Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1997 Dec;92(4):465–471. doi: 10.1046/j.1365-2567.1997.00375.x

Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells.

J Hassan 1, E Rainsford 1, D J Reen 1
PMCID: PMC1364151  PMID: 9497487

Abstract

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms.

Full text

PDF
465

Images in this article

469Figure 4
on p.469

Selected References

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

  1. Aggarwal S., Lee S., Mathur A., Gollapudi S., Gupta S. 12-Deoxyphorbol-13-O-phenylacetate 20 acetate [an agonist of protein kinase C beta 1 (PKC beta 1)] induces DNA synthesis, interleukin-2 (IL-2) production, IL-2 receptor alpha-chain (CD25) and beta-chain (CD122) expression, and translocation of PKC beta isozyme in human peripheral blood lymphocytes: evidence for a role of PKC beta 1 in human T cell activation. J Clin Immunol. 1994 Jul;14(4):248–256. doi: 10.1007/BF01552311. [DOI] [PubMed] [Google Scholar]
  2. Altman A., Mally M. I., Isakov N. Phorbol ester synergizes with Ca2+ ionophore in activation of protein kinase C (PKC)alpha and PKC beta isoenzymes in human T cells and in induction of related cellular functions. Immunology. 1992 Jul;76(3):465–471. [PMC free article] [PubMed] [Google Scholar]
  3. Berry N., Ase K., Kishimoto A., Nishizuka Y. Activation of resting human T cells requires prolonged stimulation of protein kinase C. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2294–2298. doi: 10.1073/pnas.87.6.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertotto A., Gerli R., Lanfrancone L., Crupi S., Arcangeli C., Cernetti C., Spinozzi F., Rambotti P. Activation of cord T lymphocytes. II. Cellular and molecular analysis of the defective response induced by anti-CD3 monoclonal antibody. Cell Immunol. 1990 May;127(2):247–259. doi: 10.1016/0008-8749(90)90130-j. [DOI] [PubMed] [Google Scholar]
  5. Borel J. F. Pharmacology of cyclosporine (sandimmune). IV. Pharmacological properties in vivo. Pharmacol Rev. 1990 Sep;41(3):259–371. [PubMed] [Google Scholar]
  6. Corrigan E., Kelleher D., Feighery C., Long A. Protein kinase C isoform expression in CD45RA+ and CD45RO+ T lymphocytes. Immunology. 1995 Jun;85(2):299–303. [PMC free article] [PubMed] [Google Scholar]
  7. Davis L. S., Lipsky P. E. T cell activation induced by anti-CD3 antibodies requires prolonged stimulation of protein kinase C. Cell Immunol. 1989 Jan;118(1):208–221. doi: 10.1016/0008-8749(89)90370-5. [DOI] [PubMed] [Google Scholar]
  8. Elliott J. F., Lin Y., Mizel S. B., Bleackley R. C., Harnish D. G., Paetkau V. Induction of interleukin 2 messenger RNA inhibited by cyclosporin A. Science. 1984 Dec 21;226(4681):1439–1441. doi: 10.1126/science.6334364. [DOI] [PubMed] [Google Scholar]
  9. Flanagan W. M., Corthésy B., Bram R. J., Crabtree G. R. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature. 1991 Aug 29;352(6338):803–807. doi: 10.1038/352803a0. [DOI] [PubMed] [Google Scholar]
  10. Gerli R., Bertotto A., Crupi S., Arcangeli C., Marinelli I., Spinozzi F., Cernetti C., Angelella P., Rambotti P. Activation of cord T lymphocytes. I. Evidence for a defective T cell mitogenesis induced through the CD2 molecule. J Immunol. 1989 Apr 15;142(8):2583–2589. [PubMed] [Google Scholar]
  11. Granelli-Piperno A., Inaba K., Steinman R. M. Stimulation of lymphokine release from T lymphoblasts. Requirement for mRNA synthesis and inhibition by cyclosporin A. J Exp Med. 1984 Dec 1;160(6):1792–1802. doi: 10.1084/jem.160.6.1792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hassan J., Reen D. J. Cord blood CD4+ CD45RA+ T cells achieve a lower magnitude of activation when compared with their adult counterparts. Immunology. 1997 Mar;90(3):397–401. doi: 10.1111/j.1365-2567.1997.00397.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hassan J., Reen D. J. Interleukin-1 augments the diminished interleukin-2 mRNA expression and proliferative response of neonatal T lymphocytes to anti-CD2 antibodies. Scand J Immunol. 1994 Jun;39(6):597–601. doi: 10.1111/j.1365-3083.1994.tb03418.x. [DOI] [PubMed] [Google Scholar]
  14. Hassan J., Reen D. J. Neonatal CD4+ CD45RA+ T cells: precursors of adult CD4+ CD45RA+ T cells? Res Immunol. 1993 Feb;144(2):87–92. doi: 10.1016/0923-2494(93)80064-6. [DOI] [PubMed] [Google Scholar]
  15. Höllsberg P., Melinek J., Benjamin D., Hafler D. A. Increased protein kinase C activity in human memory T cells. Cell Immunol. 1993 Jun;149(1):170–179. doi: 10.1006/cimm.1993.1145. [DOI] [PubMed] [Google Scholar]
  16. June C. H., Ledbetter J. A., Gillespie M. M., Lindsten T., Thompson C. B. T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression. Mol Cell Biol. 1987 Dec;7(12):4472–4481. doi: 10.1128/mcb.7.12.4472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kelleher D., Long A. Development and characterization of a protein kinase C beta-isozyme-deficient T-cell line. FEBS Lett. 1992 Apr 27;301(3):310–314. doi: 10.1016/0014-5793(92)80264-h. [DOI] [PubMed] [Google Scholar]
  18. Koretzky G. A., Picus J., Schultz T., Weiss A. Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2037–2041. doi: 10.1073/pnas.88.6.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krönke M., Leonard W. J., Depper J. M., Arya S. K., Wong-Staal F., Gallo R. C., Waldmann T. A., Greene W. C. Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5214–5218. doi: 10.1073/pnas.81.16.5214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lin C. S., Boltz R. C., Siekierka J. J., Sigal N. H. FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes. Cell Immunol. 1991 Apr 1;133(2):269–284. doi: 10.1016/0008-8749(91)90103-i. [DOI] [PubMed] [Google Scholar]
  21. Lucas S., Marais R., Graves J. D., Alexander D., Parker P., Cantrell D. A. Heterogeneity of protein kinase C expression and regulation in T lymphocytes. FEBS Lett. 1990 Jan 15;260(1):53–56. doi: 10.1016/0014-5793(90)80064-p. [DOI] [PubMed] [Google Scholar]
  22. Luo C., Burgeon E., Rao A. Mechanisms of transactivation by nuclear factor of activated T cells-1. J Exp Med. 1996 Jul 1;184(1):141–147. doi: 10.1084/jem.184.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Martiny-Baron G., Kazanietz M. G., Mischak H., Blumberg P. M., Kochs G., Hug H., Marmé D., Schächtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976. J Biol Chem. 1993 May 5;268(13):9194–9197. [PubMed] [Google Scholar]
  24. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  25. Oyaizu N., Chirmule N., Pahwa S. Role of CD4 molecule in the induction of interleukin 2 and interleukin 2 receptor in class II major histocompatibility complex-restricted antigen-specific T helper clones. T cell receptor/CD3 complex transmits CD4-dependent and CD4-independent signals. J Clin Invest. 1992 Jun;89(6):1807–1816. doi: 10.1172/JCI115785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pelchen-Matthews A., Parsons I. J., Marsh M. Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin-coated pits, and altered endosomal sorting. J Exp Med. 1993 Oct 1;178(4):1209–1222. doi: 10.1084/jem.178.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Petersen C. M., Christensen E. I., Andresen B. S., Møller B. K. Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells. Exp Cell Res. 1992 Jul;201(1):160–173. doi: 10.1016/0014-4827(92)90360-k. [DOI] [PubMed] [Google Scholar]
  28. Robinson A. T., Miller N., Alexander D. R. CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets. Eur J Immunol. 1993 Jan;23(1):61–68. doi: 10.1002/eji.1830230111. [DOI] [PubMed] [Google Scholar]
  29. Rothenberg E. V., Diamond R. A., Pepper K. A., Yang J. A. IL-2 gene inducibility in T cells before T cell receptor expression. Changes in signaling pathways and gene expression requirements during intrathymic maturation. J Immunol. 1990 Mar 1;144(5):1614–1624. [PubMed] [Google Scholar]
  30. Szamel M., Bartels F., Resch K. Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme, protein kinase C-beta. Eur J Immunol. 1993 Dec;23(12):3072–3081. doi: 10.1002/eji.1830231205. [DOI] [PubMed] [Google Scholar]
  31. Szamel M., Resch K. T-cell antigen receptor-induced signal-transduction pathways--activation and function of protein kinases C in T lymphocytes. Eur J Biochem. 1995 Feb 15;228(1):1–15. doi: 10.1111/j.1432-1033.1995.tb20221.x. [DOI] [PubMed] [Google Scholar]
  32. Xanthoudakis S., Viola J. P., Shaw K. T., Luo C., Wallace J. D., Bozza P. T., Luk D. C., Curran T., Rao A. An enhanced immune response in mice lacking the transcription factor NFAT1. Science. 1996 May 10;272(5263):892–895. doi: 10.1126/science.272.5263.892. [DOI] [PubMed] [Google Scholar]
  33. Zhang B. W., Zimmer G., Chen J., Ladd D., Li E., Alt F. W., Wiederrecht G., Cryan J., O'Neill E. A., Seidman C. E. T cell responses in calcineurin A alpha-deficient mice. J Exp Med. 1996 Feb 1;183(2):413–420. doi: 10.1084/jem.183.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES