Redox-sensitive events in Fas-induced apoptosis in human NK cells include ceramide generation and protein tyrosine dephosphorylation

We previously reported that intracellular oxidation-reduction (redox) regulates NK cell functions and that IL-2-activated NK cells undergo apoptosis upon contact with NK-sensitive target cells. We now report that apoptosis in activated human NK cells is also regulated by redox. Thiol deprivation increased apoptosis in NK cells induced by anti-Fas mAb or Fas ligand-transfected cells, and pretreatment of cells with N-acetyl cysteine, which increased intracellular glutathione, partially inhibited the apoptosis and reversed the effect of thiol-deficient medium, suggesting that Fas-induced apoptosis in NK cells is also redox sensitive. Thiol deprivation did not alter cell surface Fas expression, but did increase ceramide generation following Fas engagement. Although exogenous ceramides induced apoptosis of NK cells, thiol depletion had no effect on this apoptosis. Thiol deprivation increased CPP32 activation induced by Fas engagement, but not by ceramides. These findings suggest that, if ceramide is required for Fas-induced apoptosis, thiol deprivation affects the Fas-mediated signaling pathway at the generation of ceramide and/or upstream thereof. Though tyrosine phosphorylation following Fas engagement was not significantly affected by thiol deprivation, tyrosine dephosphorylation was delayed, suggesting that tyrosine phosphatases may also be redox sensitive. The notion that dephosphorylation is important in the Fas signaling pathway is supported by the finding that tyrosine phosphatase inhibitors significantly enhanced both CPP32 activity and apoptosis following Fas ligation. We conclude that events downstream of tyrosine phosphorylation and upstream of CPP32 activation, including tyrosine dephosphorylation and possibly ceramide generation, are sensitive to regulation by redox in human NK cells, requiring a reducing environment for optimal protection from apoptosis induced by Fas ligation.     

Caspase-1 is not involved in CD95/Fas-induced apoptosis in Jurkat T cells

It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene product ced-3. Using whole cells as well as an in vitro system to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed that N-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereas N-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1beta as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1beta was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.     

Inhibition of caspase activity prevents anti-IgM induced apoptosis but not ceramide generation in WEHI 231 B cells

In apoptosis induced by Reaper in Drosophila, as well as in a number of other systems, it has been suggested that the increased synthesis of ceramide might be a consequence of the activation of the caspase/ICE (Interleukin-1beta converting enzyme) protease pathway involved in cell death, implying that ceramide generation might often be the result rather than the cause of apoptosis. WEHI 231 B cells have previously been shown to undergo apoptosis following exposure to exogenous ceramide and to produce increased amounts of ceramide in response to anti-IgM crosslinking. We show here that in WEHI 231 cells a peptide inhibitor of caspase activity blocks cell death in response to both anti-IgM and exogenous ceramide. However, the induction of ceramide synthesis by WEHI 231 cells in response to anti-IgM crosslinking is not blocked by this peptide. These results indicate that antigen receptor induced ceramide generation in WEHI 231 cells does not require caspase activation, and support the view that ceramide generation in immature B cells may be the cause rather than the consequence of activation of the caspase dependent death pathway.     

Inhibition of Fas-induced apoptosis by Bcl-2

Jurkat cells express Fas, and rapidly undergo apoptosis in response to Fas ligand or an agonistic anti-Fas antibody. This apoptotic pathway is mediated by a cascade of caspases. In this report, we show that Fas activation induced the processing of caspase 8 in Jurkat cells with a time frame similar to the activation of caspase 3 and the proteolysis of nuclear proteins. Jurkat cell transformants that overexpress Bcl-2 were partially but not completely resistant to the Fas-induced apoptosis. Little processing of caspase 8 was observed upon Fas activation in these transformants. Furthermore, although caspase 8 was recruited to Fas upon Fas activation in the parental Jurkat cells, the recruitment of caspase 8 to Fas was inhibited in the transformants overexpressing Bcl-2. These results suggest that Bcl-2 inhibits Fas-induced apoptosis by preventing the formation of the death-inducing signaling complex that is composed of Fas, FADD/MORT1, and caspase 8.     

Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.     

Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation

Programmed cell death or apoptosis provides an irreversible mechanism for the elimination of excess or damaged cells. Several recent studies have implicated the activation of the interleukin 1beta-converting enzyme/Ced-3 (ICE/Ced-3) family of proteases as the "point of no return" in apoptotic cell death, while others have suggested that loss of mitochondrial membrane potential (delta psi(m)) is the ultimate determinant of cell death. The temporal relationship of these two events during apoptosis and the role of Bcl-2 proteins in inhibiting these steps has not been defined. To examine these issues, control and Bcl-x(L)-transfected Jurkat T cells were treated with Fas antibodies in the presence and absence of the ICE protease inhibitor zVAD-FMK. ICE/Ced-3 protease activity was monitored by following the cleavage of poly(ADP-ribose) polymerase (PARP) and delta psi(m) was followed by rhodamine 123 fluorescence. Although Bcl-x(L) expression did not block Fas-induced protease activation, it substantially inhibited the subsequent loss of delta psi(m) and cell death in Fas-treated cells. In contrast, zVAD-FMK blocked PARP cleavage as well as loss of delta psi(m) and cell death. Together these data demonstrate that Bcl-x(L) can maintain cell viability by preventing the loss of mitochondrial membrane potential that occurs as a consequence of ICE/Ced-3 protease activation.     

Coronavirus-induced demyelination occurs in the presence of virus-specific cytotoxic T cells

C57BI/6, but not BALB/c, mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a late onset, symptomatic demyelinating encephalomyelitis. In this report, we characterized anti-viral cytotoxic T cells in the central nervous system and spleen during the acute and chronic stages of the MHV infection. The data show that C57BI/6 mice display a cytotoxic T cell (CTL) response to the surface (S) glycoprotein and this response can be demonstrated in lymphocytes isolated from the brains and spinal cords of mice both acutely and persistently infected with MHV-JHM. Thus, the anti-S CTL activity present in the central nervous system of chronically infected animals is not sufficient to prevent the demyelinating process. BALB/c mice have been shown previously to mount a CTL response against the nucleocapsid (N) protein (Stohlman et al., 1992). Since C57BI/6 mice do not mount a response to the N protein, the role of the N-specific response in preventing the late onset disease was assessed using B10.A(18R) mice, recombinant in the H-2 locus. These mice contain the d alleles of the D and L loci and exhibit a CTL response against the N protein. However, unlike the BALB/c mice, these animals develop the late onset symptomatic disease. These results suggest that the N-specific response is partially protective against the development of the demyelinating disease, but that additional factors are also likely to be involved.     

Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling

Increased sensitivity to ionizing radiation has been shown to be due to defects in double-strand break repair and mutations in the proteins that detect DNA damage. However, it is now recognized that the cellular radiation response is complex and that radioresistance/radiosensitivity may also be regulated at different levels in the radiation signal transduction pathway. Here, we describe a direct relationship between resistance to radiation-induced apoptosis and defective ceramide signaling. Radiation sensitivity in human tumor cells correlated with the immediate accumulation of the second messenger ceramide. In the BL30A Burkitt's lymphoma line, ceramide increased 4-fold by 10 min postirradiation (10 Gy), and in the moderately sensitive HL-60 leukemia cells, ceramide accumulated 2.5-fold above basal levels. In contrast, in all radioresistant tumor cells examined, including several Burkitt's lymphoma lines (BL30K, BL29, and BL36) and the MO59K glioma cell line, ceramide did not accumulate postirradiation. The ability to abrogate ceramide production by pretreatment with the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, conferred resistance to radiation-induced apoptosis in the sensitive BL30A cells. An isogenic subline of BL30A, BL30K, was resistant to both C8-ceramide (20 microM) and ionizing radiation-induced apoptosis. Bypassing the block in radiation-induced ceramide production by the addition of exogenous ceramide was not sufficient to induce apoptosis; this suggests the existence of a second ceramide-associated signaling defect in these radioresistant cells that confers resistance to ceramide-induced apoptosis. Thus, these results provide compelling evidence that ceramide is an essential mediator of radiation-induced apoptosis and that defective ceramide signaling confers an apoptosis-resistant phenotype in tumor cells.     

Diversity and complexity of ceramide signalling in apoptosis

Sphingolipid ceramide has emerged as a lipid messenger of cell functions including differentiation and apoptosis. Diverse kinds of stresses (ultraviolet, irradiation, heat shock and hypoxia) and biological factors (TNF-alpha, IFN-gamma and Fas antibody) require ceramide generation to execute apoptosis. The review summarises the diversity and complexity of up- and downstream of ceramide signalling in apoptosis and clinical implications of ceramide-induced apoptosis.     

Wide tissue distribution of axolotl class II molecules occurs independently of thyroxin

The mouthguard is a resilient device or appliance which is placed inside the mouth to protect against injuries to the teeth, lacerations to the mouth and fractures and dislocations of the jaw. There is clear support in the scientific literature for the use of mouthguards in contact sports such as rugby. Moreover, there is evidence that mouthguards are effective in protecting against concussion and injuries to the cervical spine. There is a high level of acceptance of mouthguards by players and an increasing number are regularly wearing mouthguards. This is especially true among the elite players, but acceptance and wearing rates are moderately high among club players as well. There is strong support among players and researchers for mouthguard wearing to be made compulsory. It is generally recommended that: (i) mouthguards be worn during both practice sessions and games; (ii) the habit of wearing a mouthguard begins at an early age; (iii) mouthguards be regularly replaced while children are still growing; and (iv) adult players replace their mouthguards at least every 2 years. The selection of a mouthguard will depend on a number of factors including the age of the individual, effectiveness and cost. The type I (stock), or 'off-the-shelf', mouthguards are considered inferior when compared with the other available types, and their use is discouraged. Type II (mouth-formed) mouthguards come in 2 forms, the shell-liner version and the popular thermoplastic 'boil and bite' version. While the effectiveness of the shell-liner mouthguard was examined in one experimental study, no such research has been reported for the thermoplastic mouthguard. Type III (custom-fabricated) mouthguards are recommended for players playing in the more vulnerable positions and in the higher grades. Most experimental studies in which the effectiveness of mouthguards has been demonstrated have involved type III mouthguards.     

Interferon-gamma-induced differentiation and apoptosis of HT29 cells: dissociation of (glucosyl)ceramide signaling

Recently, (glyco)sphingolipids (SL) like ceramide (Cer) and glucosylceramide (GlcCer) have been shown to be involved in signaling pathways leading to differentiation and apoptosis in several cell types, including the colon adenocarcinoma cell line HT29. Intracellular levels of Cer can be modulated by ligands such as interferon-gamma (IFN gamma). In the present study we show that IFN gamma, depending on its concentration, has both differentiation- and apoptosis-inducing effects on HT29 cells. Since both phenomena have been related to SL-mediated signaling in other cell types, we next examined whether IFN gamma was able to induce changes in the SL levels of HT29 cells. Remarkably, no significant changes in these levels could be revealed, implying that SL are not involved in IFN gamma-induced differentiation and/or apoptosis of HT29 cells. This observation provides evidence for the hypothesis that SL-mediated signaling pathways might be more cell type specific than is generally assumed.     

Cytotoxic T cells overcome BCR-ABL-mediated resistance to apoptosis

Chronic myeloid leukemia is a disease marked by expanded clonal hematopoiesis; it is incurable by chemotherapy or radiation but is cured in a majority of patients receiving bone marrow transplantation from nonidentical sibling donors, an outcome generally attributed to a T cell-mediated graft-versus-leukemia effect. In this report, we examine the effect of the P210BCR-ABL fusion protein of the BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, on the sensitivity of mouse cell lines to apoptosis induced by chemotherapy, radiation, or activated cytotoxic T lymphocytes (CTLs). We find that, although cells expressing P210BCR-ABL by gene transfer are more resistant than their normal counterparts to apoptosis induced by chemotherapy or radiation, they are equally susceptible to apoptosis induced by alloreactive CTLs. These results show that CTLs overcome BCR-ABL-mediated resistance to apoptosis and, therefore, provide a biological correlation for the success of allogeneic bone marrow transplantation in chronic myelogenous leukemia.     

CTLA4 mediates antigen-specific apoptosis of human T cells

The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells.     

Stress signals for apoptosis: ceramide and c-Jun kinase

Mammalian systems respond to environmental stress by either adapting or undergoing programmed cell death. While there is general agreement that the caspase family of proteases serve as the effectors of the apoptotic death response, the signaling apparatus involved in the decision to activate the caspase system is less clear. In the past few years, the sphingomyelin and c-Jun Kinase (JNK)/Stress-activated Protein Kinase (SAPK) pathways have been linked to the death response in many cellular systems. These signaling systems are found throughout the animal kingdom, and ceramide signaling is conserved through yeast. Since yeast do not undergo apoptosis, the sphingomyelin pathway appears evolutionarily older than the caspase-mediated death programs. While recent reviews by several groups have broadly surveyed ceramide signaling in apoptosis, this paper examines the role of sphingomyelinases and the JNK/SAPK pathway in coordinate signaling of apoptosis.     

Fas-induced changes in cdc2 and cdk2 kinase activity are not sufficient for triggering apoptosis in HUT-78 cells

Telomerase activity is frequently associated with the malignant phenotype, and it can be considered an almost ubiquitous tumor marker. In this study, we evaluated telomerase activity in telomerase-positive human tumor cell lines exposed in vitro to antineoplastic agents. The results show that drug-induced cell killing of tumor cells is associated with a decline in detectable telomerase activity. The decrease of telomerase activity levels paralleled cell growth impairment evaluated by cell count or by measurement of cell ability to convert tetrazolium salt to colored formazan [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide assay]. The observed telomerase activity remaining after treatment with antineoplastic agents is most likely to reflect activity from the remaining viable cells. When tumor cell lines resistant to the chemotherapeutic agents temozolomide or doxorubicin were treated with these compounds, no decline of telomerase activity or cell growth was observed. The results of the present study indicate that resistance of neoplastic cells to chemotherapeutic agents can be monitored by following telomerase activity. Moreover, the test can be performed with a limited number of neoplastic cells, such as those frequently obtained from tumor biopsies. These findings provide a rationale for developing new in vitro chemosensitivity assays, and detection of telomerase activity may be a novel marker of chemotherapy failure.     

Differential TCR signaling and the generation of memory T cells

PURPOSE: Serum soluble interleukin-2 receptor level is a sensitive and quantitative marker of lymphocyte activation. We determined levels of serum soluble interleukin-2 receptor in children with reflux nephropathy to evaluate its clinical significance in the prediction for the progression of renal injuries. MATERIALS AND METHODS: Serum soluble interleukin-2 receptor values were determined in 63 children with reflux nephropathy. The group consisted of 37 boys and 26 girls 10 to 18 years old. T cells (naive and memory), B cells and macrophages were evaluated immunohistochemically in the scarred kidneys of 4 other patients (3 boys and 1 girl 5 to 16 years old) who underwent nephrectomy due to severe reflux nephropathy with little function seen on (99m)technetium-dimercapto-succinic acid (DMSA) renal scan. Levels of serum soluble interleukin-2 receptor were measured by an enzyme-linked immunosorbent assay. We simultaneously determined serum levels of creatinine and beta2-microglobulin, and urinary levels of alpha1-microglobulin and microalbumin. Individual functions of the right and left kidneys were estimated by renal dimercaptosuccinic acid uptake. RESULTS: Levels of serum soluble interleukin-2 receptor in the patients who had low total uptake of DMSA (right uptake plus left uptake) were significantly higher than those from patients with normal total uptake. Levels of serum soluble interleukin-2 receptor correlated significantly with levels of creatinine (r=0.616, p <0.0001) and beta2-microglobulin (r=0.803, p <0.0001), and levels of urinary alpha1-microglobulin (r=0.753, p <0.0001) and microalbumin (r=0.673, p <0.0001). A significant negative correlation was observed between levels of serum soluble interleukin-2 receptor and total DMSA uptake values (right uptake plus left uptake r=-0.678, p <0.0001). In the scarred kidneys leukocyte infiltrates were markedly increased in fibrosed spaces. The predominant cell type in these lesions was memory T cells. CONCLUSIONS: These results suggest that elevated levels of serum soluble interleukin-2 receptor are likely to reflect activated T cells in the kidneys of patients with reflux nephropathy and may be a useful predictor of progression of renal injury in these children.