Life and death of lymphocytes: a volume regulation affair

The loss of cell volume, termed apoptotic volume decrease (AVD) has been a hallmark feature of apoptosis. However the role of this characteristic attribute of programmed cell death has always been questioned as to whether it plays an active or passive factor during apoptosis. Here we review studies that suggest that AVD plays an active role during apoptosis and the underlying flux of ions that results in this morphological event regulates the programmed cell death process.     

Identification of apoptotic cell death in distraction osteogenesis

The purpose of this experimental work was to investigate whether apoptosis contributes to tissue remodelling during distraction bone healing. In a rabbit model of mandibular distraction osteogenesis, we quantitatively analysed the extent of apoptotic cell death in relation to differently applied mechanical loadings. Apoptotic cells were identified by means of an in situ detection assay for nuclear DNA fragmentation using a modified TUNEL procedure and by electron microscopical examination for typical morphological features of programmed cell death. TUNEL-positive cells were frequently detected in samples distracted at higher strain magnitudes. Ultrastructurally, these apoptotic cells displayed a condensed chromatin and fragmented nuclei, while the continuity of their plasma membranes remained intact. Our results clearly indicated that the discontinuous traction of osteotomized mandibles induced enhanced apoptosis. In contrast to non-distracted samples and mandibles distracted at low strain magnitudes, in which only minimal evidence of apoptotic cell death was detected, the application of hyperphysiological strain magnitudes resulted in an increased apoptosis rate. Thus, mechanical loading seems to be a triggering factor for apoptotic changes in osteoblastic cells. These findings suggest a pathophysiological role of apoptotic cell death in the control of tissue integrity during distraction osteogenesis.     

The morphology of apoptosis

The concept of apoptotic cell death as an essential part of the development and life of complex organisms has been devised in different situations and tested from various angles. This review article discusses the morphological changes during death by apoptosis. In cells undergoing apoptosis, an intracellular signalling pathway operates cell autonomously to implement the death and disposal of the cell. The similarity of the biochemical events during apoptosis in different situations is reflected by a high uniformity of morphological changes in many situations of naturally occurring or experimentally induced cell death. The unifying concept of apoptosis has been derived from the observation of this morphological consistency of dying cells almost 30 years ago. Since then, we have learned much about the intracellular signalling in the apoptotic process and the molecular background has been delineated which guides the initiation of the morphological changes. Here, an attempt is made to present the current knowledge about the molecular events in the development of these morphological alterations and to place these changes in the context of apoptotic cell death.     

Apoptosis in the lung: induction, clearance and detection

Apoptosis and other forms of programmed cell death are important contributors to lung pathophysiology. In this brief review, we discuss some of the implications of finding apoptotic cells in the lung and methods for their detection. The balance between induction of apoptosis and the normally highly efficient clearance of such cells shows that these are highly dynamic processes and suggests that abnormalities of apoptotic cell clearance may be an alternative explanation for their detection. Because recognition of apoptotic cells by other lung cells has additional effects on inflammation, immunity, and tissue repair, local responses to the dying cells may also have important consequences in addition to the cell death itself.     

Sculpturing digit shape by cell death

Physiological cell death is a key mechanism that ensures appropriate development and maintenance of tissues and organs in multicellular organisms. Most structures in the vertebrate embryo exhibit defined areas of cell death at precise stages of development. In this regard the areas of interdigital cell death during limb development provide a paradigmatic model of massive cell death with an evident morphogenetic role in digit morphogenesis. Physiological cell death has been proposed to occur by apoptosis, cellular phenomena genetically controlled to orchestrate cell suicide following two main pathways, cytochrome C liberation from the mitochondria or activation of death receptors. Such pathways converge in the activation of cysteine proteases known as caspases, which execute the cell death program, leading to typical morphologic changes within the cell, termed apoptosis. According to these findings it would be expected that caspases loss of function experiments could cause inhibition of interdigital cell death promoting syndactyly phenotypes. A syndactyly phenotype is characterized by absence of digit freeing during development that, when caused by absence of interdigital cell death, is accompanied by the persistence of an interdigital membrane. However this situation has not been reported in any of the KO mice or chicken loss of function experiments ever performed. Moreover histological analysis of dying cells within the interdigit reveals the synchronic occurrence of different types of cell death. All these findings are indicative of caspase alternative and/or complementary mechanisms responsible for physiological interdigital cell death. Characterization of alternative cell death pathways is required to explain vertebrate morphogenesis. Today there is great interest in cell death via autophagy, which could substitute or act synergistically to the apoptotic pathway. Here we discuss what is known about physiological cell death in the developing interdigital tissue of vertebrate embryos, paying special attention to the avian species.     

斑马鱼胚胎发育中细胞凋亡的研究进展

细胞凋亡是细胞在基因调控下发生的主动消亡过程,在脊椎动物胚胎发育过程中非常重要。斑马鱼作为一种十分理想的发育分子生物学研究模型,在有关细胞凋亡在诸如形态发生、性别分化等方面功能之活体在位研究中日益受到重视。目前,斑马鱼胚胎发育中主要凋亡通路研究已进行了不少工作,特别是caspase及其它凋亡调控基因在斑马鱼中已被成功克隆,通过转基因斑马鱼胚胎中胁迫诱导细胞凋亡并研究其信号通路以及斑马鱼胚胎形态发生的异常改变,为阐明这些凋亡调控基因与发育之间的关系提供了一个强有力的手段。     

Assessment of apoptosis in xenobiotic-induced immunotoxicity.

Generation of immunity is a highly complex process in which proliferation and differentiation of immune-competent cells regulated by cytokines and cell-cell interactions play a major role. Reducing the number of immune-competent cells or altering the function, selection, and differentiation of lymphocytes after xenobiotic treatment may lead to serious adverse effects. Programmed cell death, or apoptosis, is a highly regulated process by which an organism eliminates unwanted cells without eliciting an inflammatory response. However, xenobiotics are also able to trigger unwanted apoptosis or to alter the regulation of programmed cell death. Cytological characteristics of apoptosis are generally different from those seen in acute pathological cell death resulting from cell injury. The morphological characteristics of apoptosis are unique including cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, disruption of the nuclear lamina, nuclear fragmentation, and emergence of apoptotic bodies. It is now established that apoptosis plays a critical role in both development and homeostasis of the immune system: thymic selection, cytotoxicity, deletion of autoreactive cells, and regulation of the size of the lymphoid compartment. Assessment of apoptosis relies on the morphological and biochemical modifications of the dying cells. As a rule, and because an apoptotic cell rarely displays all of the characteristic apoptotic features, several criteria should be monitored in parallel including morphological examination. The techniques described in this paper have been divided into five categories: analysis of cell morphology by microscopy, identification of DNA fragmentation, determination of mitochondrial membrane potential, detection of plasma membrane changes, analysis of caspase activation.     

Modeling cell apoptosis for simulating three-dimensional multicellular morphogenesis based on a reversible network reconnection framework

Morphogenesis in multicellular organisms is accompanied by apoptotic cell behaviors: cell shrinkage and cell disappearance. The mechanical effects of these behaviors are spatiotemporally regulated within multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze 3D multicellular dynamics, 3D vertex models have been suggested, in which a reversible network reconnection (RNR) model has successfully expressed 3D cell rearrangements during large deformations. To analyze the effects of apoptotic cell behaviors on 3D multicellular morphogenesis, we modeled cell apoptosis based on the RNR model framework. Cell shrinkage was modeled by the potential energy as a function of individual cell times during the apoptotic phase. Cell disappearance was modeled by merging neighboring polyhedrons at their boundary surface according to the topological rules of the RNR model. To establish that the apoptotic cell behaviors could be expressed as modeled, we simulated morphogenesis driven by cell apoptosis in two types of tissue topology: 3D monolayer cell sheet and 3D compacted cell aggregate. In both types of tissue topology, the numerical simulations successfully illustrated that cell aggregates gradually shrank because of successive cell apoptosis. During tissue shrinkage, the number of cells in aggregates decreased while maintaining individual cell size and shape. Moreover, in case of localizing apoptotic cells within a part of the 3D monolayer cell aggregate, the cell apoptosis caused the global tissue bending by pulling on surrounding cells. In case of localizing apoptotic cells on the surface of the 3D compacted cell aggregate, the cell apoptosis caused successive, directional cell rearrangements from the inside to the surface. Thus, the proposed model successfully provided a basis for expressing apoptotic cell behaviors during 3D multicellular morphogenesis based on an RNR model framework.     

Steroid regulation of programmed cell death during Drosophila development

Steroid hormones play an important role in the regulation of numerous physiological responses, but the mechanisms that enable these systemic signals to trigger specific cell changes remain poorly characterized. Recent studies of Drosophila illustrate several important features of steroid-regulated programmed cell death. A single steroid hormone activates both cell differentiation and cell death in different tissues and at multiple stages during development. While several steroid-regulated genes are required for cell execution, most of these genes function in both cell differentiation and cell death, and require more specific factors to kill cells. Genes that regulate apoptosis during Drosophila embryogenesis are induced by steroids in dying cells later in development. These apoptosis genes likely function downstream of hormone-induced factors to serve a more direct role in the death response. This article reviews the current knowledge of steroid signaling and the regulation of programmed cell death during development of Drosophila.     

Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.     

Cell shrinkage and monovalent cation fluxes: role in apoptosis

The loss of cell volume or cell shrinkage has been a morphological hallmark of the programmed cell death process known as apoptosis. This isotonic loss of cell volume has recently been term apoptotic volume decrease or AVD to distinguish it from inherent volume regulatory responses that occurs in cells under anisotonic conditions. Recent studies examining the intracellular signaling pathways that result in this unique cellular characteristic have determined that a fundamental movement of ions, particularly monovalent ions, underlie the AVD process and plays an important role on controlling the cell death process. An efflux of intracellular potassium was shown to be a critical aspect of the AVD process, as preventing this ion loss could protect cells from apoptosis. However, potassium plays a complex role as a loss of intracellular potassium has also been shown to be beneficial to the health of the cell. Additionally, the mechanisms that a cell employs to achieve this loss of intracellular potassium vary depending on the cell type and stimulus used to induce apoptosis, suggesting multiple ways exist to accomplish the same goal of AVD. Additionally, sodium and chloride have been shown to play a vital role during cell death in both the signaling and control of AVD in various apoptotic model systems. This review examines the relationship between this morphological change and intracellular monovalent ions during apoptosis.     

Autophagy and apoptosis are redundantly required for C. elegans embryogenesis

Apoptosis, the main form of regulated (or programmed) cell death, allows the organism to tightly control cell numbers and tissue size, and to protect itself from potentially damaging cells. This type of cellular self-killing has long been assumed to be essential for early development. In the nematode Caenorhabditis elegans, however, the core apoptotic cell death pathway appears to be dispensable for embryogenesis when most developmental cell deaths take place: mutant nematodes defective for apoptosis develop into adulthood, with superficially normal morphology and behavior. Accumulating evidence indicates a similar situation in mammalian systems as well. For example, apoptosis-deficient mice can grow as healthy, fertile adults. These observations raise the possibility that alternative cell death mechanisms may compensate for the lack of apoptotic machinery in developing embryos. Interestingly, C. elegans embryogenesis can also occur without autophagy, an alternative form of cellular self-destruction (also called autophagic cell death). In an upcoming paper we report that simultaneous inactivation of the autophagic and apoptotic gene cascades in C. elegans arrests development at early stages, and the affected embryos exhibit severe morphological defects. Double-mutant nematode embryos deficient in both autophagy and apoptosis are unable to undergo body elongation or to arrange several tissues correctly. This novel function of autophagy genes in morphogenesis indicates a more fundamental role for cellular self-digestion in tissue patterning than previously thought.     

Cell volume regulation in immune cell apoptosis

The loss of cell volume is an early and fundamental feature of programmed cell death or apoptosis; however, the mechanisms responsible for cell shrinkage during apoptosis are poorly understood. The loss of cell volume is not a passive component of the apoptotic process, and a number of experimental findings from different laboratories highlight the importance of this process as an early and necessary regulatory event in the signaling of the death cascade. Additionally, the loss of intracellular ions, particularly potassium, has been shown to play a primary role in cell shrinkage, caspase activation, and nuclease activity during apoptosis. Thus, an understanding of the role that ion channels and plasma membrane transporters play in cellular signaling during apoptosis may have important physiological implications for immune cells, especially lymphocyte function. Furthermore, this knowledge may also have an impact on the design of therapeutic strategies for a variety of diseases of the immune system in which apoptosis plays a central role, such as oncogenic processes or immune system disorders. The present review summarizes our appreciation of the mechanisms underlying the early loss of cell volume during apoptosis and their association with downstream events in lymphocyte apoptosis.     

Interdigital cell death function and regulation: new insights on an old programmed cell death model

Interdigital cell death (ICD) is the oldest and best-studied model of programmed cell death (PCD) in vertebrates. The classical view of ICD function is the separation of digits by promotion of tissue regression. However, in addition, ICD can contribute to digit individualization by restricting interdigital tissue growth. Depending on the species, the relative contribution of either regression or growth-restricting functions of ICD to limb morphogenesis may differ. Under normal conditions, most cells appear to die by apoptosis during ICD. Accordingly, components of the apoptotic machinery are found in the interdigits, though their role in the initiation and execution of cell death is yet to be defined. Fgf8 has been identified as a survival factor for the distal mesenchymal cells of the limb such that ICD can initiate following specific downregulation of Fgf8 expression in the ectoderm overlying the interdigital tissue. On the other hand, Bmps may promote cell death directly by acting on the interdigital tissue, or indirectly by downregulating Fgf8 expression in the ectoderm. In addition, retinoic acid can activate ICD directly or through a Bmp-mediated mechanism. Interactions at different levels between these factors establish the spatiotemporal patterning of ICD activation. Defining the regulatory network behind ICD activation will greatly advance our understanding of the mechanisms controlling PCD in general.     

Apoptosis in mammalian eye development: lens morphogenesis, vascular regression and immune privilege

Formation of the mammalian eye requires a complex series of tissue interactions that result in an organ of exquisite sensory capability. The early steps in eye development involve extensive cell death associated with morphogenesis. Later, suppression of programmed cell death is essential for tissue differentiation and in the adult, the immune privileged status of the eye is maintained in part through factors that induce inflammatory cell apoptosis. Experimental evidence suggests that suppression of apoptosis in cells of the lens lineage by fibroblast growth factors is one component of their action during lens morphogenesis. Fibroblast growth factors are also required for normal lens fiber-cell differentiation. This includes a degenerative step for organelles that is presumably an adaptation for the clearance of light scattering elements from the optic axis. The process of organelle degeneration may be related to apoptosis in a few of its features. Actively-induced apoptosis becomes important for eye development as the temporary ocular vasculatures regress. This too, is presumably an adaptation for the disposal of cells that would disturb the passage of light to the retina. Ocular macrophages appear to be essential for the induction of apoptosis in the endothelial cells comprising the ocular vasculatures. In the adult, inflammatory cells entering the eye are exposed to the pro-apoptotic agents transforming growth factor-beta2 and Fas ligand. The expression of these molecules in the eye, and their action in killing inflammatory cells, has evolved as a means of preventing inflammation and subsequent loss of vision. Thus, the eye offers a unique and versatile system for studying the role of programmed cell death in lens development, vascular regression and immune privilege.     

Death penalty for keratinocytes: apoptosis versus cornification

Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.     

Autophagy: paying Charon's toll

Autophagy (literally self-eating) has diverse cytoprotective functions but also is linked to programmed cell death (PCD). In this issue, Qu et al. (2007) analyze the role of autophagy in PCD during development. Unexpectedly they find that autophagy induces apoptotic cells to present a signal to ensure their clearance, which is crucial for mouse embryonic morphogenesis.     

Caspase-independent cytochrome c release is a sensitive measure of low-level apoptosis in cell culture models

Age-associated loss of tissue function and several chronic diseases may derive in part from the cumulative effects of subtle changes in the level of apoptotic cell death. Because apoptosis is rapid and undetectable once complete, small changes in its incidence are difficult to detect, even in well-controlled cell cultures. We describe a new apoptosis assay that provides greater sensitivity than conventional assays because it measures the accumulation of apoptotic cells. Human and mouse fibroblasts and human mammary epithelial cells that initiated apoptosis were preserved for 3 days by inhibiting caspase activity using the chemical inhibitor Q-VD-OPH (QVD). Cells suspended in the process of apoptosis were scored by immunostaining for cytochrome c, which redistributed from mitochondria in healthy cells to the cytoplasm in dying cells. This caspase-independent cytochrome c release (CICR) assay was more sensitive than several conventional assays when apoptosis was induced by actinomycin D, and detected cumulative background levels of apoptosis over a 3-day interval. Using this assay, we show that normal fibroblasts undergo very little apoptosis upon X-irradiation, indicating dominance of the senescence response in this cell type. Further, apoptosis increased subtly but measurably when human mammary epithelial and skin fibroblast cells entered crisis, indicating that cell death during crisis is largely non-apoptotic.