Mechanisms of Cell Injury and Death: Ischaemia, Accident, or Suicide?, Study notes of Pathology

Download Mechanisms of Cell Injury and Death: Ischaemia, Accident, or Suicide? and more Study notes Pathology in PDF only on Docsity! British Journal of Anaesthesia 1996; 77: 3-10 Mechanisms of cell injury and death J. P. Coss, R. S. Horcuxiss, I. E. KARL AND T. G. BUCHMAN Control of the rate of cell death relative to the rate of cell division maintains organ integrity and physio- logical homeostasis. Cell death is valuable for the organism because it removes terminally injured or unwanted cells that utilize valuable substrates and nutrients. Likewise, cell death also has value for the species, as it provides a mechanism for eliminating terminally injured individuals who consume necess- ary societal resources or harbour toxic pathogens. Recent advances in cellular biology have contributed. substantially to our understanding of the processes of cell injury and death, and have provided the molecular tools necessary to control it. This paper reviews cell injury and adaptation; mechanisms of cell death; the roles of cell injury and death in the pathophysiology of organ dysfunction; and implica- tions for prevention and therapy of multiple organ dysfunction syndrome (MODS). Cell injury and adaptation Cell injury occurs as a result of physical, chemical or biological insults or as a result of vital substrate deficiency (table 1). The cellular response to these injuries is adaptive, designed to restore homeostasis and protect the cell from further injury. Although characteristic changes in gene transcription occur, it is not the relative amount but the pattern of transcription that changes, with emphasis directed towards transcription of “vital” genes. The responses induced by cellular injury fall into four main patterns: the ischaemic/anoxic, oxidative, heat shock and acute phase responses. These are reviewed. briefly below. ISCHAEMIC/ANOXIC STRESS RESPONSE [13] Because of the unusual high efficiency of the cardiopulmonary system to transport oxygen, cells in higher animals have not developed elaborate cellular pathways to adapt to hypoxia and are thus relatively sensitive to ischaemia [13, 18]. As a consequence, lack of oxygen dramatically increases the need for anaerobic glycolysis to maintain intracellular ATP (Br. J. Anaesth. 1996; 77: 3-10) Key words Cells, apoptosis. Cells, necrosis. Cells, death. Complications, sepsis. reserves. The associated changes in gene expression include decreased total protein synthesis, induction of hypoxia-associated proteins (e.g. glyceraldehyde- 3-phosphate dehydrogenase, a glycolytic enzyme), induction of the heat shock response (see below), and induction of glucose-regulated proteins. If these changes are inadequate to prevent ATP depletion, membrane ion pumps fail and membrane integrity is lost. Increased intracellular Ca* occurs and a variety of degradative processes are initiated, leading to cytoplasmic swelling and eventual cell death. OXIDATIVE STRESS RESPONSE [9] Agents that provoke oxidative injury include pro- ducts of oxidative metabolism (especially from the mitochondria) and those released from activated phagocytes. Collectively, they are called reactive oxygen species (ROS), and include superoxide, hy- drogen peroxide, hydroxyl radical (the generation of which depends on availability of ferrous ion and superoxide) and nitric oxide. Iron, which is essential for DNA synthesis and oxidative metabolism, also catalyses the Fenton and Haber-Weiss reactions, converting superoxide to molecular oxygen and hydrogen peroxide to the hydroxyl radical. Under normal conditions, free iron is compartmentalized by the protein ferritin, which “protects” the cell by keeping iron in a non-reactive crystallized core as ferric ion (Fe**). The results of the oxidative reactions of ROS include de-energization of mito- chondria and loss of energy stores, peroxidation and disruption of lipid membranes, and direct DNA damage. The adaptive response of the cell to oxidative stress includes both enzymatic and non-enzymatic capacities and induction of oxidative stress response proteins [9]. Superoxide dismutase reduces super- oxide to molecular oxygen and hydrogen peroxide; catalase catalyses the conversion of this hydrogen peroxide to oxygen and water. Reduced glutathione (which reacts with hydrogen peroxide in the presence of glutathione peroxidase to form water and oxidized glutathione), vitamin C and vitamin E constitute the J.PERREN copB*, MD, TIMOTHY G. BUCHMAN, PHD, MD (Department of Surgery); RICHARD S. HOTCHKISS, MD (Depart- ment of Anesthesia); IRENE KARL, PHD (Department of Medicine); Washington University School of Medicine, St Louis, MO, USA. *Address for correspondence: Campus Box 8109, Department of Surgery, 660 South Euclid Ave, St Louis, MO 63110-1093, USA. 4 Table 1 Mechanisms of cell injury (1) Physical (a) ionizing radiation (b) temperature (©) mechanical trauma (2) Chemical (a) drugs (b) poisons (3) Biological (a) enzymes (b) cytokines (©) viral infection (@) cell-mediated (4) Critical substrate deficiency (a) oxygen (b) glucose major intracellular reducing (non-enzymatic) agents. Several proteins are induced under oxidative stress, the most important of which may be metallothionein, which binds not only heavy metals but also ROS via the free sulphydryl groups of its many cysteine residues. ROS also activate the transcriptional factors AP-1 and NF«B, both of which promote transcription of cytokines and have been associated with induction of apoptotic cell death (see below). If these adaptive responses are inadequate to prevent depletion of cellular glutathione, then cell protein thiol groups become the remaining reducing agents, leading to loss of critical enzymatic function and cell death. HEAT SHOCK RESPONSE [10, 20] The highly conserved cellular response to heat, known as the heat shock response, is associated with induction of heat shock proteins (HSP). The most extensively studied are the HSP 70 family, which includes stress-induced HSP 72. Interestingly, HSP also are induced by a variety of other cellular insults, including ischaemia/reperfusion, oxidative stress, exposure to heavy metals (e.g. arsenite) and in- fection. For this reason, the heat shock response is called simply the “stress response”, and HSP are known as “stress proteins”. Control of HSP tran- scription is mediated by heat shock element ac- tivation in the heat shock protein promoter region. HSP aid in the proper folding of newly translated proteins, and are referred to as “molecular chaper- ones”. Increased production of HSP is believed to provide an adaptive advantage to stressed cells by increasing the fidelity of protein synthesis and aiding refolding of damaged or denatured proteins. ACUTE PHASE RESPONSE [26] Although “acute phase response” can refer to the response of any tissue to injury, its use is commonly restricted to the dramatic change in the pattern of hepatocyte protein synthesis. The most important stimuli are interleukin 1 (IL-1), tumour necrosis factor (TNF), and IL-6, products of macrophage/ monocyte activation. The hepatocyte response to these cytokines is an outpouring of plasma proteins British Journal of Anaesthesia that function to maintain the homeostasis of the organism. They include C-reactive protein, fibrino- gen, complement, the metal binding proteins hapto- globin and ferritin, and plasminogen activator in- hibitor and many others. The acute phase response (activation) of endothelial cells includes increased surface expression of adhesion, selectin and integrin molecules that facilitate leucocyte adhesion and release of IL-1, IL-6, IL-8 and platelet activating factor (PAF). Mechanisms of cell death If the genetic and metabolic adaptive responses described above are inadequate for a given injury, the cell will die. Increased interest in the mechanisms responsible for cell death, however, has made it clear that morphological classification of cell death requires revision. At least two types of cell death have been described. In the first, cell damage is manifested by cytoplasmic swelling, plasma mem- brane blebbing, dilation of the endoplasmic reticu- lum and mitochondria, dissolution of chromatin and, finally, interruption of membrane integrity. The other, less frequently observed type of cell death was described by Kerr [16] and called shrinkage necrosis. It is manifested by cytoplasmic shrinkage, larger plasma membrane buds and nuclear chromatin condensation. Both types of cell death result in the generation of necrotic debris that is engulfed by phagocytic cells. TERMINOLOGY The morphological changes described above are not restrictive. Nuclear condensation and mitochondrial swelling, for example, have been observed in both swelling and shrinkage types of cell death. There is also clear evidence of significant overlap between them at the molecular level (see below) [11, 14, 18]. Moreover, there is considerable imprecision in the literature regarding the terminology of cell death. Historically, necrosis has been the term used to refer to cell death in general. The term apoptosis (coined by Kerr, Wyllie, and Currie from the Greek word meaning “dropping off”, as in leaves dropping from a tree [17]) is now used specifically to describe death manifested by cell shrinkage. Apoptosis is respon- sible for the ordered, normal cell death of intestinal epithelial cells and blood neutrophils. Programmed cell death, which refers to the ordered death of cells during embryogenesis, however, is also characterized by cell shrinkage. Apoptosis and programmed cell death are frequently, but in- correctly, used interchangeably. The question of what to call non-apoptotic cell death has been problematic. Necrosis, a leading contender, is also used to refer to the process of removal of the cellular remains of apoptosis (so-called “post-apoptotic necrosis” [18]) [11, 32]. This degree of imprecision led Farber recently to remark that “there is no field of basic cell biology and cell pathology that is more confusing and unintelligible than is the area of apoptosis versus necrosis” [11]. Consequently, several Mechanisms of cell injury and death Figure 3 Normal cultured porcine aortic endothelial cells (A, x 360 magnification) stained with nuclear binding dye Hoechst 33342. The nuclei are smooth and ovoid. Nuclei from cells challenged with endotoxin followed by arsenite are compact and fragmented (“apoptotic bodies”, B, x 360 magnification). Table 2 Comparison of characteristics of oncosis and apoptosis Oncosis—death by swelling Apoptosis—death by shrinkage (1) Swelling of organelles and cytoplasm (2) Small dense chromatin clumps which are not sharply defined (3) Swelling and eventual disintegration of organelles (e.g. mitochondria and endoplasmic reticulum) (4) Early focal disruption of the plasma membrane with blebbing (5) DNA agarose gel electrophoresis demonstrates smear pattern indicative of randomized breakdown (6) Cells rupture and contents are released causing inflammatory response (1) Shrinkage of cytoplasm (2) Large, dense, often crescent-shaped aggregates of chromatin and nuclear fragmentation (3) Organelles maintain structural integrity (4) Plasma membrane maintains integrity early; later stage characterized by budding of membrane, frequently containing organelles (5) DNA agarose gel electrophoresis demonstrates “ladder” pattern of discrete internucleosomal breakdown (6) Cells are either ingested whole by phagocytic cells, or they break into membrane-bound fragments (apoptotic bodies) which then are ingested 1), end-labelling of DNA fragments (fig. 2), DNA agarose gel electrophoresis and binding of nuclear- specific dyes (fig. 3). Despite the relatively clear distinction which can be made morphologically (oncosis versus apoptosis is compared in table 2), examination at the molecular level demonstrates considerable overlap. For example, both oncosis and apoptosis have been associated with increases in intracellular Ca?*, which activate a number of enzymes including phospho- lipases, endonucleases, proteases and protein kinases. In addition, the adaptive response to ischaemic, inflammatory, oxidant and heat-induced stress in- volve changes in gene expression that are shared by both types of cell death. Further, the same type of injury can result in cell death by either mode. Indeed, Kerr’s original description of apoptosis (“shrinkage necrosis”) was based upon differ- entiation of shrunken necrotic cells from the more common swollen necrotic cells in ischaemic liver tissue [16]. Pathophysiology of organ dysfunction Studies of cellular responses in vivo indicate that injury activates the programmes of stress gene expression reviewed above. For example, the hepato- cellular response to ischaemic injury in a porcine model of MODS includes activation of the acute phase, oxidative, and heat shock responses [7]. In 8 contrast, neighbouring hepatic endothelial cells in this model did mot stain for heat shock gene expression (in situ hybridization), suggesting that there are cell-specific differences in the genetic response to similar injury. A subsequent investi- gation conducted in vitro under more controlled conditions explored the time-course and interaction of these cellular stress responses. Human hepato- blastoma (HepG2) cell lines were treated with inducers of the acute phase response (IL-1 and TNF) and heat shock responses separately and in combination. The results indicated that shock- induced expression of these responses were distinct, exclusive and prioritized [6]. Specifically, the heat shock stress response, the most primitive and highly conserved of stress gene responses, was capable of extinguishing expression of other genetic pro- grammes [5, 28]. These data suggest that hepatocyte injury of sufficient severity to trigger the heat shock response can prevent production of system-stabil- izing acute phase proteins and other adaptive responses. The liver’s response to such a systemic injury, from the host’s perspective, would appear to be inadequate and indicative of failure. Hepatic dysfunction in this model of MODS, thus, would result from execution of primitive, protective genetic responses that give individual cells priority over the organism. The sequence of stress gene programme activation also appears to be important. In one in vitro model, the endothelial cell response to arsenite, an inducer of the heat shock response, and to endotoxin, an inducer of the acute phase response, were studied. The results indicated that induction of the heat shock response could protect against endotoxin- induced cytotoxicity, but reversing the order— endotoxin challenge followed by induction of the heat shock response—increased cell death by apop- tosis [5]. The effect of the heat shock response was not the result of a detrimental effect of the heat shock proteins themselves, but rather to the heat shock- induced inhibition of further protein synthesis [5]. An important hypothesis drawn from these data is that two independent programmes of stress gene expression can interact, depending on the sequence, to either protect the cell or trigger cell death by suicide (apoptosis). This may explain the absence of endothelial cells expressing heat shock proteins after shock in vivo ([7], discussed above), as endothelial cell apoptosis may have resulted in their subsequent removal by phagocytes before in situ staining. Thus, endothelial cell dysfunction and death in this model result from the adverse interaction of two usually beneficial patterns of stress responses. The mech- anism responsible may involve alterations in the cellular redox potential and changes in nuclear transcription factor activity [unpublished observa- tions]. For example, in the cultured endothelial cell model, reducing agents such as N-acetyl-1-cysteine and dithiothreitol block induction of endotoxin/ arsenite-induced endothelial cell apoptosis, impli- cating ROS in the induction of apoptotic cell death [1, 2]. This raises the possibility that the adaptive response produced by oxidative stress could modu- late the interaction of the acute phase and heat shock British Journal of Anaesthesia responses, and may participate in determining the fate of the cell. Apoptosis may also contribute to failure of organs in other settings, in particular, septic shock. Injection of Gram-negative or Gram-positive bacteria [34], endotoxin [34, 36], or caecal ligation and puncture [4] induced apoptosis in rodent thymocytes. Zhang and colleagues [36] reported that mice injected with lipopolysaccharides had apoptosis not only in the thymus, but also in the spleen, bone marrow and endothelial cells. Norimatsu and associates injected swine intravenously with 0.5 mg kg”! Escherichia coli endotoxin and noted that cells in both thymus and mesenteric lymph nodes underwent apoptosis [24]. In a recent report, Rogers and colleagues demon- strated apoptosis in the liver of mice infected with Listeria monocytogenes [27]. Apoptosis has also been reported in rabbit heart [12] and rat kidney [29] after ischaemia/reperfusion injury. Collectively, these investigations suggest that apoptotic cell death may be a principal mechanism responsible for organ dysfunction and death as a consequence of shock- induced injury. Conclusions—from basic science to therapy and prevention Cell injury occurs as a result of physical, chemical or biological insults or from vital substrate deficiency. These insults induce expression of adaptive stress response gene programmes that include the ischaemic/hypoxic stress, oxidative stress, heat shock and acute phase responses. If the severity of the injury exceeds the ability of the cell to adapt, then cell death occurs. The processes of cell death include cell death by ischaemia, accident or suicide. Morphologically, ischaemic and accidental deaths are usually manifested by cytoplasmic swelling (oncosis) and suicidal cell death by cytoplasmic shrinkage (apoptosis). However, this terminology lacks precision, as some “accidental” causes of cell death, such as heat, can also induce apoptosis [18]. The molecular machinery responsible for cell death and the boundaries between apoptosis and other modes of cell death remain to be determined [14]. The sequence and interaction of the acute phase, heat shock and oxidative stress responses appear to be a determinant of the fate of the cell. The pattern of cell responses is injury- and tissue-dependent and may help to explain differences in the course and nature of organ failure. Specifically, it appears that stimuli sufficient to trigger the heat shock response can precipitate cell (and organ) failure by blocking initiation of other programmes of stress gene ex- pression (e.g. the system stabilizing acute phase response of hepatocytes). In other cells (e.g. the endothelium), the sequence of acute phase followed by heat shock response may result directly in apoptotic cell death. The function of shock-induced apoptosis is not known, but it may contribute to control of immune cell proliferation in thymus, spleen, bone marrow, and lymph nodes and modu- lation of the inflammatory response. MODS is the result of cellular responses to a stimulus of either enormous magnitude or distri- Mechanisms of cell injury and death bution. The stimulus itself frequently does not result directly in cell death, but rather the magnitude of the appropriate response from stimulated cells collec- tively is injurious to the organism. For instance, small doses of i.v. lipopolysaccharide (endotoxin) in humans are not directly cytotoxic, but lead to the release of the intercellular mediators, such as cyto- kines, that amplify the stimulus and trigger organ dysfunction [30]. Unfortunately, attempts to at- tenuate the magnitude of this response by attacking the circulating mediators themselves (e.g. anti- endotoxin and anti-cytokine therapies) have failed to improve patient survival in randomized, prospective trials [22]. These negative data forced a broad re- examination of the treatment strategies for the systemic inflammatory response syndrome (SIRS) and MODS and a change in focus from extracellular signals (i.e. the mediators) to the intracellular responses to these signals [8]. Recent symposia have addressed this important issue (e.g. “The Future of Sepsis Research”, August 1995, Bethesda, MD, USA, sponsored by the American College of Chest Physicians, National Institute of Allergy and In- fectious Disease, National Heart, Lung, and Blood Institute and National Institutes of Health, USA). A NEW THERAPEUTIC STRATEGY: MODULATION OF CELLULAR RESPONSES TO INJURY In summary, the data presented above are consistent with a new hypothesis on the cellular origin of MODS, one that has emerged as a result of a change in focus from the extracellular to the intracellular response to injury. They suggest that there are distinct, exclusive, and prioritized genetic pro- grammes expressed in response to cell injury that are specific to cell type and injury. The effects of these stress response gene programmes are usually cyto- protective, but when activated in sequence, they can precipitate apoptotic cell death. Recent success in modulating induction of apoptosis with reducing agents [1], anti-oxidants [2], inhibitors of protein synthesis [5], anti-TNF antibody [34] and steroid antagonists [3] suggests that pharmacological control in the clinical setting is possible. 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