Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1988 Dec 1;168(6):2309–2318. doi: 10.1084/jem.168.6.2309

Specific inhibition of lymphokine biosynthesis and autocrine growth using antisense oligonucleotides in Th1 and Th2 helper T cell clones

PMCID: PMC2189147  PMID: 2974066

Abstract

T helper cells have recently been divided into two subsets. The Th1 subset secretes and responds to IL-2 in an autocrine manner. The Th2 subset upon mitogen or antigen stimulation releases IL-4. Here we describe a novel technology that allowed us to confirm this distinction. We have used synthetic oligonucleotides complementary to the 5' end of mouse IL-2 and IL-4 to specifically block the biosynthesis of IL-2 or IL-4 in two murine helper T cell clones from the Th1 or Th2 subset. We show that the antisense IL-2 oligonucleotide inhibited the proliferation of the Th1 clone and had no effect on the Th2 clone. In parallel experiments, the antisense IL-4 oligonucleotide blocked the proliferation of the Th2 clone and not the proliferation of the Th1 clone. The inhibition was significantly reversed in both cases by the addition of the relevant lymphokine (IL-2 in the case of the Th1 clone, IL-4 in the case of the Th2 clone). Northern analysis, using cDNA probes specific for the two lymphokines, showed a decrease in the steady-state level of the relevant lymphokine mRNA, suggesting the specific degradation of the mRNA by an RNase H-like enzymatic activity. This strategy, which allows the specific blockade of the biosynthesis of a lymphokine, could be useful for future studies on the role of each T helper subset in physiological immune responses.

Full Text

The Full Text of this article is available as a PDF (623.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baserga S. J., Benz E. J., Jr Nonsense mutations in the human beta-globin gene affect mRNA metabolism. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2056–2060. doi: 10.1073/pnas.85.7.2056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blake K. R., Murakami A., Miller P. S. Inhibition of rabbit globin mRNA translation by sequence-specific oligodeoxyribonucleotides. Biochemistry. 1985 Oct 22;24(22):6132–6138. doi: 10.1021/bi00343a015. [DOI] [PubMed] [Google Scholar]
  3. Büsen W., Peters J. H., Hausen P. Ribonuclease H levels during the response of bovine lymphocytes to concanavalin A. Eur J Biochem. 1977 Mar 15;74(1):203–208. doi: 10.1111/j.1432-1033.1977.tb11382.x. [DOI] [PubMed] [Google Scholar]
  4. Cazenave C., Loreau N., Thuong N. T., Toulmé J. J., Hélène C. Enzymatic amplification of translation inhibition of rabbit beta-globin mRNA mediated by anti-messenger oligodeoxynucleotides covalently linked to intercalating agents. Nucleic Acids Res. 1987 Jun 25;15(12):4717–4736. doi: 10.1093/nar/15.12.4717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Benedetti A., Pytel B. A., Baglioni C. Loss of (2'-5')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections. Proc Natl Acad Sci U S A. 1987 Feb;84(3):658–662. doi: 10.1073/pnas.84.3.658. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  6. Eckhardt H., Lührmann R. Blocking of the initiation of protein biosynthesis by a pentanucleotide complementary to the 3' end of Escherichia coli 16 S rRNA. J Biol Chem. 1979 Nov 25;254(22):11185–11188. [PubMed] [Google Scholar]
  7. Fernandez-Botran R., Sanders V. M., Oliver K. G., Chen Y. W., Krammer P. H., Uhr J. W., Vitetta E. S. Interleukin 4 mediates autocrine growth of helper T cells after antigenic stimulation. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9689–9693. doi: 10.1073/pnas.83.24.9689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Green P. J., Pines O., Inouye M. The role of antisense RNA in gene regulation. Annu Rev Biochem. 1986;55:569–597. doi: 10.1146/annurev.bi.55.070186.003033. [DOI] [PubMed] [Google Scholar]
  9. Harel-Bellan A., Ferris D. K., Vinocour M., Holt J. T., Farrar W. L. Specific inhibition of c-myc protein biosynthesis using an antisense synthetic deoxy-oligonucleotide in human T lymphocytes. J Immunol. 1988 Apr 1;140(7):2431–2435. [PubMed] [Google Scholar]
  10. Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
  11. Holt J. T., Gopal T. V., Moulton A. D., Nienhuis A. W. Inducible production of c-fos antisense RNA inhibits 3T3 cell proliferation. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4794–4798. doi: 10.1073/pnas.83.13.4794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holt J. T., Redner R. L., Nienhuis A. W. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol Cell Biol. 1988 Feb;8(2):963–973. doi: 10.1128/mcb.8.2.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Inoue H., Hayase Y., Iwai S., Ohtsuka E. Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 1987 May 11;215(2):327–330. doi: 10.1016/0014-5793(87)80171-0. [DOI] [PubMed] [Google Scholar]
  14. Izant J. G., Weintraub H. Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science. 1985 Jul 26;229(4711):345–352. doi: 10.1126/science.2990048. [DOI] [PubMed] [Google Scholar]
  15. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  16. Jayaraman K., McParland K., Miller P., Ts'o P. O. Selective inhibition of Escherichia coli protein synthesis and growth by nonionic oligonucleotides complementary to the 3' end of 16S rRNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1537–1541. doi: 10.1073/pnas.78.3.1537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kawasaki E. S. Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes. Nucleic Acids Res. 1985 Jul 11;13(13):4991–5004. doi: 10.1093/nar/13.13.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaye J., Gillis S., Mizel S. B., Shevach E. M., Malek T. R., Dinarello C. A., Lachman L. B., Janeway C. A., Jr Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: interleukin 1 is required for the expression of receptors for interleukin 2. J Immunol. 1984 Sep;133(3):1339–1345. [PubMed] [Google Scholar]
  19. Knecht D. A., Loomis W. F. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. doi: 10.1126/science.3576221. [DOI] [PubMed] [Google Scholar]
  20. Kurt-Jones E. A., Hamberg S., Ohara J., Paul W. E., Abbas A. K. Heterogeneity of helper/inducer T lymphocytes. I. Lymphokine production and lymphokine responsiveness. J Exp Med. 1987 Dec 1;166(6):1774–1787. doi: 10.1084/jem.166.6.1774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lemaitre M., Bayard B., Lebleu B. Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site. Proc Natl Acad Sci U S A. 1987 Feb;84(3):648–652. doi: 10.1073/pnas.84.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lichtman A. H., Kurt-Jones E. A., Abbas A. K. B-cell stimulatory factor 1 and not interleukin 2 is the autocrine growth factor for some helper T lymphocytes. Proc Natl Acad Sci U S A. 1987 Feb;84(3):824–827. doi: 10.1073/pnas.84.3.824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mosmann T. R., Cherwinski H., Bond M. W., Giedlin M. A., Coffman R. L. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986 Apr 1;136(7):2348–2357. [PubMed] [Google Scholar]
  24. Smith C. C., Aurelian L., Reddy M. P., Miller P. S., Ts'o P. O. Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5. Proc Natl Acad Sci U S A. 1986 May;83(9):2787–2791. doi: 10.1073/pnas.83.9.2787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stephenson M. L., Zamecnik P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):285–288. doi: 10.1073/pnas.75.1.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wickstrom E. L., Bacon T. A., Gonzalez A., Freeman D. L., Lyman G. H., Wickstrom E. Human promyelocytic leukemia HL-60 cell proliferation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide targeted against c-myc mRNA. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1028–1032. doi: 10.1073/pnas.85.4.1028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zamecnik P. C., Goodchild J., Taguchi Y., Sarin P. S. Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4143–4146. doi: 10.1073/pnas.83.12.4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zamecnik P. C., Stephenson M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):280–284. doi: 10.1073/pnas.75.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES