Apoptosis and necrosis: Detection, discrimination and phagocytosis

Abstract

Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.

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.