Regulation of T cell apoptosis by calcium and zinc ions

Sammanfattning: Apoptosis is a major form of cell death involved in fundamental processes in the mammalian immunesystem. Although extensive studies have been carried out to explore the mechanisms involved in lymphocyteapoptosis, a defined signaling pathway is still unclear. The aims of the study were: 1) to set up bettermodels to investigate the role of imbalanced ion signaling (a change in intracellular Ca2+ or Zn2+) in T cellapoptosis; 2) to study the regulation and molecular mechanism of T cell apoptosis induced by an elevationof cytosolic free Ca2+ ([Ca2+];) and chelation of intracellular Zn2+; and 3) to examine the role of Ca2+ andZn2+ in the regulation of apoptosis during T cell development. In this study, we established that a selective increase in [Ca2+]; triggers apoptosis in rat thymocytes. Weused an endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin, which has been shown to increase[Ca2+]; by causing the release of Ca2+ from intracellular stores and promoting Ca2+ influx across the plasmamembrane. We found that the thapsigargin-induced increase in [Ca2+]; preceded DNA fragentation andformation of apoptotic nuclei, and served as an essential step leading to apoptosis. However, to initiateapoptosis, the increase in [Ca2+]; must be sustained for 2 h. A transient increase in thymocyte [Ca2+]; duesolely to the release of Ca2+ from intracellular stores was insufficient to trigger apoptosis. The formation ofhigh molecular weight DNA fragments occurs prior to internucleosomal fragmentation and is an earlymarker of apoptosis. We showed that Ca2+-dependent mechanisms, including endonuclease activation, areinvolved in this early step of chromatin cleavage. We then established another model in which chelation of intracellular Zn2+ triggers apoptosis in TIymphocytes. We chose the membrane-permeable heavy metal chelator, N,N,N'N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), to induce the characteristic features of apoptosis. We found that TPEN-inducedapoptosis is not affected by altering intracellular or extracellular Ca2+ concentration, suggesting that itoccurs by a Ca2+-independent mechanism. The susceptibility of T cells at different stages of development to apoptosis induced by thapsigarginand TPEN, and its regulation by the immunosuppressive drugs cyclosporin A and FK506, was also studied.Our results demonstrated that the susceptibility and regulation of T cell apoptosis differ both (a) betweendifferent stimuli in the same cells and (b) between the same stimuli in cells of different developmentalstages. Cyclosporin A and FK506 block apoptosis associated with an increase in [Ca2+]; in T cell hybridomaand activated T cells, but not in immature thymocytes. This suggests that calcineurin (the Ca2+-activatedprotein phosphatase targeted by both immunosuppressive drugs) is critically involved in Ca2+-mediatedapoptosis in activated T cells but not in immature thymocytes. Proteases, particularly interleukin-1beta-converting enzyme (ICE)-like proteases, have been shown toplay an important role in apoptosis. We found that both thapsigargin and TPEN induced an increase in ICE-like protease activity and a degradation in poly(ADP-ribose) polymerase (PARP) and lamin prior to DNAcleavage, which were all inhibited by an inhibitor of ICE-like proteases. However, an inhibitor of Ca2+-regulated serine protease, only inhibited thapsigargin- but not TPEN-induced cleavages of PARP, lamin andchromatin. When a cell-free system was used to further study the early signaling in TPEN-inducedapoptosis, we found that TPEN was unable to induce DNA fragmentation in isolated thymocyte nuclei. Onthe contrary, cytoplasmic extract from TPEN-treated Jurkat cells resulted in extensive DNA fragmentationin isolated nuclei. The apoptosis-inducing activity in this extract was partially reduced by the inhibitors ofICE-like proteases. Our results demonstrate that activation of cytosolic proteases, including ICE-likeproteases, is essential for the induction of apoptosis by thapsigargin and TPEN. In conclusion, this thesis provides strong evidence that either a selective increase in [Ca2+];, or chelationof intracellular Zn2+, triggers apoptosis in T cells. The lack of involvement of Ca2+ in the latter systemsuggests that TPEN induces apoptosis by a distinct mechanism. The difference in susceptibility of T cells toapoptosis at different stages of development and its regulation by immunosuppressive drugs further indicatethat diverse signaling pathways can be initiated in different cell types and by different inducers. However,the finding that ICE-like proteases are involved in both systems suggests that at some point, apoptotic deathpathways converge.Doctoral Thesis1996 Shunai JiangISBN 91-628-1957-7