IL-2 Deprivation-induced Apoptosis Research Articles

TSC-22 Promotes Interleukin-2-Deprivation Induced Apoptosis in T-Lymphocytes.

Originally described as a TGF-β-inducible gene, tsc-22 (Transforming growth factor-beta Stimulated Clone 22) encodes a transcriptional regulator affecting biological processes such as cell growth, differentiation, or apoptosis. Along with GILZ (Glucocorticoid-Induced Leucine Zipper), TSC-22 belongs to the evolutionary conserved TSC-22 Domain family. We previously showed that, in T-lymphocytes, GILZ expression was induced upon IL-2 withdrawal, delaying apoptosis through down-regulation of the pro-apoptotic protein BIM expression. The aim of this work was then to elucidate the respective roles of GILZ and TSC-22 upon IL-2 deprivation-induced apoptosis. We report here that these two highly homologous genes are concomitantly expressed in most human tissues and in primary T-lymphocytes and that expression of TSC-22 promotes T-lymphocytes apoptosis by inhibiting GILZ functions. Indeed, we demonstrated that TSC-22 expression in the murine lymphoid CTLL-2 cell line promoted IL-2 deprivation-induced apoptosis. BIM expression and caspases-9 and -3 activities were markedly increased in TSC-22 expressing clones compared to control clones. Analysis of GILZ expression revealed that TSC-22 prevented the induction of the GILZ protein upon IL-2 deprivation, by inhibiting gilz mRNA transcription. These results suggested that TSC-22 could counteract the protective effect of GILZ on IL-2-deprivation-induced apoptosis. Moreover, TSC-22-induced inhibition of GILZ expression was also found in CTLL-2 cells treated with glucocorticoids or TGF-β. In the human NKL cell line deprived of IL-2, TSC-22 showed the same effect and thus may represent a potent repressor of GILZ expression in IL-2-dependent cells, independently of the cell type, or the stimulus, leading to an increase of IL-2-deprived T-cells apoptosis. J. Cell. Biochem. 117: 1855-1868, 2016. © 2016 Wiley Periodicals, Inc.

Induction of PP2A Bβ, a regulator of IL-2 deprivation-induced T-cell apoptosis, is deficient in systemic lupus erythematosus

The activity and substrate specificity of the ubiquitously expressed phosphatase PP2A is determined by the type of regulatory (B) subunit that couples to the catalytic/scaffold core of the enzyme. We determined that the Bβ subunit (PPP2R2B) is expressed in resting T cells, its transcription is down-regulated during T-cell activation, and up-regulated in conditions of low IL-2. Specifically, high levels of PP2A Bβ were produced during IL-2 deprivation-induced apoptosis, whereas Fas ligation had no effect. Forced expression of the Bβ subunit in primary human T cells was sufficient to induce apoptosis, whereas silencing using siRNA protected activated T cells from IL-2 withdrawal-induced cell death. Because T-cell apoptosis is known to be altered in T cells from patients with systemic lupus erythematosus, we analyzed the regulation of PP2A Bβ in this autoimmune disease. We found that levels of PP2A Bβ did not increase upon IL-2 deprivation in 50% of the patients. Remarkably, this defect was accompanied by resistance to apoptosis. Importantly, kinetics of cell death were normal in cells of patients that up-regulated PP2A Bβ in a normal manner. We have identified a unique role for the phosphatase PP2A, particularly the holoenzyme formed by PP2A Bβ. Bβ appears to trigger apoptosis of T cells in the absence of IL-2 and probably contributes to the termination of a no-longer-needed immune response. We propose that defective production of PP2A Bβ upon IL-2 deprivation results in apoptosis resistance and longer survival of autoreactive T cells, in a subset of SLE patients.

Cascade of transcriptional induction and repression during IL-2 deprivation-induced apoptosis

Apoptosis of mature T lymphocytes is an essential process for maintaining immune system homeostasis. However, the details of the molecular signaling pathways leading to T cell apoptosis are poorly understood. We used cDNA microarrays containing 15,630 murine genes to study the gene expression profile in T lymphocytes at different time points of IL-2 withdrawal. Comparison of the gene expression profiles revealed that 2% of the genes were affected by cytokine starvation. Interestingly, the apoptotic program rather seems to activate gene expression in the early phase of cell death. On the contrary, transcription was strongly repressed in later stages of apoptosis. Self-organizing map clustering of the 270 differentially expressed transcripts revealed specific temporal expression patterns supporting the idea that IL-2 deprivation triggers a tightly regulated transcriptional program to induce cell death. To validate microarray results, changes in gene expression following IL-2 deprivation were confirmed for selected genes by Northern blot. In addition, the signaling pathways created can explain the molecular events leading to T cell apoptosis, even if the T cell line used in this study might not reflect individual T cell subpopulations expressing different level of IL-2 receptor or IL-2 dependence. Taken together, these results provide novel insights into the temporal regulation of gene expression during T lymphocyte death.

Identification of PP1α as a Caspase-9 Regulator in IL-2 Deprivation-Induced Apoptosis

One of the mechanisms that regulate cell death is the reversible phosphorylation of proteins. ERK/MAPK phosphorylates caspase-9 at Thr(125), and this phosphorylation is crucial for caspase-9 inhibition. Until now, the phosphatase responsible for Thr(125) dephosphorylation has not been described. Here, we demonstrate that in IL-2-proliferating cells, phosphorylated serine/threonine phosphatase type 1alpha (PP1alpha) associates with phosphorylated caspase-9. IL-2 deprivation induces PP1alpha dephosphorylation, which leads to its activation and, as a consequence, dephosphorylation and activation of caspase-9 and subsequent dissociation of both molecules. In cell-free systems supplemented with ATP caspase-9 activation is induced by addition of cytochrome c and we show that in this process PP1alpha is indispensable for triggering caspase-9 as well as caspase-3 cleavage and activation. Moreover, PP1alpha associates with caspase-9 in vitro and in vivo, suggesting that it is the phosphatase responsible for caspase-9 dephosphorylation and activation. Finally, we describe two novel phosphatase-binding sites different from the previously described PP1alpha consensus motifs, and we demonstrate that these novel sites mediate the interaction of PP1alpha with caspase-9.

Rescue by cytokines of apoptotic cell death induced by IL-2 deprivation of human antigen-specific T cell clones.

The control of cell survival and cell death is of central importance in tissues with high cell turnover such as the lymphoid system. We have examined the effect of cytokines on IL-2 deprivation-induced apoptosis of human antigen-specific T helper clones with different cytokine production profiles. We found that IL-2, interferon-alpha (IFN-alpha), and IFN-beta inhibited IL-2 deprivation apoptosis in Th0, Th1, and Th2 clones. We also found that IL-2 protects T cell clones from IL-2 deprivation apoptosis accompanying active proliferation and enhanced expression of P53, Rb and Bcl-xL proteins. In contrast, IFN-alpha/beta rescued T cell clones from apoptosis without active proliferation, and expression of apoptosis-associated proteins tested so far was unaffected. This may be due to the fact that T cells treated with IL-2 contained those located in S + G2/M phases of the cell cycle, whereas the vast majority of T cells treated with IFN-alpha/beta were located in G0/G1 phase. IFN-alpha/beta specifically induced tyrosine phosphorylation and translocation into nucleus of signal transducers and activators of transcription (STAT) 2 protein in the T cell clones. In addition, over-expression of STAT2 by transfection of the cDNA prevented apoptosis of the T cell clones. Our present study shows that IFN-alpha and -beta mediate anti-apoptotic effect through other pathways than that of IL-2 in growth factor deprivation apoptosis.

Bcl-2 targets protein phosphatase 1 alpha to Bad.

The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1alpha is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1alpha and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1alpha to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1alpha. Disruption of Bcl-2/PP1alpha association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1alpha to Bad.

Rescue of human T cells by interleukin-9 (IL-9) from IL-2 deprivation-induced apoptosis: correlation with alpha subunit expression of the IL-9 receptor.

Interleukin-9 (IL-9) is a Th2-derived cytokine that uses the gamma-chain of the IL-2 receptor for signaling. Therefore, the responsiveness of human Th1 and Th2 cell clones to IL-9 was measured by examining the ability of this cytokine to prevent apoptosis induced by IL-2 deprivation. A time course study demonstrated that both subsets of T cell clones underwent apoptosis with similar kinetics when deprived of IL-2 and that viability could be maintained by the addition of either IL-4 or IL-7. Interestingly, IL-9 prevented apoptosis in only 2 (Th2) of 14 clones tested. Analysis of IL-9R alpha subunit expression on 18 T cell clones revealed that IL-9 responsiveness was directly proportional to the expression of the high-affinity receptor. IL-9 responsiveness was also dependent on long-term culturing because neither freshly isolated nor 3-day phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) expressed IL-9R alpha. In summary, the data showed that IL-9 can rescue only a small subset of Th2 cells from apoptosis induced by growth factor withdrawal and that expression of IL-9R alpha is required for the antiapoptotic signals mediated by this cytokine.

CD 95-independent mechanisms of IL-2 deprivation-induced apoptosis in activated human lymphocytes.

Growth factor deprivation-induced apoptosis plays an important role in several cellular systems. However, knowledge of the molecular mechanisms involved are restricted to a few murine models or tumor cell lines. Therefore, we aimed studying signaling pathways leading to apoptosis in activated human peripheral T cells after IL-2 withdrawal. Lymphoblasts from patients with CD 95 (Fas/APO-1)-deficiency revealed that functional CD95 was not required to induce apoptosis after IL-2 withdrawal. Moreover, apoptosis induction in response to various cytotoxic stimuli was found to be mediated in the absence of functional CD95 but was affirmatorily influenced by IL-2 signaling. Immunoblots showed no downregulation of Bcl-2 or Bcl-xL and no upregulation of Bax, whereas decreased mitochondrial membrane potential was readily measurable 24 h after cytokine deprivation. Tetrapeptide inhibitors showed limited efficacy in preventing apoptosis whereas the caspase inhibitor zVAD-FMK potently blocked induction of apoptosis. Cleavage of different fluorogenic substrates revealed multiple caspase enzyme activities in lymphoblasts, which were not negatively affected by the fas mutation. Starting at 8 h after IL-2 withdrawal, upregulation of active caspase-3 but not of caspase-8 could be detected. Taken together, our data argue for molecular mechanisms of cytokine deprivation-induced apoptosis in activated human lymphocytes independent of CD95.

Resistance of Crohn’s Disease T Cells to Multiple Apoptotic Signals Is Associated with a Bcl-2/Bax Mucosal Imbalance

AbstractCrohn’s disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2+, and Bax+ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-α and nitric oxide. TUNEL+ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68+ and myeloperoxidase+, but no CD45RO+, apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivation-induced apoptosis. CD T cells were also more resistant to Fas- and nitric oxide-mediated apoptosis, whereas TNF-α failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2+, but fewer Bax+, cells, while UC mucosa contained fewer Bcl-2+, but more Bax+, cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.

IL-2 deprivation triggers apoptosis which is mediated by c-Jun N-terminal kinase 1 activation and prevented by Bcl-2.

A variety of environmental stresses, as well as inflammatory cytokines, induce activation of c-Jun N-terminal kinases. We describe here that IL-2 deprivation-induced apoptosis in TS1alphabeta cells does not modify c-Jun protein levels and correlates Bcl-2 downregulation and an increase in JNK1, but not JNK2, activity directly related to the induction of apoptosis. Indeed, downregulation of JNK1 expression using antisense oligonucleotides inhibits apoptosis induced by IL-2 withdrawal. Overexpression of Bcl-2 promotes cell survival and blocks JNK1 activation as well as apoptosis caused by IL-2 deprivation. This suggests that inhibition of the JNK1 signaling pathway may be a mechanism through which Bcl-2 promotes cell survival and prevents apoptosis triggered by growth factor withdrawal.

IL-12 Inhibits Apoptosis Induced in a Human Th1 Clone by gp120/CD4 Cross-Linking and CD3/TCR Activation or by IL-2 Deprivation

The aim of our work was to study apoptosis as a possible mechanism of CD4+ lymphocyte depletion in AIDS patients and to test whether IL-12 could limit this phenomenon. As an in vitro model, we used a human IL-2-dependent Th1 clone from an uninfected individual. We found that CD4 cross-linking, obtained either by mouse anti-CD4 mAb plus goat anti-mouse or by recombinant gp120 plus anti-gp120 mAb, followed by activation with immobilized anti-CD3 or anti-TCR mAb, induced apoptosis at early times (15-25% apoptotic cells at 4 hr), whereas IL-2 deprivation required longer times (20-40 hr) to induce apoptosis. Both CD4 cross-linking and IL-2 deprivation-induced apoptosis appeared to be PTK-dependent and were inhibited by either IL-2 or IL-12. Our data suggest that in vivo CD4/gp120 interactions could directly prime the apoptosis of Th1 lymphocytes and that IL-2 and IL-12 could be used to prevent this phenomenon.