., Kemmer K., Shakhov A., Jongeneel V., Beutler B. Constitutive activity of the tumor necrosis factor promoter is canceled by the 3' untranslated region in nonmacrophage cell lines; a trans-dominant factor overcomes this suppressive effect. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):673–677. doi: 10.1073/pnas.89.2.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. MILLER J. F. Immunological function of the thymus. Lancet. 1961 Sep 30;2(7205):748–749. doi: 10.1016/s0140-6736(61)90693-6. [DOI] [PubMed] [Google Scholar]
  26. Nossal G. J., Pike B. L. Functional clonal deletion in immunological tolerance to major histocompatibility complex antigens. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3844–3847. doi: 10.1073/pnas.78.6.3844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Oliff A., Defeo-Jones D., Boyer M., Martinez D., Kiefer D., Vuocolo G., Wolfe A., Socher S. H. Tumors secreting human TNF/cachectin induce cachexia in mice. Cell. 1987 Aug 14;50(4):555–563. doi: 10.1016/0092-8674(87)90028-6. [DOI] [PubMed] [Google Scholar]
  28. Pauli U., Beutler B., Peterhans E. Porcine tumor necrosis factor alpha: cloning with the polymerase chain reaction and determination of the nucleotide sequence. Gene. 1989 Sep 1;81(1):185–191. doi: 10.1016/0378-1119(89)90350-8. [DOI] [PubMed] [Google Scholar]
  29. Peppel K., Crawford D., Beutler B. A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity. J Exp Med. 1991 Dec 1;174(6):1483–1489. doi: 10.1084/jem.174.6.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Piguet P. F., Grau G. E., Allet B., Vassalli P. Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs.-host disease. J Exp Med. 1987 Nov 1;166(5):1280–1289. doi: 10.1084/jem.166.5.1280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ranges G. E., Figari I. S., Espevik T., Palladino M. A., Jr Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha. J Exp Med. 1987 Oct 1;166(4):991–998. doi: 10.1084/jem.166.4.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rothenberg E., Lugo J. P. Differentiation and cell division in the mammalian thymus. Dev Biol. 1985 Nov;112(1):1–17. doi: 10.1016/0012-1606(85)90114-9. [DOI] [PubMed] [Google Scholar]
  33. Salgame P., Abrams J. S., Clayberger C., Goldstein H., Convit J., Modlin R. L., Bloom B. R. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science. 1991 Oct 11;254(5029):279–282. doi: 10.1126/science.254.5029.279. [DOI] [PubMed] [Google Scholar]
  34. Schmid D. S., Tite J. P., Ruddle N. H. DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1881–1885. doi: 10.1073/pnas.83.6.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shalaby M. R., Espevik T., Rice G. C., Ammann A. J., Figari I. S., Ranges G. E., Palladino M. A., Jr The involvement of human tumor necrosis factors-alpha and -beta in the mixed lymphocyte reaction. J Immunol. 1988 Jul 15;141(2):499–503. [PubMed] [Google Scholar]
  36. Sherry B. A., Gelin J., Fong Y., Marano M., Wei H., Cerami A., Lowry S. F., Lundholm K. G., Moldawer L. L. Anticachectin/tumor necrosis factor-alpha antibodies attenuate development of cachexia in tumor models. FASEB J. 1989 Jun;3(8):1956–1962. doi: 10.1096/fasebj.3.8.2721856. [DOI] [PubMed] [Google Scholar]
  37. Sung S. S., Jung L. K., Walters J. A., Chen W., Wang C. Y., Fu S. M. Production of tumor necrosis factor/cachectin by human B cell lines and tonsillar B cells. J Exp Med. 1988 Nov 1;168(5):1539–1551. doi: 10.1084/jem.168.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thivolet J., Monier J. C., Ruel J. P., Richard M. H. Antinuclear autoantibodies in Swiss mice thymectomized at birth. Nature. 1967 Jun 10;214(5093):1134–1136. doi: 10.1038/2141134a0. [DOI] [PubMed] [Google Scholar]
  39. Tovey M. G., Content J., Gresser I., Gugenheim J., Blanchard B., Guymarho J., Poupart P., Gigou M., Shaw A., Fiers W. Genes for IFN-beta-2 (IL-6), tumor necrosis factor, and IL-1 are expressed at high levels in the organs of normal individuals. J Immunol. 1988 Nov 1;141(9):3106–3110. [PubMed] [Google Scholar]
  40. Tracey K. J., Beutler B., Lowry S. F., Merryweather J., Wolpe S., Milsark I. W., Hariri R. J., Fahey T. J., 3rd, Zentella A., Albert J. D. Shock and tissue injury induced by recombinant human cachectin. Science. 1986 Oct 24;234(4775):470–474. doi: 10.1126/science.3764421. [DOI] [PubMed] [Google Scholar]
  41. Ulich T. R., Guo K., del Castillo J. Endotoxin-induced cytokine gene expression in vivo. I. Expression of tumor necrosis factor mRNA in visceral organs under physiologic conditions and during endotoxemia. Am J Pathol. 1989 Jan;134(1):11–14. [PMC free article] [PubMed] [Google Scholar]

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