Innate apoptotic immunity: the calming touch of death.

Apoptotic cell death is an essential and highly ordered process that contributes to both the development and the homeostasis of multicellular organisms. It is associated with dramatic biochemical and cell biological events within the dying cell, including fragmentation of the nucleus and the redistribution of intracellular proteins and membrane lipids. It has long been apparent that phagocytic clearance of the cell corpse is an integral part of the apoptotic process; apoptotic clearance also may be essential in tissue homeostasis. During the cell death process, apoptotic cells acquire new cell surface determinants for specific recognition by responder phagocytes and suppression of immune responsiveness. Recent studies indicate that these determinants are well conserved throughout metazoan evolution; remarkably, their recognition shows no species-specific restriction. Professional and non-professional phagocytes recognize and respond to apoptotic cells similarly, notably with the immediate-early transcriptional repression of a variety of specific genes including those encoding inflammatory cytokines. Secondary responses following engulfment of the apoptotic corpse, utilizing several distinct mechanisms, enhance and sustain this apoptotic suppression. In this review, we highlight the central role of apoptotic cells in innate homeostatic regulation of immunity.

Caspase-dependent Cdk activity is a requisite effector of apoptotic death events.

The caspase-dependent activation of cyclin-dependent kinases (Cdks) in varied cell types in response to disparate suicidal stimuli has prompted our examination of the role of Cdks in cell death. We have tested the functional role of Cdk activity in cell death genetically, with the expression of dominant negative Cdk mutants (DN-Cdks) and Cdk inhibitory genes. Here we demonstrate that Cdk2 activity is necessary for death-associated chromatin condensation and other manifestations of apoptotic death, including cell shrinkage and the loss of adhesion to substrate. Susceptibility to the induction of the cell death pathway, including the activation of the caspase cascade, is unimpaired in cells in which Cdk2 activity is inhibited. The direct visualization of active caspase activity in these cells confirms that death-associated Cdk2 acts downstream of the caspase cascade. Cdk inhibition also does not prevent the loss of mitochondrial membrane potential and membrane phospholipid asymmetry, which may be direct consequences of caspase activity, and dissociates these events from apoptotic condensation. Our data suggest that caspase activity is necessary, but not sufficient, for the full physiological cell death program and that a requisite function of the proteolytic caspase cascade is the activation of effector Cdks.

T-cell regulation. Tails of phosphorylation and T-cell activation.

How do quantitative differences in T-cell signal transduction lead to qualitatively different responses? Recent work demonstrates that even well-established regulatory paradigms are open to question.

Glucocorticoids and chromosomal position modulate murine mammary tumor virus transcription by affecting efficiency of promoter utilization.

The rate of transcription of murine mammary tumor virus (MTV) sequences in MTV-infected rat hepatoma tissue culture cells is strongly affected by both glucocorticoid hormones and the chromosomal position of provirus integration. We have characterized MTV RNAs produced in J2.17 and M1.54, independent isolates containing, respectively, 1 and 10 proviruses integrated at distinct chromosomal loci. M1.54, but not J2.17, synthesized MTV RNA in the absence of glucocorticoids; the rate of hormone-stimulated viral gene transcription in M1.54 was 50- to 100-fold higher than in J2.17. In each case in which MTV genes were expressed (J2.17 induced, M1.54 basal and induced), the viral RNAs produced were indistinguishable. RNA blotting revealed accumulation of two transcripts, 7.8 and 3.8 kilobases; the latter was likely produced from the former by RNA splicing. Sites used for transcription initiation, polyadenylation, and splicing have been identified from the sizes of end-labeled hybridization probes protected from digestion with mung bean nuclease; the unique initiation and polyadenylation sites were both encoded within the MTV long-terminal-repeat sequence. The efficiencies of splicing and of utilization of the polyadenylation signal did not appear to vary as functions of chromosomal position or hormonal stimulation. Differences in rates of viral gene transcription were reflected in the differential accumulation of the 5'-terminal 136 nucleotides of MTV RNA. Thus, glucocorticoids and chromosomal position appeared to affect solely the efficiency of utilization of the MTV promoter, leaving unchanged the sites of initiation, splicing, and polyadenylation, as well as the efficiencies of the latter two processes.

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.

Commitment and effector phases of the physiological cell death pathway elucidated with respect to Bcl-2 caspase, and cyclin-dependent kinase activities.

Physiological cell deaths occur ubiquitously throughout biology and have common attributes, including apoptotic morphology with mitosis-like chromatin condensation and prelytic genome digestion. The fundamental question is whether a common mechanism of dying underlies these common hallmarks of death. Here we describe evidence of such a conserved mechanism in different cells induced by distinct stimuli to undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern of requisite components. We have dissected the role of cysteine proteases (caspases) in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream interleukin-1beta-converting enzyme-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing the activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. In addition to caspase 3-like proteases, the effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. The conservation of the cell death mechanism is not reflected in the molecular identity of its individual components, however. For example, we have detected different cyclin-Cdk pairs in different instances of cell death. The ordered course of events that we have observed in distinct cases reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.

Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes.

We examined virally transformed murine fibroblast clones as targets for cytotoxic T lymphocyte (CTL)-triggered lysis and genome digestion. Strikingly, while all clones were essentially equivalent in the ability to be lysed, one clone, SV3T3-B2.1, failed to exhibit genome digestion associated with CTL attack. Other aspects of the physiological cell death process, including loss of adhesion and nuclear envelope breakdown (lamin phosphorylation and solubilization), were not altered in this clone. The absence of genome digestion associated with CTL-induced cell death correlated with the absence of endodeoxyribonuclease activity in the nuclei of that clone. Characterization of the activity affected identifies a calcium-dependent, DNase I-like endonuclease of approximately 40 kDa, normally present constitutively in all cell nuclei, as the enzyme responsible for genome digestion associated with CTL-mediated cell death. These observations indicate that neither genome digestion per se nor its consequences [such as activation of poly(ADP-ribose) polymerase] are essential for cell death resulting from the triggering of this cell suicide process.

The cell cycle-regulatory CDC25A phosphatase inhibits apoptosis signal-regulating kinase 1.

CDC25A phosphatase promotes cell cycle progression by activating G(1) cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts. In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening. ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal protein kinase-stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses. Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm. The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A. This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1. Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli. Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression. These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses.

Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex.

The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.

Distinct modes of macrophage recognition for apoptotic and necrotic cells are not specified exclusively by phosphatidylserine exposure.

The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead cells. This clearance is believed to reveal an innate immunity, associated with inflammation in cases of pathological but not physiological cell deaths. Using objective and quantitative measures to assess these processes, we find that macrophages bind and engulf native apoptotic and necrotic cells to similar extents and with similar kinetics. However, recognition of these two classes of dying cells occurs via distinct and noncompeting mechanisms. Phosphatidylserine, which is externalized on both apoptotic and necrotic cells, is not a specific ligand for the recognition of either one. The distinct modes of recognition for these different corpses are linked to opposing responses from engulfing macrophages. Necrotic cells, when recognized, enhance proinflammatory responses of activated macrophages, although they are not sufficient to trigger macrophage activation. In marked contrast, apoptotic cells profoundly inhibit phlogistic macrophage responses; this represents a cell-associated, dominant-acting anti-inflammatory signaling activity acquired posttranslationally during the process of physiological cell death.

Chemopreventive isothiocyanates induce apoptosis and caspase-3-like protease activity.

Isothiocyanates exert strong anticarcinogenic effects in a number of animal models of cancer, presumably by modulation of xenobiotic-metabolizing enzymes, such as by inhibition of cytochrome P-450 and/or by induction of phase II detoxifying enzymes. Here, we report that phenethyl isothiocyanate and other structurally related isothiocyanates, phenylmethyl isothiocyanate, phenylbutyl isothiocyanate, and phenylhexyl isothiocyanate, but not phenyl isothiocyanate induced apoptosis in HeLa cells in a time- and dose-dependent manner. Treatment with apoptosis-inducing concentrations of isothiocyanates also caused rapid and transient induction of caspase-3/CPP32-like activity. Furthermore, these isothiocyanates, except phenyl isothiocyanate, stimulated proteolytic cleavage of poly(ADP-ribose) polymerase, which followed the appearance of caspase activity and preceded DNA fragmentation. Pretreatment with a potent caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde inhibited isothiocyanate-induced caspase-3-like activity and apoptosis. These results suggest that isothiocyanates may induce apoptosis through a caspase-3-dependent mechanism. The induction of apoptosis by isothiocyanates may provide a distinct mechanism for their chemopreventive functions.

Exploiting death: apoptotic immunity in microbial pathogenesis.

Innate immunity typically is responsible for initial host responses against infections. Independently, nucleated cells that die normally as part of the physiological process of homeostasis in mammals (including humans) suppress immunity. Specifically, the physiological process of cell death (apoptosis) generates cells that are recognized specifically by viable cells of all types and elicit a profound transient suppression of host immunity (termed 'innate apoptotic immunity' (IAI)). IAI appears to be important normally for the maintenance of self-tolerance and for the resolution of inflammation. In addition, pathogens are able to take advantage of IAI through a variety of distinct mechanisms, to enable their proliferation within the host and enhance pathogenicity. For example, the protist pathogen Leishmania amazonensis, at its infective stage, mimics apoptotic cells by expressing apoptotic-like protein determinants on the cell surface, triggering immunosuppression directly. In contrast, the pathogenic bacterium Listeria monocytogenes triggers cell death in host lymphocytes, relying on those apoptotic cells to suppress host immune control and facilitate bacterial expansion. Finally, although the inhibition of apoptotic cell death is a common attribute of many viruses which facilitates their extended replication, it is clear that adenoviruses also reprogram the non-apoptotic dead cells that arise subsequently to manifest apoptotic-like immunosuppressive properties. These three instances represent diverse strategies used by microbial pathogens to exploit IAI, focusing attention on the potency of this facet of host immune control. Further examination of these cases will be revealing both of varied mechanisms of pathogenesis and the processes involved in IAI control.

Differential activation of MAPK and ICE/Ced-3 protease in chemical-induced apoptosis. The role of oxidative stress in the regulation of mitogen-activated protein kinases (MAPKs) leading to gene expression and survival or activation of caspases leading to apoptosis.

Chemical-induced oxidative stress to a cell can signal many cellular responses which include proliferation, differentiation, hemeostasis, apoptosis or necrosis. To better understand the underlying molecular mechanisms after exposure to chemicals, we investigated the signal transduction pathways, in particular the mitogen-activated protein kinase (MAPK) pathway and the ICE/Ced-3 protease (caspase) pathway, activated by different agents. Butylated hydroxyanisol (BHA) and its metabolite, t-butyl-hydroquinone (tBHQ), both are well known phenolic antioxidants used in food preservatives, strongly activated c-Jun N-terminal kinase 1 (JNK1) and/or extracellular signal-regulated protein kinase 2 (ERK2) in a dose- and time-dependent fashion. Pretreatment with free radical scavengers N-acetyl-L-cysteine (NAC), glutathione (GSH), or vitamin E, inhibited ERK2 activation and, to a much lesser extent, JNK 1 activation by BHA and tBHQ, implicating the role of oxidative stress. Under conditions where JNK1 and ERK2 were activated, BHA also activated transcription factors nuclear factor kappa B (NF-kappaB), activated-protein-1 (AP-1), and anti-oxidant response element (ARE), leading to induction of genes such as c-jun, and c-fos. At relatively high concentrations, BHA and tBHQ stimulated proteolytic activity of ICE/Ced3 cysteine proteases, and caused apoptosis, which was blocked by pretreatment with NAC. Further increase in concentrations lead to rapid cell death predominantly occurred via necrosis. Some naturally occurring phytochemicals, such as phenylethyl isothiocyanate (PEITC), green tea polyphenols (GTP), and sulfarophane, which have been shown to be potent inducers of Phase II enzymes, also differentially regulated the activities of JNK, ERK, or CPP-32, in a time- and dose-dependent manner. Our data, together with the work of others, enable us to propose a model in which low concentrations of these chemicals (e.g., BHA, PEITC) activate MAPKs leading to induction of gene expression (e.g., c-jun, c-fos, GSI) which may protect the cells against toxic insults and enhance cell survival. At relatively high concentrations, these agents activated both MAPKS, and the ICE/Ced-3 caspase pathway, leading to apoptosis. The exact mechanisms by which MAPK and caspases are activated by these agents are currently unknown, but may involve oxidative modification of glutathione (GSH) and/or protein thiols, and/or generation of secondary messengers, ceramide and calcium, which further activate downstream events. Taken together, our results suggest that chemicals including phenolic antioxidants activate MAPK pathways which may lead to the induction of genes producing protection and survival mechanisms, as well as the ICE/Ced-3 protease pathway, leading to apoptosis. The balancing amongst these pathways may dictate the fate of the cells upon exposure to chemicals.

Molecular mapping of the physiological cell death process. Mitochondrial events may be disordered.

The mitochondrion plays a central role in Bcl-2-inhibitable physiological cell deaths. The detailed order of mitochondrial and other events during cell death in vivo remains ambiguous, however. As part of an effort to explore this issue, we have asked whether mitochondrial dissolution during physiological cell death occurs in an orderly and concerted process. Here, we describe the characterization of two elements of mitochondrial disintegration on the level of individual cells. Using a novel cytofluorimetric approach, we have assessed simultaneously the release of cytochrome c (specifically a fluorescently tagged transfected construct) from mitochondria and the dissipation of mitochondrial membrane potential. Our results indicate that mitochondrial disintegration does not follow a strictly ordered process and is not concerted. We are extending these studies to further characterize mitochondrial events in the context of Bcl-2 family members and place them definitively within the context of the caspase cascade.

Apoptotic morphology reflects mitotic-like aspects of physiological cell death and is independent of genome digestion.

Several hallmarks characterize what has come to be recognized as a common physiological process of cell death. In particular, the two defining characteristics are the apoptotic morphology of cell shrinkage and chromatin condensation originally described by Kerr et al. [(1972) Br. J. Cancer, 26:239-256] and the prelytic digestion of genomic DNA of the dying cell, as noted first by Wyllie [(1980) Nature, 284:555-556] and Russell et al. [(1982) J. Immunol., 128:2087-2094]. Many suicidal stimuli are able to modulate this process; each of these suicidal inducers activates cell death via a specific pathway. While it remains to be established, we hypothesize that a single mechanism of physiological cell death pertains in all cases [Ucker (1991) New Biol., 3:103-109; Ucker et al. (1994) Immunol. Rev., 142:273-299]. The various modulatory processes act afferently on this single effector pathway. We have examined the significance of the hallmarks of physiological cell death in an effort to elucidate critical mechanistic elements of the cell death process. Here we describe our recent studies of genome digestion. Our work has centered on the characterization of a set of fibroblastic cell clones that vary in their ability to undergo genome digestion associated with physiological cell death induced by cytotoxic T lymphocytes (CTL) and other stimuli. Our results demonstrate that genome digestion is dispensable for physiological cell death and that apoptotic morphology is independent of genome digestion. Our data suggest further that apoptotic morphology is reflective of mitotic-like aspects of the cell death process.

Cytotoxic T lymphocytes and glucocorticoids activate an endogenous suicide process in target cells.

Cytotoxic T lymphocytes (CTL) induce a cytolytic process in target cells which, like the glucocorticoid-mediated cytolysis of immature thymocytes, effects a rapid and characteristic degradation of chromosomal DNA. I have explored the possibility that these two lethal processes share a common pathway by studying the susceptibility of glucocorticoid-resistant mutants to CTL-mediated killing. Here, I report that an unusual thymoma mutant, which has normal hormone receptor activity, is resistant to both glucocorticoids and CTL. The failure to be killed by CTL is not due to an inability of this 'deathless' mutant to be recognized. Further, a single-step reversion can restore sensitivity to both glucocorticoids and CTL. The genetic locus thus identified may reveal one element of an endogenous suicide pathway that can be triggered by different effectors. Unlike complement-mediated lysis, the processes of glucocorticoid- and CTL-mediated cytolysis seem to require that target cells be active in their own death.

Death by suicide: one way to go in mammalian cellular development?

Cell deaths occur selectively in many types of tissues throughout development. These physiological deaths appear to follow an orderly process of internal cellular disintegration that is distinct from the process observed in cell death resulting from trauma. Studies of a variety of physiological cell deaths have revealed that this process appears generally to involve the active participation of the dying cell in its own death. In other words, physiological cell death seems to be a process of induced cellular self-destruction, or cell suicide. Whether a single, genetically determined mechanism is utilized in all cell suicides remains to be established. Nonetheless, while genome digestion and intracellular calcium rises are dissociable from, and thus neither necessary nor sufficient for, cell death, control of the cell cycle may be critical in all cases of induced cell suicide. It is proposed here that physiological cell death occurs through a process of abortive mitosis.

Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.