Apoptosis and necrosis: Detection, discrimination and phagocytosis
Introduction
Homeostasis is maintained in multicellular organisms by a balance between cell proliferation and cell death. Several types of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II), necrosis or oncosis (type III), mitotic catastrophe, anoikis, excitotoxicity, Wallerian degeneration, and cornification of the skin [1]. The final fate of almost any dying/dead cells regardless of death type is engulfment by non-professional or professional phagocytes [2]. This clearance process is of outmost importance for the development and homeostasis of organisms because defective or inefficient clearance may contribute to several human pathologies, including systemic lupus erythematosus [3], cystic fibrosis [4] and chronic obstructive pulmonary disease [5].
Cells undergoing apoptosis show typical, well-defined morphological changes, including plasma membrane blebbing, chromatin condensation with margination of chromatin to the nuclear membrane, karyorhexis (nuclear fragmentation), and formation of apoptotic bodies [6]. Apoptosis has been characterized by several biochemical criteria, including different kinetics of phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane [7], [8], changes in mitochondrial membrane permeability [9], release of intermembrane space mitochondrial proteins [10], and caspase-dependent activation and nuclear translocation of a caspase-activated DNase resulting in internucleosomal DNA cleavage [11]. Identification of these morphological and biochemical markers of apoptosis makes it possible to distinguish it from other forms of cell death.
Cells undergoing death associated with autophagy are characterized by the presence of double membrane autophagic vacuoles. Autophagy is foremost a survival mechanism that is activated in cells subjected to nutrient or obligate growth factor deprivation. When cellular stress continues, cell death may continue by autophagy alone, or else it often becomes associated with features of apoptotic or necrotic cell death [12]. Specific biochemical markers have been determined for cell death associated with autophagy, such as delocalization of GFP-LC3 to the autophagosomes [13] or the lipidation of LC3, as detected by band shift in western blots [14], [15].
In contrast, necrosis is characterized by rapid cytoplasmic swelling and is therefore also often referred to as oncosis. It culminates in rupture of the plasma membrane and organelle breakdown [16]. Necrosis has long been described as a consequence of extreme physicochemical stress, such as heat, osmotic shock, mechanical stress, freeze thawing and high concentration of hydrogen peroxide. In these conditions, cell death occurs quickly due to the direct effect of the stress on the cell, and therefore this cell death process has been described as accidental and uncontrolled. However, many different cellular stimuli (TNF on certain cell lines, dsRNA, IFN-γ, ATP depletion, ischemia) have been shown to induce a necrotic process that follows defined steps and signaling events reminiscent of a true cell death program [17]. This induced necrotic cell death results from extensive crosstalk between several biochemical and molecular events at different cellular levels, and it is as controlled and programmed as apoptosis (reviewed in [18]). It is important to distinguish necrosis from other forms of cell death, particularly because it is often associated with unwarranted loss of cells in human pathologies [18], [19], [20]. It can also lead to local inflammation due to release from dead cells of intracellular factors, the so-called damage associated molecular patterns that alert the innate immune system. However, there is no clear biochemical definition of necrotic cell death and consequently no positive biochemical makers that unambiguously discriminate necrosis from apoptosis. Another problem is that even the interpretation of dying cell morphology may be complex, because in the absence of phagocytosis apoptotic cells proceed to a stage called secondary necrosis, which shares many features of primary necrosis. In this review we describe a selection of techniques for discrimination of necrosis from apoptosis, and we stress that accurate discrimination is only possible through an integrated approach based on several morphological and biochemical parameters.
Section snippets
Methods for analysis of cell morphology
In this section we will describe the use of time-lapse microscopy, flow fluorocytometry and transmission electron microscopy as means for distinguishing between apoptotic and necrotic cell death by analyzing cell morphology. Throughout this article we will use as an example the L929sA fibrosarcoma cell line, in which apoptotic as well as necrotic cell death can be induced. Necrotic cell death is induced in L929sA cells by stimulation of TNFR1, whereas in L929sA cells transfected with human Fas
Methods for analysis of cell surface markers
Phosphatidylserine (PS) is an aminophospholipid that resides in the inner leaflet of the plasma membrane of living cells. During apoptotic cell death, PS is actively externalized to the outer surface of the plasma membrane, where its presence is required for recognition and engulfment of dying cells [2]. Several possible mechanisms for PS exposure have been proposed, including a coordinate increase in phospholipid flip–flop due to inactivation of the aminophospholipid translocase; flip refers
Methods for analysis of intracellular markers
In this section we will describe the use of intracellular markers, such as fragmentation of DNA, activation of caspases, cleavage of Bid into its truncated pro-apoptotic form (tBid), and release of cytochrome c; all these can help distinguish between apoptosis and necrosis. None of these events occurs in TNFR1-induced necrotic cell death [8]. In addition to determining the activation pattern of different caspases, the cleavage pattern of a variety of substrates, such as PARP and ICAD, can also
Methods for analysis of extracellular markers
If apoptotic cells are not taken up by phagocytosis, they become secondary necrotic cells with many features of primary necrotic cells (discussed in Section 3.1). For example, the plasma membrane becomes permeabilized, with consequent leakage of large amounts of intracellular contents, which have many biological effects, e.g. induction of immune responses and chemoattraction of antigen presenting cells [47]. Detection of lactate dehydrogenase (LDH) release may provide an easy method for
Methods for analysis of cell–cell interactions
Recognition and clearance of dying cells is a complex and dynamic process coordinated by the interplay between ligands on dying cell, bridging molecules, and receptors on engulfing cells [2]. Dying cells can be cleared by professional phagocytes, such as macrophages and immature DCs, or by non-professional phagocytes, such as endothelial cells, fibroblasts, smooth cells, and epithelial cells. Efficient clearance of cells undergoing either apoptotic or necrotic cell death is crucial for normal
Concluding remarks
Distinguishing necrosis from apoptosis should never be based on either morphological or biochemical criteria alone, but rather should take into account and integrate all available data. Accumulating evidence supports the concept that necrotic cell death is programmed. Therefore, unraveling molecular players and defining biochemical pathways in necrosis will provide us with powerful and specific methods for necrotic cell identification and help us to distinguish it positively from other forms of
Acknowledgments
We thank Wim Drijvers for the artwork and Dr. Amin Bredan for editing the manuscript. Dr. Dmitri V. Krysko is paid by a postdoctoral fellowship from the BOF (Bijzonder Onderzoeksfonds 01P05807), Ghent University, and Dr. Tom Vanden Berghe is paid by a postdoctoral fellowship from FWO (Fonds Wetenschappelijk Onderzoek—Vlaanderen). This work has been supported by Flanders Institute for Biotechnology (VIB) and several Grants from the European Union (EC Marie Curie Training and Mobility Program,
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