Target cell death triggered by cytotoxic T lymphocytes: a target cell mutant distinguishes passive pore formation and active cell suicide mechanisms.

The role of the target cell in its own death mediated by cytotoxic T lymphocytes (CTL) has been controversial. The ability of the pore-forming granule components of CTL to induce target cell death directly has been taken to suggest an essentially passive role for the target. This view of CTL-mediated killing ascribes to the target the single role of providing an antigenic stimulus to the CTL; this signal results in the vectoral degranulation and secretion of pore-forming elements onto the target. On the other hand, by a number of criteria, target cell death triggered by CTL appears fundamentally different from death resulting from membrane damage and osmotic lysis. CTL-triggered target cell death involves primary internal lesions of the target cell that reflect a physiological cell death process. Orderly nuclear disintegration, including lamin phosphorylation and solubilization, chromatin condensation, and genome digestion, are among the earliest events, preceding the loss of plasma membrane integrity. We have tested directly the involvement of the target cell in its own death by examining whether we could isolate mutants of target cells that have retained the ability to be recognized by and provide an antigenic stimulus to CTL while having lost the capacity to respond by dying. Here, we describe one such mutant, BW87. We have used this CTL-resistant mutant to analyze the mechanisms of CTL-triggered target cell death under a variety of conditions. The identification of a mutable target cell element essential for the cell death response to CTL provides genetic evidence that target cell death reflects an active cell suicide process similar to other physiological cell deaths.     

Induction of allogeneic cytolytic T lymphocytes by partially purified membrane glycoproteins

It has been previously shown that P815 (H-2^d) purified plasma membranes can induce cytolytic activity from primed C57BL/6 (H-2^b) spleen cells. The secondary cytolytic T lymphocyte (CTL) inducing activity is retained when these P815 plasma membranes are solubilized in deoxycholate. Evidence is now presented that the cell surface antigens responsible for CTL induction can be partially purified in active form and these antigens can be incorporated into reconstituted membranes and phospholipid vesicles. The active antigens have the properties expected for H-2 molecules on lentil lectin chromatography and gel filtration.     

Purines inhibit poly(ADP-ribose) polymerase activation and modulate oxidant-induced cell death.

Purines such as adenosine, inosine, and hypoxanthine are known to have potent antiinflammatory effects. These effects generally are believed to be mediated by cell surface adenosine receptors. Here we provide evidence that purines protect against oxidant-induced cell injury by inhibiting the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Upon binding to broken DNA, PARP cleaves NAD+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins such as histones and PARP itself. Overactivation of PARP depletes cellular NAD+ and ATP stores and causes necrotic cell death. We have identified some purines (hypoxanthine, inosine, and adenosine) as potential endogenous PARP inhibitors. We have found that purines (hypoxanthine > inosine > adenosine) dose-dependently inhibited PARP activation in peroxynitrite-treated macrophages and also inhibited the activity of the purified PARP enzyme. Consistently with their PARP inhibitory effects, the purines also protected interferon gamma + endotoxin (IFN/LPS) -stimulated RAW macrophages from the inhibition of mitochondrial respiration and inhibited nitrite production from IFN/LPS-stimulated macrophages. We have selected hypoxanthine as the most potent cytoprotective agent and PARP inhibitor among the three purine compounds, and investigated the mechanism of its cytoprotective effect. We have found that hypoxanthine protects thymocytes from death induced by the cytotoxic oxidant peroxynitrite. In line with the PARP inhibitory effect of purines, hypoxanthine has prevented necrotic cell death while increasing caspase activity and DNA fragmentation. As previously shown with other PARP inhibitors, hypoxanthine acted proximal to mitochondrial alterations as hypoxanthine inhibited the peroxynitrite-induced mitochondrial depolarization and secondary superoxide production. Our data imply that purines may serve as endogenous PARP inhibitors. We propose that, by affecting PARP activation, purines may modulate the pattern of cell death during shock, inflammation, and reperfusion injury.     

Features of target cell lysis by class I and class II MHC-restricted cytolytic T lymphocytes

The lytic activity of influenza virus-specific murine cytolytic T lymphocyte (CTL) clones that are restricted by either H-2K/D (class I) or H-2I (class II) major histocompatibility (MHC) locus products was compared on an influenza virus-infected target cell expressing both K/D and I locus products. With the use of two in vitro measurements of cytotoxicity, conventional 51Cr release, and detergent-releasable radiolabeled DNA (as a measure of nuclear disintegration in the early post-lethal hit period), we found no difference between class I and class II MHC-restricted CTL in the kinetics of target cell destruction. In addition, class II MHC-restricted antiviral CTL failed to show any lysis of radiolabeled bystander cells. Killing of labeled specific targets by these class II MHC-restricted CTL was also efficiently inhibited by unlabeled specific competitor cells in a cold target inhibition assay. In sum, these data suggest that class I and class II MHC-restricted CTL mediate target cell destruction by an essentially similar direct mechanism.     

Staurosporine induced poly (ADP-ribose) polymerase independent cell death in Dictyostelium discoideum

In the present study D. discoideum has been used as a model organism to understand the role of poly (ADP-ribose) polymerase (PARP) in caspase independent paraptotic cell death pathways. D. discoideum lacks caspases and Bcl-2 family proteins; nevertheless it has 9 potential genes for PARP. PARP has been known to get activated in various cell death associated diseases. In this study kinetics of cell death induced by staurosporine (STS), a bacterial alkaloid, was established to unravel the role of PARP. It was found that STS induced cell death in D. discoideum did not involve PARP activation, however it involved cathepsin D. Results indicated that an alternative mechanism may be existing in D. discoideum that lacks Bcl-2 family proteins for STS induced cell death that evades Bax involvement.     

Demonstration of a calcium influx in cytolytic T lymphocytes in response to target cell binding

By using the Ca2+-sensitive dye indo-1, an antigen-specific increase in intracellular Ca2+ in cloned cytolytic T lymphocytes (CTL) was measured under conditions that were permissive for T cell-mediated cytolysis. To synchronize lethal hit delivery in a suspension of effector and target cells, a modification of the cation pulse method in which Ca2+ is added to preformed conjugates of CTL and target cells was used. Conjugate formation was unaffected by the absence of extracellular Ca2+ under these conditions. Lytic activity of these cloned CTL was markedly reduced in the absence of extracellular Ca2+ and was restored upon Ca2+ repletion. When indo-1-loaded CTL were preincubated with target cells in the absence of extracellular Ca2+, a marked antigen-specific increase in indo-1 fluorescence, indicative of an increase in intracellular Ca2+, was observed after repletion of extracellular Ca2+. This increase in intracellular Ca2+ was shown to be due solely to changes in the CTL and not the target cell within the time course of the experiment, and results from the influx of extracellular Ca2+. Antibody to the T cell receptor for antigen also evokes a similar increase in intracellular Ca2+ in CTL under these conditions. This method provides a means for the direct examination of the response of CTL to cellular antigen as well as soluble antibody and is a versatile and valuable tool for the study of CTL function.     

Role of poly(ADP-ribose) polymerase activity in imatinib mesylate-induced cell death

Imatinib targets Bcr-Abl, the causative event of chronic myelogenous leukemia (CML), and addresses leukemic cells to growth arrest and cell death. The exact mechanisms responsible for imatinib-induced cell death are still unclear. We investigated the role of poly(ADP-ribose) polymerase (PARP) activity in imatinib-induced cell death in Bcr-Abl-positive cells. Imatinib leads to a rapid increase of poly(ADP-ribosyl)ation (PAR) preceding loss of integrity of mitochondrial membrane and DNA fragmentation. The effect of imatinib on PAR can be mimicked by inhibition of phosphatidylinositol 3-kinase (PI3-K) implicating a central role of the PI3-K pathway in Bcr-Abl-mediated inhibition of PAR. Importantly, inhibition of PAR in imatinib-treated cells partially prevented cell death to an extent comparable to that observed after caspase inhibition. Simultaneous blockade of both caspases and PAR revealed additive cytoprotective effects indicating that both pathways function in parallel. In conclusion, our results suggest that in addition to the well-documented caspase-dependent pathway, imatinib also induces a PARP-mediated death process.     

Oxygen radicals induce poly(ADP-ribose) polymerase-dependent cell death in cytotoxic lymphocytes

Cytotoxic T cells and NK cells will acquire features of apoptosis when exposed to oxygen radicals, but the molecular mechanisms underlying this phenomenon are incompletely understood. We have investigated the role of two enzyme systems responsible for execution of cell death, caspases and the poly(ADP-ribose) polymerase (PARP). We report that although human cytotoxic lymphocytes were only marginally protected by caspase inhibitors, PARP inhibitors completely protected lymphocytes from radical-induced apoptosis and restored their cytotoxic function. The radical-induced, PARP-dependent cell death was accompanied by nuclear accumulation of apoptosis-inducing factor and a characteristic pattern of large-fragment DNA degradation. It is concluded that the PARP/apoptosis-inducing factor axis is critically involved in oxygen radical-induced apoptosis in cytotoxic lymphocytes.     

Increased expression of poly(ADP-ribose) polymerase-1 contributes to caspase-independent myocyte cell death during heart failure

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in regulating genome stability, cell cycle progression, and cell survival. However, overactivation of PARP has been shown to contribute to cell death and organ failure in various stress-related disease conditions. In this study, we examined the role of PARP in the development and progression of cardiac hypertrophy. We measured the expression of PARP in mouse hearts with physiological (swimming exercise) and pathological (aortic banding) cardiac hypertrophy as well as in human heart samples taken at the time of transplantation. PARP levels were elevated both in swimming and banded mice hearts and demonstrated a linear positive correlation with the degree of cardiac hypertrophy. A dramatic increase (4-fold) of PARP occurred in 6-wk banded mice, accompanied by apparent signs of ventricular dilation and myocyte cell death. PARP levels were also elevated (2- to 3-fold) in human hearts with end-stage heart failure compared with controls. However, we found no evidence of caspase-mediated PARP cleavage in either mouse or human failing hearts. Overexpression of PARP in primary cultures of cardiac myocytes led to suppression of gene expression and robust myocyte cell death. Furthermore, data obtained from the analysis of PARP knockout mice revealed that these hearts produce an attenuated hypertrophic response to aortic banding compared with controls. Together, these results demonstrate a role for PARP in the onset and progression of cardiac hypertrophy and suggest that some events related to cardiac hypertrophy growth and progression to heart failure are mediated by a PARP-dependent mechanism.     

Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.

The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Accessory cell requirements for the generation of cytolytic T lymphocytes.

A system is presented that may simplify the study of accessory cell requirements for CTL generation. Cortisone resistant (CR) thymocytes containing alloreactive CTL precursors do not respond to allogeneic tumor cells unless non-T accessory cells are added to culture. In addition, splenic T cells do not respond to allogeneic tumor cells in the absence of non-T accessory cells. These accessory cells share several properties of macrophages.     

Involvement of poly(ADP-ribose) polymerase activity in regulating Chk1-dependent apoptotic cell death

The activity of poly(ADP-ribose) polymerase (PARP) is highly stimulated following DNA damage resulting in formation of DNA nicks and strand breaks. This leads to modification of numerous proteins, including itself, using NAD(+) as substrate and to exhaustion of intracellular ATP. A highly cytotoxic concentration of the DNA methylating agent methyl methanesulfonate (MMS) results in cellular ATP depletion and cell death primarily by necrosis in both wild-type and DNA polymerase beta null mouse fibroblasts. The loss of ATP can be prevented by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN), and now cells die by an energy-dependent apoptotic pathway. We find that inhibition of PARP activity transforms a sub-lethal exposure to MMS into a highly cytotoxic event. Under this condition, ATP is not depleted and cell death is by apoptosis. The caspase inhibitor, Z-VAD, shifts the mechanism of cell death to necrosis indicating a caspase-dependent component of the apoptotic cell death. Co-exposure to the Chk1 inhibitor UCN-01 also produces a decrease in apoptotic cell death, but now there is an increase in viable cells and an enhancement in long-term survival. Taken together, our results suggest that inhibition of PARP activity, induced as a result of low dose MMS exposure, signals via a Chk1-dependent pathway for cell death by apoptosis.     

Murine melanoma cell differentiation and melanogenesis induced by poly(ADP-ribose) polymerase inhibitors.

Murine melanoma cells treated with the melanocyte-stimulating hormone (MSH) family of peptides undergo differentiation characterized by enhanced melanogenesis and altered morphology. These effects are mediated via the adenylate cyclase-cAMP pathway leading to activation of protein kinase A (PKA). We have discovered that inhibition of a post-translational modification of chromatin proteins, viz. poly(ADP-ribosylation), also induces melanogenesis and differentiation in these cells. A range of competitive inhibitors (benzamide and its derivatives) of the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP; EC 2.4.2.30) was utilized, and their ability to induce melanogenesis reflected their potency as PADPRP inhibitors. These compounds induced melanogenesis at low doses (20 microM-2 mM) which did not affect cell growth or viability. Induction of melanogenesis was not attributable to inhibition of cyclic nucleotide phosphodiesterase by these compounds. MSH treatment caused a transient rise in cAMP levels (up to 200-fold by 5 min and returning to near basal levels by 5 h). It also stimulated PKA activity up to 5-fold, and the temporal kinetics of this activation mirrored the changes in cAMP levels. In comparison, the PADPRP inhibitors had no effect on either of these processes. These data constitute a novel demonstration of a cAMP-independent mechanism for the induction of melanoma cell differentiation, including melanogenesis.     

Cytoplasmic poly(ADP-ribose) polymerase during the HeLa cell cycle.

Poly(ADP-ribose) polymerase, an enzyme that has reportedly been confined to the nucleus of eukaryotic cells, has been found in the cytoplasm of HeLa cells. The enzyme activity is stimulated more than 30-fold by the addition of both DNA and histones. These two macromolecules are absolutely necessary for maximal activity and they act in a synergistic manner. The product of the reaction was characterized as poly(ADP-ribose) by its acid insolubility, its insensitivity to hydrolysis by DNase, RNase, spleen phosphodiesterase or Pronase and by release of 5′-AMP and 2′-(5″-phosphoribosyl)-5′-AMP by incubation with snake venom phosphodiesterase. A covalent attachment between histone F1 and poly(ADP-ribose) has been established by using the cytoplasmic enzyme. The enzyme is primarily associated with ribosomes, both free ribosomes and those found in polysomes. Inhibition of protein synthesis in the intact cell reduced the level of activity in the cytoplasm. The enzyme can be removed from the ribosomes by centrifugation through sucrose gradients containing 0.6 m ammonium chloride. A relationship between this enzyme and DNA replication is suggested by the fact that the specific activity in the cytoplasm parallels the rate of DNA synthesis during the HeLa cell cycle.     

Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death

Poly(ADP-ribose) polymerase (PARP) is responsible for post-translational modification of proteins in the response to numerous endogenous and environmental genotoxic agents. PARP and poly(ADP-ribosyl)ation are proposed to be important for the regulation of many cellular processes such as DNA repair, cell death, chromatin functions and genomic stability. Activation of PARP is one of the early DNA damage responses, among other DNA sensing molecules, such as DNA-PK, ATM and p53. The generation and characterization of PARP deficient mouse models have been instrumental in defining the biological role of the molecule and its involvement in the pathogenesis of various diseases including diabetes, stroke, Parkinson disease, general inflammation as well as tumorigenesis, and have, therefore, provided information for the development of pharmaceutical strategies for the treatment of diseases.     

Inactivation of poly (ADP-ribose) polymerase by hypochlorous acid.

The effects of the phagocyte-derived reactive oxidants hydrogen peroxide (H2O2) and hypochlorous acid (HOC1) on the activity of poly(ADP-ribose) polymerase (pADP RP), an enzyme involved in DNA repair, and on the induction and repair of DNA strand breaks in human mononuclear leukocytes (MNL) have been investigated in vitro. Exposure of MNL to reagent H2O2 was accompanied by DNA damage and activation of pADP RP. Addition of reagent HOCl (25 microM) was not associated with DNA strand breaks. However, when combined with 150 microM H2O2, HOCl potentiated H2O2-mediated DNA damage, and compromised the repair process. Furthermore, HOCl caused a dose-related decrease in the activity of pADP RP in both control and H2O2-exposed MNL. Interactions between the phagocyte-derived reactive oxidants H2O2 and HOCl are probably involved in the etiology of inflammation-related cancer.     

Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes

It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed.