Thymocyte apoptosis by glucocorticoids and cAMP.
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Animals↗
Biomedical subjects
D J McConkey
Publications and source records attributed to D J McConkey.
Calcium and cyclosporin A in the regulation of apoptosis.
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Animals↗
Co-receptor (CD4/CD8) engagement enhances CD3-induced apoptosis in thymocytes. Implications for negative selection.
Negative selection of self-reactive immature T cells is mediated by TCR engagement and is thought to occur via apoptosis (programmed cell death). The requirement for the co-receptors CD4 and CD8 in negative selection has been demonstrated, but the biochemical mechanisms underlying their involvement in this process remain undefined. Here we present evidence that co-receptor engagement dramatically enhances CD3-induced endonuclease activation and cell death characteristic of apoptosis in immature thymocytes. The responses are associated with increased tyrosine phosphorylation of a number of cellular substrates, including the gamma isoform of phospholipase C, and with increased association of tyrosine phosphoproteins, including the protein tyrosine kinase p56lck, with the TCR complex. Co-receptor engagement also potentiated CD3-mediated Ca2+ increases via a mechanism dependent upon tyrosine kinase activation. Sustained Ca2+ availability was found to be necessary for endonuclease activation and apoptosis to occur. We suggest that CD4 and CD8 may participate in negative selection by enhancing TCR/CD3-induced tyrosine kinase activation and sustained Ca2+ increases that lead to endonuclease activation and apoptosis in self-reactive CD4+ CD8+ thymocytes.
Amino Acid Sequence↗
Signal transduction pathways to apoptosis.
Recent work has demonstrated that a number of signalling events, including cytosolic Ca(2+) rises, cAMP accumulation, activation of protein kinase C, activation of protein tyrosine kinases, and production of ceramide, regulate apoptosis in diverse model systems. However, in some cells these signals promote apoptosis, whereas in others they block the response. This review discusses these observations and proposes explanations for how a given set of signal transduction systems might be involved in multiple cellular responses.
Journal Article↗
The regulation of apoptosis in thymocytes.
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Animals↗
Signalling and chromatin fragmentation in thymocyte apoptosis.
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Animals↗
Cyclic AMP potentiates glucocorticoid-induced endogenous endonuclease activation in thymocytes.
The present study was undertaken to determine whether specific interactions between cAMP and glucocorticoids regulate apoptosis in thymocytes. Incubation of murine thymocytes with agents that elevate the cAMP level resulted in enhancement of glucocorticoid-induced Ca2+ increases, DNA fragmentation, and cell death compared to levels observed in thymocytes treated with steroid alone. cAMP did not affect DNA fragmentation in thymocytes treated with Ca2+ ionophore, a compound that induces endonuclease activation via an independent mechanism. Treatment with cAMP also increased glucocorticoid potency by lowering the concentration of steroid required for induction of apoptosis. The mechanism of cAMP action appeared to involve the glucocorticoid receptor, since the glucocorticoid antagonist RU-486 abrogated the cAMP response in animals treated with the adenosine analog NECA in vivo. Analysis of cellular glucocorticoid binding and receptor protein levels revealed modest cAMP-stimulated increases that appeared insufficient to account for the effects of cAMP on endogenous endonuclease activation, suggesting the possible involvement of a posttranslational mechanism in the response. These results demonstrate that cAMP and glucocorticoids synergize to promote apoptosis in thymocytes via a mechanism that appears to involve modification of glucocorticoid receptor activity.
Adenosine↗
Overexpression of CD3 eta during thymic development does not alter the negative selection process.
To elucidate the role of CD3 eta in thymic development and to determine whether CD3 eta is involved in the negative selection process, CD3 eta was overexpressed > 100 fold in transgenic (tg) mice using a Thy-1 promoter and regulatory elements. CD3 eta was readily observed in the majority of cortical thymocytes and in a fraction of medullary thymocytes in tg mice by immunohistochemical staining with an anti-CD3 eta-specific mAb. In contrast, endogenous CD3 eta levels were too low to detect in normal littermates. Flow cytometric analysis demonstrated an increased level of TCR on thymocytes with intermediate TCR density in tg animals and parallel biochemical studies showed a marked increased in TCR-associated CD3 zeta-eta heterodimers and CD3 eta-eta homodimers relative to controls. Despite this change in surface TCR phenotype, there was no significant alteration in the total numbers or proportion of CD4+CD8+ double-positive or CD4+CD8- or CD4-CD8+ single-positive thymocytes or peripheral T cells. Percentages of SP V beta 5, V beta 6, and V beta 8 thymocytes in tg animals were unaltered compared to normal littermates when backcrossed either to C57BL/6 (H-2b) or DBA/2 (H-2d) backgrounds. Furthermore, induction of DNA fragmentation with anti-CD3 epsilon mAb treatment in vivo was not significantly different for tg and normal littermates. Collectively, these data imply that CD3 eta is not a limiting component of the negative selection process.
Animals↗
Cellular signaling in cell death.
Apoptosis is a process of selective cell deletion involving endogenous endonuclease activation that mediates most examples of programmed cell death. Recent work has demonstrated that the process is regulated within the dying cell by signal transduction pathways, including increases in cytosolic ionized calcium concentrations, activation of protein kinases A and C, and stimulation of the nuclear enzyme poly(adenosine 5'-diphosphate-ribose) polymerase. In addition, oncogenes, tumor suppressor genes, and transcription factors also regulate induction of cell death. Evidence also indicates that apoptosis mediates a number of instances of pathologic cell death and, in some of these examples, the biochemical mechanisms responsible appear to mimic the cell-signaling mechanisms controlling the process in physiologic situations.
Animals↗
The CD3 zeta cytoplasmic domain mediates CD2-induced T cell activation.
CD2-mediated T lymphocyte activation requires surface expression of CD3-Ti, the T cell receptor (TCR) for antigen major histocompatibility complex protein. Given the importance of CD3 zeta in TCR signaling, we have directly examined the ability of the CD3 zeta cytoplasmic domain to couple CD2 to intracellular signal transduction pathways. A cDNA encoding a chimeric protein consisting of the human CD3 zeta cytoplasmic domain (amino acid residues 31-142) fused to the CD8 alpha extracellular and transmembrane domains (amino acid residues 1-187) was transfected into a CD2+CD3-CD8- variant of the human T cell line Jurkat. The resulting transfectants expressed the CD8 alpha/CD3 zeta chimeric receptor at the cell surface in the absence of other TCR subunits. Stimulation of these transfectants with anti-T11(2) + anti-T11(3) monoclonal antibodies (mAbs) initiated both a prompt cytosolic free calcium ([Ca2+]i) rise and protein tyrosine kinase activation. Stimulation with either intact anti-T11(2) + anti-T11(3) mAbs or purified F(ab')2 fragments resulted in interleukin 2 (IL-2) secretion. In contrast, control cell lines transfected with a cDNA encoding wild-type CD8 alpha, and thus lacking surface expression of the CD3 zeta cytoplasmic domain, failed to show any [Ca2+]i rise, protein tyrosine kinase activation, or IL-2 secretion after identical stimulation. These data directly establish the CD3 zeta cytoplasmic domain as a necessary and sufficient component of the CD3-Ti complex involved in T lymphocyte activation through CD2. Moreover, they show that CD2 signaling can function in the absence of Fc receptors.
Antigens, Differentiation, T-Lymphocyte↗
CD3 zeta dependence of the CD2 pathway of activation in T lymphocytes and natural killer cells.
In T lymphocytes, signal transduction through the CD2 adhesion molecule requires surface expression of the CD3-Ti T-cell receptor (TCR) complex. In contrast, in natural killer (NK) cells, CD2 functions in the absence of a TCR. Because the TCR on T lymphocytes and the CD16 low-affinity IgG Fc receptor (Fc gamma receptor type III) complex on NK cells share a common CD3 zeta subunit, we reasoned that CD3 zeta may be critical for CD2 signaling in T lymphocytes and NK cells. Here we show that transfection of a cDNA encoding a transmembrane form of CD16 into TCR- variants of the Jurkat T-cell line results in CD16 expression in association with endogenous CD3 zeta homodimers and restores CD2 signaling function. To test directly the role of CD3 zeta in CD2 triggering, we then transfected human CD2 into each of two murine T-T hybridomas that express TCRs containing either a full-length CD3 zeta subunit or a truncated CD3 zeta subunit incapable of transducing signals. Despite expression of equivalent surface levels of TCR, CD2-mediated signaling is seen only in the T cells containing wild-type CD3 zeta. These findings show that (i) CD16 on NK cells is functionally equivalent to the TCR on T lymphocytes for coupling CD2 to signaling pathways and (ii) CD2 signal transduction depends upon the CD3 zeta subunit of the TCR complex and, by inference, the CD3 zeta subunit of the CD16 complex.
Animals↗
Phosphorylation of multiple CD3 zeta tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation.
T lymphocyte activation resulting from antigen recognition involves a protein tyrosine kinase pathway which triggers phosphorylation of several cellular substrates including the CD3 zeta subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3 eta, is an alternatively spliced product of the same gene locus as CD3 zeta. CD3 eta lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3 zeta and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phosphorylation studies herein demonstrates that Tyr-132 is required for the formation of pp21. Moreover, the differential phosphorylation of CD3 zeta versus CD3 eta is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59fyn; p59fyn but not p56lck or p62yes is associated with each of the three TCR isoforms containing CD3 zeta 2, or CD3 eta 2, or CD3 zeta-eta. This association occurs through components of the TCR complex distinct from CD3 zeta or CD3 eta. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3 zeta residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3 zeta Tyr----Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3 zeta upon TCR stimulation as reflected by alteration in its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis may affect subsequent events such as receptor desensitization, receptor movement, and/or protein associations.
Amino Acid Sequence↗
Cellular signaling in thymocyte apoptosis.
Induction of apoptosis (programmed cell death) in response to T cell receptor triggering is now thought to be involved in the process of negative selection in the thymus, and current work is therefore aimed at investigating how apoptosis is regulated within the cells. To this end, recent work has implicated several of the well-known signal transduction pathways already known to regulate T cell activation in the regulation of apoptosis in thymocytes. In particular, elevations of the cytosolic Ca2+ level or increases in cAMP can trigger thymocyte apoptosis, whereas activation of protein kinase C appears to inhibit apoptosis in response to either induction pathway. Moreover, crosslinking of Thy-1, CD4, or CD8 leads to potentiation of T cell receptor-mediated cell death, effects that appear to involve protein tyrosine kinase activation. These observations may be relevant to the question of how T cell receptor occupancy can mediate both differentiation and death during intrathymic T cell development.
Animals↗
Differential signal transduction via T-cell receptor CD3 zeta 2, CD3 zeta-eta, and CD3 eta 2 isoforms.
The T-cell antigen receptor (TCR) consists of an antigen-binding heterodimer, termed Ti, which is noncovalently associated with the invariant CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3 zeta and -eta subunits form either homodimeric or heterodimeric structures in turn associated with the other components of the TCR complex. This feature increases the structural complexity of TCRs by creating "isoforms." Both CD3 zeta and -eta are thought to play an important role in signal transduction triggered by antigen/major histocompatibility complex. To compare signaling functions of TCR isoforms, MA5.8, a CD3 zeta-eta- variant of the cytochrome c-specific, I-Ek-restricted T-cell hybridoma 2B4.11, was stably transfected with cDNAs encoding CD3 zeta and/or CD3 eta, and resulting clones were characterized. The findings indicate that signals inducing Ca2+ mobilization, phosphatidylinositol turnover, and interleukin 2 production are each transmitted by the above TCR isoforms. In contrast, tyrosine phosphorylation of the CD3 zeta subunit but not the CD3 eta subunit follows TCR stimulation. Given the general importance of tyrosine phosphorylation for receptor signaling, it is likely that this difference between TCR isoforms plays a regulatory role in T-lineage function by qualitatively or quantitatively altering signaling events.
Animals↗
Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells.
Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.
Antibodies, Monoclonal↗
Role of calcium in toxic and programmed cell death.
An uncontrolled and sustained increase in cytosolic Ca2+ concentration has been implicated as an early event in the development of anoxic or toxic cell injury. More recently it has become clear that an elevation of cytosolic Ca2+ is also involved in programmed cell death in the immune system. Here, we review some of our recent studies and provide further evidence for the role of Ca2+ in cell killing.
Animals↗
Agents that elevate cAMP stimulate DNA fragmentation in thymocytes.
Increases in the cAMP level are often inhibitory in mature T lymphocytes and may be involved in the development of tolerance to self Ag. In this report, agents inducing an increase in the cAMP level by independent mechanisms were found to stimulate DNA fragmentation, characteristic of a suicide program known as apoptosis, in isolated thymocytes. Data obtained with cAMP analogs known to act synergistically to stimulate protein kinase A suggested that the latter directly mediated endonuclease activation. Agents previously shown to stimulate protein kinase C and to inhibit Ca2(+)-dependent, TCR-mediated thymocyte apoptosis, including IL-1, also blocked both DNA fragmentation and cell death in response to cAMP, suggesting interactions ("cross-talk") between the two protein kinase systems. As it has been proposed that apoptosis mediates negative cell selection in the thymus, our results indicate that cAMP may play a role in the development of functional mature T lymphocytes.
Animals↗
Interleukin 1 inhibits T cell receptor-mediated apoptosis in immature thymocytes.
T cell receptor signaling has been implicated in an intrathymic process of cell selection in which potentially harmful autoreactive precursors are deleted before they emigrate to the periphery. Recent work has shown that immature thymocytes undergo apoptosis, or programmed cell death, upon stimulation via the T cell antigen receptor, and it has been suggested that this mechanism may mediate deletion of the self-reactive clones during T cell development. Here we report that the accessory cell-derived T cell growth hormone interleukin-1 prevents T cell receptor-mediated thymocyte apoptosis by a mechanism that appears to involve protein kinase C activation. This effect may be relevant to the positive signaling that spares the appropriate precursors during the generation of functional T lymphocytes.
Antibodies, Monoclonal↗