Molecular Mechanisms and Pathophysiology of Necrotic Cell Death

Current Molecular Medicine 2008, 8, 207-220 207

1566-5240/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.

Molecular Mechanisms and Pathophysiology of Necrotic Cell Death

Nele Vanlangenakker1,2, Tom Vanden Berghe1,2, Dmitri V. Krysko1,2, Nele Festjens3,4 and Peter Vandenabeele*,1,2

1Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, 9052 Ghent,

Belgium 2Department of Molecular Biology, Ghent University, 9052 Ghent, Belgium

3Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium

4Department of Biochemistry, Physiology and Microbiology, Laboratory for Protein Biochemistry and Biomo-

lecular Engineering, Ghent University, 9052 Ghent, Belgium

Abstract: Necrotic cell death has long been considered an accidental and uncontrolled mode of cell death. But

recently it has become clear that necrosis is a molecularly regulated event that is associated with pathologies

such as ischemia-reperfusion (IR) injury, neurodegeneration and pathogen infection. The serine/threonine ki-

nase receptor-interacting protein 1 (RIP1) plays a crucial role during the initiation of necrosis induced by li-

gand-receptor interactions. On the other hand, ATP depletion is an initiating factor in ischemia-induced necrotic

cell death. Common players in necrotic cell death irrespective of the stimulus are calcium and reactive oxygen

species (ROS). During necrosis, elevated cytosolic calcium levels typically lead to mitochondrial calcium over-

load, bioenergetics effects, and activation of proteases and phospholipases. ROS initiates damage to lipids,

proteins and DNA and consequently results in mitochondrial dysfunction, ion balance deregulation and loss of

membrane integrity. Membrane destabilization during necrosis is also mediated by other factors, such as acid-

sphingomyelinase (ASM), phospholipase A2 (PLA2) and calpains. Furthermore, necrotic cells release immu-

nomodulatory factors that lead to recognition and engulfment by phagocytes and the subsequent immunologi-

cal response. The knowledge of the molecular mechanisms involved in necrosis has contributed to our under-

standing of necrosis-associated pathologies. In this review we will focus on the intracellular and intercellular

signaling events in necrosis induced by different stimuli, such as oxidative stress, cytokines and pathogen-

associated molecular patterns (PAMPs), which can be linked to several pathologies such as stroke, cardiac fai-

lure, neurodegenerative diseases, and infections.

Keywords: Necrosis, RIP1, mitochondrial Ca2+

overload, ROS, clearance of necrotic cells, phagocyte response, neurodegenerative disorders, ischemia-reperfusion injury.

INTRODUCTION

Three major morphological types of cell death have been described [1]. Type I or apoptotic cell death is mediated by caspases and characterized by cellular shrinkage, chromatin condensation and DNA degrada-tion (reviewed in [2]). Type II cell death is associated with the formation of autophagic vacuoles inside the dying cell (reviewed in [3]). Type III or necrotic cell death is characterized by cellular swelling, plasma membrane rupture and the subsequent loss of the in-tracellular contents (reviewed in [4]). For a long time necrosis has been considered to be an accidental, un-controlled form of cell death, but evidence is accumula-ting that execution of necrotic cell death may be carried out by a set of signal transduction pathways and exe-cution mechanisms [4]. Some studies have demonstra-ted the occurrence of necrotic cell death in develop-ment and maintenance of homeostasis. For example, Apaf-1-deficient embryos show delayed development of digits, which are formed through the induction of ne-crosis of interdigital cells [5,6]. During homeostasis,

*Address correspondence to this author at the Technologiepark 927, B-9052 Zwijnaarde (Ghent), Belgium; Tel: 0032.9.3313760; Fax: 0032.9.3313609, E-mail: [email protected]

necrosis of chondrocytes facilitates longitudinal growth of bones [7] and necrosis of specific epithelial cells of the human colon supports the turnover of the large in-testine [8]. In general, necrosis is mainly associated with pathologies such as IR injury, neurodegeneration and more recently also with certain pathogen infec-tions.

METABOLIC CHANGES DURING NECROSIS

Necrotic cell death occurs generally in response to physico-chemical stress, including hypoxia, ischemia, hypoglycemia, extreme temperature changes, and nu-trient deprivation [9,10]. In this paragraph we will focus on metabolic changes that lead to necrotic cell death in IR injury. Ischemia due to obstruction of blood flow to a tissue results in inadequacy of oxygen and nutrient supply, accumulation of metabolic waste products, and consequent impairment of energy metabolism. If pro-longed, this can result in cell death. Restoration of the blood flow, called reperfusion, particularly after prolon-ged ischemia, increases cell death, a phenomenon known as ‘reperfusion injury’. When oxygen supply is limited, oxidative phosphorylation is inhibited and mito-chondrial ATP production is thereby blocked. The im-

208 Current Molecular Medicine, 2008, Vol. 8, No. 3 Vanlangenakker et al.

portance of the intracellular ATP concentration in cell death was initially demonstrated by Leist and cowor-kers, who observed in conditions of reduced ATP levels a switch from apoptosis to necrosis in human T cells exposed to apoptotic triggers [11]. During progressive ischemia, several components of the mitochondrial electron transport chain and the adenine nucleotide translocator (ANT) are damaged, and complex V hy-drolyses ATP. This leads to impairment of mitochon-drial ATP generation and increase in free phosphate [12]. To maintain ATP production, cells resort to anae-robic glycolysis using glycogen stores and the remai-ning glucose in the surrounding tissue fluid [13], which can lead to accumulation of lactic acid and drop in in-tracellular pH. In ischemic conditions, however, ATP production through glycolysis is much less efficient be-cause of the limited supplies of oxygen and glucose. The cell counteracts the pH drop by activating the Na

+/H

+ antiporter, causing increased levels of intracel-

lular Na+. This excess Na

+ cannot be pumped out by

the Na+/K

+ ATPase due to the reduced ATP levels

[14,15]. Consequently, the cell loads with extracellular Ca

2+ through the reversal of the Na

+/Ca

2+ antiporter.

The cytosolic Ca2+

concentration can also be elevated by the release of Ca

2+ from the endoplasmic reticulum

(ER). Subsequently, Ca2+

enters the mitochondria via a uniporter, now known to be a channel, and a Na

+/Ca

2+

antiporter pumps it out again. As mitochondrial matrix Ca

2+ concentration increases, the activity of the

Na+/Ca

2+ antiporter becomes saturated. If the extrami-

tochondrial Ca2+

level increases beyond a certain value, the mitochondria can no longer regulate their matrix Ca

2+ concentration, and mitochondrial Ca

2+

overload ensues [9,16-18].

At the onset of reperfusion, intracellular pH is resto-red and the reintroduction of oxygen results in a burst of ROS generated by the affected mitochondria. The sustained Ca

2+ level can stimulate mitochondrial dehy-

drogenases with concomitant increase in NADH levels. The augmented NADH concentration contributes to an increased mitochondrial ROS production [19]. Aug-mented ROS generation can result in DNA damage, which causes hyperactivation of poly(ADP-ribose) po-lymerase-1 (PARP-1), which catalyzes poly(ADP)-ribosylation of a variety of proteins. This hyperactiva-tion depletes the cytosolic NAD

+ pool and thereby in-

hibits glycolysis and ATP production [20,21]. Oxidative stress and normalized pH, combined with calcium over-load and other factors, such as high phosphate con-centrations and low adenine nucleotide levels, trigger the mitochondria to undergo a permeability transition (Fig. (1)) [14,15,18,22]. Opening of the mitochondrial permeability transition pore (MPTP) causes mitochon-drial permeability transition (MPT), accompanied by mitochondrial inner membrane depolarization, matrix swelling and outer mitochondrial membrane rupture (reviewed in [23]). MPTP is a multiprotein megachannel connecting the mitochondrial matrix with the cytosol, and consists of, together with other molecules, the vol-tage-dependent anion channel (VDAC, present on the outer membrane), ANT (present on the inner mito-

chondrial membrane) and cyclophilin D (CypD, present in the mitochondrial matrix) [24]. CypD seems to be the leading figure in this multiprotein complex, because treatment with cyclosporine A (CsA) decreases sensiti-vity to MPT and necrotic cell death induced by Ca

2+

overload or oxidative stress; CypD-null cells also ex-hibit decreased sensitivity [25-27]. However, these data should be interpreted cautiously and in terms of the role of CypD in cell death, not in MPT, because MPTP can still open in the absence of CypD, provided that a permissive Ca

2+ load is attained [28-31]. The crucial

role of VDAC and ANT in MPTP [32,33] is under review again, because mitochondria of mice deficient in VDAC or ANT still exhibit MPT in response to Ca

2+ overload

[34,35]. Besides an increase in Ca2+

, ATP depletion and ROS production, other metabolic changes are in-volved in necrotic cell death, such as a disproportionate production of nitric oxide (NO) and enhanced formation of advanced glycation end (AGE) products. Literature describing these metabolic changes in detail can be found elsewhere [36,37].

NECROSIS INDUCED BY LIGAND RECEPTOR INTERACTION

Besides IR, necrotic cell death can also be induced when ligands bind to their receptors to initiate signaling cascades. In this field of research, the induction of ne-crosis by death receptors, such as tumor necrosis fac-tor receptor 1 (TNFR1), Fas and TRAIL receptor, has been intensively studied. In most cell types, these re-ceptors mediate apoptotic cell death and their functions often require the presence of transcription or translation inhibitors [38]. However, in some cell lines and primary cells, the presence of caspase inhibitors blocks apopto-tic cell death and reveals an alternative cell death pathway, namely necrosis [39]. One of the proteins recruited to the TNFR1 is the serine-threonine kinase RIP1, which is a crucial initiator in death receptor-mediated necrosis (reviewed in [40]). In the presence of caspase inhibitors, FasL- and TNF-induced necrotic cell death is abrogated in T cells deficient in RIP1 [41]. Moreover, dimerization of the kinase domain of RIP1 induces necrotic cell death [42]. The cellular targets of the RIP1 kinase remain unknown and the subject of active research.

More recently, pathogen recognition receptors (PRRs) were also shown to induce necrotic cell death. The PRR receptor family includes the membrane-associated Toll-like receptors (TLRs), the cytosolic NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs), all of which are involved in the sensing of PAMPs such as bacterial RNA, flagellin or fragments of peptidoglycan, and leading to initiation of inflammation and/or cell death [43-45]. Recognition of synthetic dsRNA by TLR3 induces necrotic cell death in human Jurkat cells and murine L929 fibrosarcoma cells [46]. TLR4 triggering by lipopolysaccharide leads to non-apoptotic death of macrophages when caspase-8 acti-vity is inhibited [47]. Similarly, the gram-negative bacte-rium Shigella flexneri triggers necrosis in neutrophils

Molecular Mechanisms and Pathophysiology of Necrosis Current Molecular Medicine, 2008, Vol. 8, No. 3 209

through the action of type III secretion systems, specific toxins and actin polymerization [48]. In addition, this bacterium causes mitochondrial damage leading to necrosis in human monocyte-derived macrophages [49]. Monocytes of patients carrying disease-associated mutations in cryopyrin (NLR member) ex-hibit excessive necrotic cell death, and Shigella flexneri infection induces cryopyrin-dependent macrophage necrosis with features similar to death caused by mu-tant cryopyrin. This necrotic cell death apparently does not rely on caspase-1 and IL-1 [50]. Necrotic cell death also occurs during viral infections, but it is not clear yet whether or not the above-described sensors are involved. HIV-1 kills CD4(+) T lymphocytes by ne-crosis [51-53]. Also, infection of Vero cells (kidney epi-thelial cells from the African green monkey) with high doses of the West Nile virus induces necrotic cell death associated with rapid loss of plasma membrane integri-ty and abundant budding of the progeny virus particles at the cell surface [54]. Necrotic-like cell death was also observed in human Jurkat cells and in pancreatic -cells after infection with Coxsackievirus B [55,56].

RIP1 MEDIATED ROS SIGNALING IN NECRO-SIS

Many signals converge on RIP1 and induce necrotic cell death (reviewed in [40]). In this paragraph we will mainly focus on TNF-induced RIP1-mediated necrosis.

Several studies revealed the molecular link between RIP1 and ROS production in necrotic cell death. Mito-chondria are the major intracellular source of ROS, which are mainly generated at complexes I and III of the respiratory chain. The accumulation of mitochon-drial ROS is RIP1-dependent [57] and can be blocked by the lipophilic ROS scavenger butylated hydroxyani-sole (BHA) and by complex I inhibitors [39,57-59]. The-se observations argue for a contribution of mitochon-drially generated ROS in necrosis. Therefore, it is con-ceivable that RIP1 directly or indirectly targets the mi-tochondria. Indeed, it has been demonstrated that TNF stimulation leads to localization of RIP1 to the mito-chondria and reduces the interaction between ANT and CypD, resulting in ATP depletion and induction of ne-crosis. Moreover, overexpression of CypD blocked TNF-induced necrotic cell death [60] but enhanced ne-crosis triggered by Ca

2+ overload or oxidative stress

[61], suggesting the involvement of different signaling mechanisms in necrosis, depending on the stimulus. In addition, cells deficient in CypD are as sensitive as wild type cells to TNF-induced cell death but are less sensi-tive to necrotic cell death induced by Ca

2+ overload or

oxidative stress [28-30,60].

A connection between RIP1 and a non-mitochondrial source of ROS is the plasma membrane-associated NADPH oxidase-1 (Nox1). Knockdown of Nox1, the major NADPH oxidase responsible for TNF-

Fig. (1). Mechanisms of mitochondrial calcium overload and subsequent mitochondrial permeability transition.

Depletion of oxygen and glucose during ischemia induce the shutdown of oxidative phosphorylation and result in ATP depletion.

The consequent increase in anaerobic glycolysis lowers pH. To counteract this pH drop, the cell activates the Na+/H

+-antiporter,

causing an increase in Na+. The excess Na

+ cannot be pumped out via the Na

+/K

+-ATPase because of reduced ATP availability.

So the cell becomes loaded with Ca2+

due to reversal of the Na+/Ca

2+-antiporter. In addition, cytosolic Ca

2+ can also be elevated

by Ca2+

influx from the ER. Calcium entering the mitochondria via the Ca2+

-uniporter cannot be pumped out by the mitochondrial

Na+/Ca

2+-antiporter due to saturation, and the result is mitochondrial calcium overload. Reperfusion reintroduces oxygen, the

damaged mitochondria produce a burst of ROS, and intracellular pH is restored. These changes, in combination with the cal-

cium overload and other factors, induce MPTP.


induced generation of superoxide anions, delays ne-crosis at least in mouse fibroblasts. Upon TNF stimula-tion Nox1 gets activated through a RIP1-dependent signaling complex containing TRADD, NOXO1 and the small GTPase Rac1 [62,63].

ROS MEDIATED DNA DAMAGE IN NECROSIS

ROS can initiate damage to proteins, nucleic acids and lipids (Fig. (2)) [64,65]. ROS can attack the disul-fide bonds (-S-S-) and thiol groups (-SH) of proteins and thereby change their functions. Oxidation of the thiol group of ANT by ROS has been shown to promote MPT [66]. Another effect of ROS during necrosis could be the modification of Ca

2+ channels of the ER and

plasma membrane [67].

Mitochondrial DNA is particularly susceptible to oxi-dative damage because of the absence of protective histones, limited base excision repair mechanisms, and close proximity to the electron transport chain, all of which lead to mitochondrial genomic instability and consequent respiratory dysfunction [68]. Genomic DNA damage by ROS causes the DNA damage response, including activation of p53 and PARP-1. It has been shown that necrotic cell death induced by alkylating DNA damage does not depend on p53 [69]. As already mentioned above, hyperactivation of PARP-1 inhibits glycolysis [20,21]. Besides causing ATP depletion, PARP-1 can also induce release of apoptosis-inducing

factor (AIF) from the mitochondrial intermembrane spa-ce in response to extensive DNA damage [70]. The release of AIF from the mitochondria promotes com-plex I destabilization and concomitant ROS production and is associated with generation of large DNA frag-ments (~50 kbp) upon translocation to the nucleus [71]. Accordingly, hydrogen peroxide and IR-induced necro-sis are reduced by downregulation of AIF [69,72], as well as by inhibition or genetic knockout of PARP-1 [70,73] (reviewed in [74]).

ROS MEDIATED LIPID PEROXIDATION IN NE-CROSIS

Other important targets susceptible to ROS activity are the polyunsaturated fatty acids in the cellular mem-branes. Reactive aldehydes derived from lipid peroxi-dation can compromise the membrane function by interacting with both the protein and the lipid moieties in the membrane (Fig. (2)) [75]. In mitochondria, the lipid peroxidation products negatively affect oxidative phosphorylation, inner membrane barrier properties, maintenance of mitochondrial membrane potential, and the mitochondrial Ca

2+ buffering capacity, contributing

in this way to necrosis [76]. Lipid peroxidation-mediated destabilization of both the plasma membrane and intra-cellular membranes, such as those of the lysosomes and the ER, results in leakage of proteases or efflux of Ca

2+ into the cytosol, both of which participate in necro-

Fig. (2). ROS damages lipids, proteins and DNA.

ROS initiates damage to DNA, proteins and lipids. PARP-1 is hyperactivated in response to DNA damage, leading to ATP de-

pletion and AIF translocation. Translocation of AIF from the mitochondria promotes complex I destabilization and concomitant

ROS production. In the nucleus, AIF causes further damage to the DNA. Oxidation of complex I, V and ANT by ROS results in

mitochondrial dysfunction and subsequent ATP depletion. Lipid peroxidation induced by ROS may lead to loss of membrane

integrity and disruption of ion homeostasis.


tic cell death processes. Moreover, ROS generation is auto-amplified through several reactions. Interaction of hydrogen peroxide with the superoxide anion in a Haber-Weiss reaction (1) or with iron in a Fenton reac-tion (2) generates the highly reactive hydroxyl radical, resulting in enhanced lipid peroxidation. The Fenton reaction is favored in lysosomes as they are rich in iron and reducing equivalents (such as cysteine), have a low pH (4.5-5.5) and do not contain catalases and glu-tathione peroxidases to detoxify hydrogen peroxide, which can enter the lysosomes by diffusion [77-79]. Lysosomes seem to be the only place where large amounts of redox-active iron temporarily reside before entering the cytosol for use in anabolic processes or for storage in ferritin (reviewed in [80]).

H2O2 + •O2- O2 + OH

- + HO• (1)

H2O2 + Fe2+

OH- + HO• + Fe

3+ (2)

Redox-active iron is a threat to the cells and needs to be cautiously transported and stored in a non-redox-active form, such as transferrin and ferritin. In this res-pect, recent data suggest that TNF-induced ROS for-mation is increased due to c-Jun N-terminal kinase 1 (JNK1)-dependent ferritin degradation, which results in an increase in the labile iron pool [81]. In addition, ferri-tin-deficient cells, storing a small amount of iron, were more resistant to elevation of the labile iron pool, and consequently showed no enhancement of ROS produc-tion and less cell death upon TNFR1 stimulation. RIP1 was indicated to be upstream of the induction of the labile iron pool in response to TNF [82]. Lysosomal membrane permeabilization (LMP) induced by lipid peroxidation as well as ensuing cell death were preven-ted by chelation of labile iron by desferrioxamine (DFO) when cells were exposed to oxidative stress, sugges-ting that oxidative stress per se is not detrimental but requires in some conditions the presence of redox-active iron [83-85].

MECHANISMS OF LMP IN NECROSIS

Besides lipid peroxidation, other factors contribute to LMP. One of the molecules that have been shown to regulate LMP is sphingosine. In response to cell death triggers, the lysosomal enzyme ASM is activated and converts sphingomyelin, the major sphingolipid present in cellular membranes, into ceramide. Accumulation of ceramide has been demonstrated in TNF-induced ne-crosis and is RIP1-dependent [86]. An increase in ce-ramide was also observed in Fas-induced necrosis in human T cells mouse B cells [87]. Ceramide has been shown to block mitochondrial respiratory chain complex III, thereby increasing the production of ROS [88,89]. Moreover, synthetic ceramide analogs induce necrotic-like cell death in human T cells [90]. Ceramide can be further converted into sphingosine by ceramidase. Owing to its lysosomotropic properties, sphingosine accumulates within the lysosomes, where it can per-meabilize the membrane by a detergent-like mecha-nism, causing LMP in a dose-dependent manner [91].

Lysosomal membranes can also be destabilized by calpains. This family of Ca

2+-dependent cysteine pro-

teases can be divided into two subfamilies, μ- and m-calpains, which require micromolar and millimolar con-centrations of Ca

2+ for their activation, respectively [92].

It is not only oxidative stress that can enhance calpain activation by increasing the Ca

2+ concentration, calpain

itself can provide a positive feedback loop when it pro-teolytically modulates the plasma membrane Na

+/Ca

2+

exchanger, leading to irreversible deregulation of Ca

2+

[93,94]. The ‘calpain-cathepsin’ hypothesis was propo-sed as a key mediator of necrosis in primate hippo-campal neurons [95]. Cathepsins in healthy cells reside mainly in lysosomes, where they participate in many biological processes [96,97]. After ischemia, activated μ-calpains localize at the lysosomal membrane and compromise its integrity, releasing cathepsins from the lysosomes into the cytosol; these digest target proteins and thereby lead to cell death. Ischemic neuronal cell death following calpain-mediated release of cathepsins could be inhibited with the cathepsin inhibitors CA-074 and E-64c [95,98-100]. The importance of calpains and cathepsins in necrosis was also suggested in a study in C. elegans reporting that necrotic cell death requires calcium-regulated CLP-1, TRA-3 and aspartyl protea-ses ASP-3 and ASP-4, homologues of mammalian cal-pain proteases and cathepsins [101].

Another important mechanism for amplification of LMP is mediated by activated PLA2 [102]. PLA2 enzy-mes can be classified into three major subfamilies: Ca

2+-independent PLA2 (iPLA2), secretory PLA2

(sPLA2), and cytosolic PLA2 (cPLA2). The latter has been associated mainly with necrosis, and is an intra-cellular enzyme responsible for the release of arachi-donic acid (AA) upon Ca

2+-mediated translocation to

cellular membranes. Next, lipoxygenases (LOX) can convert AA into lipid hydroperoxides that lead to mem-brane disruption. We have recently shown that activa-tion of a PLA2/LOX pathway contributes to TNF-induced necrotic cell death of L929 cells [58]. In L929 cells, PLA2 was activated upon TNF treatment, and cPLA2 overexpression in TNF-resistant L929 variants sensitized them to TNF-induced necrotic cell death [103,104]. Moreover, cPLA2 activity seemed to be ne-cessary for ceramide generation in TNF-induced ne-crosis in L929 cells [105]. A contribution of cPLA2 to TNF/zVAD-fmk-induced lethal shock has also been demonstrated in vivo [106]. Furthermore, mice deficient in cPLA2 exhibited reduced injury after brain ischemia [107].

More evidence showing the important role of LMP in necrotic cell death is provided by overexpression or knockdown of several guardians of lysosomal mem-brane integrity. Heat shock protein 70 (Hsp70) can pro-tect against LMP by delaying necrosis induced by TNF, heat shock or oxidative stress [108-113]. Moreover, mitochondria overexpressing Hsp70 show a decrease in ROS generation and lipid peroxidation upon IR [114]. On the other hand, RNAi-mediated knockdown of Hsp70 enhanced the labile iron pool in the absence of external stimuli, but this pool was increased considera-


bly in response to oxidative stress compared to paren-tal cells. Although the molecular details of how Hsp70 affects lysosomal membrane stability remain unclear, it is plausible that its ability to stabilize lysosomal mem-branes may be related to its effect on iron homeostasis [113]. Recent data show that the C-terminal portion of Hsp70 is sufficient for neuroprotection in response to IR. This indicates that neither the ability to fold denatu-red proteins nor interactions with co-chaperones nor other proteins that bind the amino-terminal half of Hsp70 are essential for neuronal ischemic protection [115].

RELEASE OF IMMUNOMODULATORY FAC-TORS BY NECROTIC CELLS

Clearance of dying cells by professional phagocytes involves attraction of phagocytes to the site of dying cells, recognition of dying cells, and their engulfment, followed by the phagocytic responses [116,117]. These aspects of cell-cell communication differ according to the type of cell death. Uptake of necrotic cells is delay-ed and less efficient, and occurs only after loss of membrane integrity [118]. This indicates that necrotic cells release their intracellular contents before they are cleared by phagocytes (Fig. (3)). Indeed, high mobility group box 1 protein (HMGB-1), a DNA binding molecu-le involved in chromatin structure and transcriptional regulation, is released by necrotic dying cells but re-mains bound to DNA in apoptotic cells even during se-condary necrosis [119,120]. Extracellular HMGB-1 acti-vates macrophages and dendritic cells (DC) [121,122] and promotes neutrophil recruitment [123]. Necrotic cells also release heat shock proteins (gp96, Hsp70, Hsp90) [124] and large amounts of uric acid [125], both of which have a pro-inflammatory effect through TLR2 and TLR4 [126,127]. Long time known as a pro-inflammatory molecule when crystallized in the joints in gout, uric acid was lately shown to activate the inflammasome, which is involved in the matura-tion of the inflammatory cytokine IL-1 [128]. In ge-neral, it is conceivable that upon loss of membrane integrity many other intracellular substances that are normally confined within cells are released from necro-tic cells. Among them are mRNA [129,130], genomic DNA (double-stranded) [131], nucleotides (ATP) and nucleosides (adenosine) [132-134]. Once released, these molecules exert immunostimulatory effects on macrophages and DC. However, nucleotides and nu-cleosides are unstable and therefore might act only transiently in the local environment.

Necrotic cells can also induce an inflammatory res-ponse by active secretion of inflammatory cytokines. It was found that necrotic cells actively release the pro-inflammatory cytokine IL-6 due to upregulation of NF-

B and p38MAPK [135]. These observations are in

agreement with the notion that necrotic cells retain their ability to synthesize proteins until the membrane com-pletely loses its integrity [56,136,137].

RECOGNITION AND ENGULFMENT OF NE-CROTIC CELLS

Recognition of necrotic cells by phagocytes is much less understood than recognition of apoptotic cells (Fig. (3)). Nevertheless, despite what was considered a pa-radigm, it has been demonstrated that necrotic cells with preserved plasma membrane integrity also exter-nalize phosphatidylserine [138], which is involved in their recognition and internalization [118,139]. Several macrophage receptor systems implicated in the en-gulfment of apoptotic cells also contribute to the uptake of necrotic cells. The thrombospondin-CD36- v 3 com-plex and CD14 are involved in the engulfment of heat-induced necrotic cells by human monocyte-derived macrophages [140]. Several bridging molecules can bind to surface molecules of dying and/or phagocytic cells, thereby supporting and facilitating their clearan-ce. Interaction with C1q, C-reactive protein [141] and thrombospondin [142] depend on the stage of cell death [143]. C1q is also required for effective uptake of necrotic cell-derived chromatin by macrophages [144]. Moreover, C3, C4, mannose binding lectin, pentraxin-3 and histidine-rich glycoprotein can bind both secondary and primary necrotic cells [145-150]. However, it re-mains uncertain whether certain bridging molecules specifically bind to and promote the clearance of only necrotic cells and not of apoptotic cells.

Binding and recognition of necrotic cells induces in the phagocytes sustained signaling, actin rearrange-ment and

internalization processes. In contrast to apop-

totic cells, which are engulfed by formation of tight-fitting phagosomes resembling a zipper-like mecha-nism, necrotic material is internalized by formation of spacious, fluid-filled macropinosomes [151]. Uptake of necrotic material by macropinocytosis (Fig. (3)) is ac-companied by macrophage ruffling and co-localization of fluid phase tracers within the same compartment [152].

IMMUNOLOGICAL CONSEQUENCES

The anti-inflammatory phagocyte responses to apoptotic cell uptake have often been contrasted to the pro-inflammatory effects of necrotic cells. Indeed, it has been shown that lysed neutrophils stimulate production of macrophage-inflammatory protein 2 (MIP-2), IL-8 and TNF (Fig. (3)) [153]. Necrotic cells can activate NF- B in macrophages and may induce expression of genes involved in inflammation and tissue-repair, e.g. the neutrophil-specific chemokine gene vascular en-dothelial growth factor (VEGF) [154]. Interestingly, VEGF was expressed near necrotic cells in various disease models [155,156], including the vicinity of ne-crotic foci in tumors [157]. A strict dichotomy between the immunomodulatory effects of apoptotic and necrotic cells is oversimplified: the effects also depend on the type of necrotic stimulus and the dying cell type. In this respect, it has been demonstrated that the uptake of necrotic Jurkat cells (ATP depleted or lysed) clearly inhibited E. coli-induced TNF secretion by human mo-nocyte-derived macrophages to an extent comparable


Fig. (3). Phagocytosis of necrotic cells.

Engulfment of necrotic cells can be divided into several phases. A: First, necrotic cells release chemotactic factors that recruit

phagocytes to the site of cell death. Necrotic cells, before they are cleared, may also release factors that modulate macrophages

and dendritic cells. Next, phagocytes recognize and bind to the necrotic cells via bridge molecules and receptors. B: Following

recognition, necrotic material is internalized by the macrophages by macropinocytosis with formation of membrane ruffles. C:

Inside the macrophages, necrotic material is co-localized with fluid phase markers within the same macropinosomes (hatched).

Finally, ingested necrotic material is degraded and a macrophage response is produced. (CD14, lipopolysaccharide receptor;

CD36, thrombospondin receptor; v 3/5 vitronectin receptor integrins).

to the uptake of apoptotic cells, whereas heat-killed Jurkat cells failed to inhibit the pro-inflammatory reac-tion of macrophages [139]. We have observed that phagocytosis of necrotic L929 fibrosarcoma cells oc-curs without activation of NF- B (Krysko and Vandena-

beele, data not published) or cytokine induction [118]. In another experimental setting, coincubation with ne-crotic cells sensitize the pro-inflammatory responses of activated macrophages, but they are unable to trigger macrophage activation by themselves [158]. All the


above-mentioned data indicate that the interaction between necrotic cells and phagocytes is complex and involves an array of macrophage reactions from neutral to pro-inflammatory (Fig. (3)). It is noteworthy that neither apoptotic nor necrotic cells are easily found in tissues because they are efficiently cleared by profes-sional and/or non-professional phagocytes. However, when clearance of dying cells is defective or ineffective, it may contribute to the persistence of inflammation, excessive tissue injury and development of several diseases, such as chronic obstructive pulmonary di-sease [159], diabetes [160], atherosclerosis [161] and systemic lupus erythematosus [162].

PATHOPHYSIOLOGICAL CHANGES TO-WARDS NECROSIS-LINKED PATHOLOGIES

Associated with necrotic cell death are several pat-hologies, such as neurodegeneration, ischemia-reperfusion and infection. Excitotoxicity is implicated in stroke and many neurodegenerative disorders, e.g. Alzheimer’s (AD), Huntington’s (HD) and Parkinson’s disease (PD). Excitotoxicity is the pathological process by which nerve cells are damaged and killed by the excitatory neurotransmitter glutamate and by similar substances. Overactivation of glutamate receptors (NMDA, AMPA and kainate receptors) by excitotoxins such as NMDA and kainic acid (both mimic glutamate) as well as pathologically high concentrations of gluta-mate allow high levels of Ca

2+ to enter the cell. This

initial Ca2+

influx is followed by a second uncontrolled increase due to release from the ER and/or by influx of extracellular Ca

2+, leading to necrotic-like cell death of

neurons [163,164]. Mitochondrial calcium overload and the subsequent MPT when the blood flow is restored are involved in necrotic cell death [28-30]. The impor-tant role of mitochondrial Ca

2+ overload in necrosis was

confirmed in rat neurons: Ca2+

uptake in mitochondria, not the elevated cytosolic Ca

2+ levels, accounted for

the excitotoxicity [164,165]. In addition, in primates ex-cessive Ca

2+ levels resulting from ischemia induce cal-

pain-mediated cathepsin release from the lysosomes, which in turn leads to necrotic cell death of neurons [95,98-100]. Besides contributing to LMP, calpains also cleave the plasma membrane Na

+/Ca

2+ antiporter du-

ring brain ischemia, and their inhibition could prevent Ca

2+ overload and rescue neurons from excitotoxic

death [93]. Calpain-mediated impairment of the Na+/K

+

ATPase activity was also demonstrated in myocardial cell death following ischemia [166].

Besides the role of mitochondrial Ca2+

overload in neurodegenerative diseases, oxidative stress also con-tributes to these conditions. Oxidative stress and mito-chondrial dysfunction occur early in all major neurode-generative diseases, and there is strong evidence that it has a causal role in pathogenesis. Indeed, oxidative damage in AD brains has been shown to occur before the onset of significant plaque pathology [167], to acti-vate signaling pathways [168,169] and inactivate criti-cal molecules [170,171], thereby contributing to the AD pathology. Decreased mitochondrial complex I activity

and oxidative stress have been observed in brains of patients with HD and PD [172,173]. Post mortem stu-dies have revealed that the overall levels of oxidative damage to proteins, lipids, and DNA are elevated in AD and PD brains [174-178]. Oxidative changes to proteins such as amyloid peptide in AD and -synuclein in PD, might result in increased protein misfolding, impai-red degradation and subsequent aggregate formation, which contributes to neurodegeneration [177]. During normal aging, the brain accumulates iron, copper and zinc, which increase vulnerability to oxidative stress. Thus, despite the presence of abundant antioxidants, the brain becomes more vulnerable to ROS generated via the Fenton reaction [179]. PD is associated with an increase in neuromelanin-bound iron in degenerating neurons of living PD patients and in post mortem brains. This abnormal protein-metal interaction increa-ses the oxidative stress in the neurons and contributes to neuronal damage [180]. Another aberrant protein-metal interaction is described in AD: accumulation of amyloid peptide in the presence of iron and copper leads to efficient generation of ROS (reviewed in [181]). In agreement with the protective effect against necrosis provided by iron chelation and ROS scavenging, stu-dies with metal-protein attenuating compounds (e.g. clioquinol) [182,183], iron chelators (e.g. DFO) [184,185] and natural antioxidants (e.g. curcumin, ginkgo) [186-188] seem promising for future treatment of patients with neurodegenerative diseases. The im-portance of ROS and redox active iron in necrosis was also shown in rat cardiac myocytes. Trolox, a water soluble analogue of vitamin E, able to prevent lipid pe-roxidation, and dipyridyl, an iron chelator, both protec-ted against hydrogen peroxide-induced lipid peroxida-tion and the ensuing necrotic cell death. This study points to potentially promising therapeutic interventions for heart damage following acute myocardial infarction [189].

As described above, MPT can occur in response to TNF, Ca

2+ overload and oxidative stress, and thereby

contribute to necrosis. Inhibiting MPTP opening by re-ducing oxidative stress or Ca

2+ overload can protect

against reperfusion injury. Indeed, functional recovery of rat hearts after IR injury was improved when they were treated with the free radical scavenger, propofol, which lowers oxidative stress. Mitochondria from pro-pofol-treated hearts exhibited increased respiratory chain activity and less sensitivity to calcium-induced MPTP opening [190]. The pharmacological inhibitor, dantrolene, a blocker of the sarcoplasmic reticulum Ca

2+ release channel, also reduced IR injury in rat

hearts [190,191]. As mentioned, MPTP is composed of several proteins, including VDAC, ANT and CypD. Blocking MPTP opening by using agents such as CsA and sanglifehrin-A at reperfusion protects the heart against IR injury [192-194]. CsA-treated animals exhibi-ted less necrotic cardiomyocyte death and required a significantly higher Ca

2+ overload to induce MPTP ope-

ning than controls [192]. The crucial role of CypD in necrotic cell death was confirmed in mice lacking CypD, which were protected against heart and brain


failure upon IR and Ca2+

influx [28-30,195,196]. Mice overexpressing CypD showed mitochondrial swelling and spontaneous cell death [28]. IR injury in brain and heart can be decreased with the chemical compound necrostatin-1 (Nec-1), which blocks RIP1-mediated necrotic cell death [42,197]. Moreover, a recent study shows that Nec-1, given at reperfusion, significantly limited infarct size in wild type mice but not in CypD-deficient mice, indicating that the cardioprotective effect of Nec-1 requires CypD to delay the opening of MPTP in rat cardiomyocytes subjected to oxidative stress [198].

Besides blocking MPTP and preventing lipid peroxi-dation, overexpression of Hsp70 can also delay necro-sis [108,109]. Overexpressing Hsp70 in vivo can pro-tect against myocardial and cerebral ischemia [199-201]. Upregulation of Hsp70 by pre-treatment with gel-danamycin can protect the brain against stroke in vivo and astrocytes against ischemia in vitro [202]. These data indicate that Hsp70 is protective against neuronal

injury in vivo, but it should be noted that the protective effect may depend on the nature and severity of the insult, cell type, and dose-dependent effects of Hsp70 overexpression [203]. In addition, it was recently de-monstrated that Hsp70 transgenic mice are protected against TNF-induced lethal shock. Addition of zinc to the drinking water of mice induced expression of Hsp70 in several organs and conferred protection against TNF-induced death. The protective effect of zinc was completely absent in mice lacking the Hsp70-inducible gene, suggesting that zinc protects against TNF in a Hsp70-dependent way [204].

CONCLUDING REMARKS

As discussed above, necrotic cell death is not due to a single well-described signaling pathway but results from the interplay between several signaling events (Fig. (4)). Although the necrotic process is associated with many biochemical parameters, there is still no

Fig. (4). An overview of necrosis induced by ligand-receptor interaction and by depletion of oxygen and glucose.

Necrotic cell death results from the interplay between several signaling cascades. RIP1 is a central initiator of death receptor-

induced necrosis. RIP1 has been implicated in the activation of plasma membrane-associated Nox1 during TNF-induced necro-

sis. Upon TNF stimulation, RIP1 localizes to the mitochondria, where it reduces the interaction between CypD and ANT, which

results in ATP depletion and subsequent necrosis. cPLA2, activated in response to TNF stimulation, induces formation of lipid

hydroperoxides, which in turn disrupt organelle and cell integrity. It has been suggested that the TNF-induced increase in ROS

formation is due to JNK1-mediated ferritin degradation in the lysosomes. In addition, ceramide generation, also RIP1-

dependent, contributes indirectly to LMP. When the supplies of oxygen and glucose are limited, the cytosolic Ca2+

concentration

increases and mitochondrial Ca2+

overload can occur. Reintroduction of oxygen induces a burst of ROS and MPT. The elevated

Ca2+

concentration activates calpains, which localize at the lysosomal membrane, cause LMP, and subsequent release into the

cytosol of cathepsins that will digest target proteins leading to cell death. (See text for details). (* localized at the plasma

membrane).


clear biochemical marker to define necrotic cell death. We propose that the definition of cell death type should be based on three different criteria: morphological cha-racteristics, biochemical characteristics, and interac-tions with phagocytes [205]. The increase in knowledge of the molecular mechanisms of necrotic cell death creates new opportunities for experimental therapy. This is exemplified by Degterev and coworkers, who used a synthetic inhibitor (Nec-1) to demonstrate the crucial role of RIP1-mediated necrotic cell death during ischemic brain injury in mice [42]. Lowering oxidative stress is another interesting target for therapeutic inter-vention. Antioxidant therapy with curcumin and ginkgo in patients with neurodegenerative disorders [186,188] and with trolox and propofol in mice with cardiac IR injury [189,190] has proven to be modestly successful. A more complete approach to antioxidant therapy would be to decrease ROS production and to upregula-te the endogenous antioxidant defense network. As mitochondria are the major source of intracellular ROS, the ideal drug therapy needs to be targeted to the mito-chondria. Recent preclinical studies of small antioxidant peptides (Szeto-Schiller peptides), selectively targeted to and concentrated in mitochondria, support their po-tential use for IR injury and neurodegenerative disor-ders [15,206]. Iron chelation therapy (e.g. DFO, dipyri-dyl) has also been demonstrated to reduce ROS gene-ration and the necrotic insult in vitro [85,189]. Although a controlled study of DFO in AD had some promising results, concerns about toxicity and brain delivery have limited trials of traditional chelators [184,185]. Treat-ment with metal-protein attenuating compounds (e.g. clioquinol) has been proposed as a new therapeutic strategy for neurodegenerative diseases [182,183]. Another key element implicated in necrosis is distur-bance of Ca

2+ homeostasis. Treatment with dantrolene

in rats [191] and use of LTCC blockers and -adrenergic receptor antagonists in mice [196] pre-vented Ca

2+ overload and cardiac myocyte necrosis

and thus it may represent new cardioprotective thera-pies. Moreover, future studies aimed at understanding the attraction signals released from dying cells, the specific mechanisms of recognition and internalization, and the phagocytic responses will enable us to exploit the immunomodulatory properties of necrotic cell clea-rance for development of treatments in which a strong immune response is needed, such as anti-cancer the-rapy [207].

ACKNOWLEDGEMENTS

We thank A. Bredan for editing the manuscript. This research has been supported by Flanders Institute for Biotechnology (VIB) and several grants. European grants: EC Marie Curie Training and Mobility Program, FP6 ApopTrain, MRTN-CT-035624; EC RTD Integrated Project, FP6 Epistem, LSHB-CT-2005-019067; EC RTD Integrated Project, Apo-Sys, FP7-200767. Belgian grants: Interuniversity attraction poles, IAP 6/18. Fle- mish grants: Fonds Wetenschappelijke Onderzoek Vlaanderen, 3G.0218.06; Ghent University grants: BOF-GOA – 12.0505.02 and UGent-cofinancing EU

project (grant I/00001/02). Peter Vandenabeele is full professor at Ghent University, Tom Vanden Berghe and Nele Festjens are supported by a FWO postdoctoral fellowship. Dmitri V. Krysko is paid by a postdoctoral fellowship from the BOF (Bijzonder Onderzoeksfonds 01P05807), Ghent University. Nele Vanlangenakker is supported by a BOF grant (01D21005) from Ghent University for a Ph.D. fellowship.

ABBREVIATIONS

AA = Arachidonic acid

AD = Alzheimer’s disease

AIF = Apoptosis-inducing factor

ANT = Adenine nucleotide translocator

ASM = Acid-sphingomyelinase

BHA = Butylated hydroxyanisole

CsA = Cyclosporin A

CypD = Cyclophilin D

C1q = Complement protein C1q

DC = Dendritic cells

DFO = Desferrioxamine

ER = Endoplasmic reticulum

HD = Huntington’s disease

HMGB-1 = High mobility group box-1

HRG = Histidine-rich glycoprotein

Hsp70 = Heat shock protein 70

IR = Ischemia-reperfusion

JNK1 = c-Jun N-terminal kinase 1

LMP = Lysosomal membrane permeabilization

LOOH = Lipid hydroperoxides

LOX = Lipoxygenase

MBL = Mannose-binding lectin

MPT = Mitochondrial permeability transition

MPTP = Mitochondrial permeability transition pore

Nec-1 = Necrostatin-1

NLR = NOD-like receptor

Nox1 = NADPH oxidase-1

PAMP = Pathogen-associated molecular pattern

PARP-1 = Poly(ADP-ribose) polymerase-1

PLA2 = Phospholipase A2

PD = Parkinson’s disease

PRR = Pathogen recognition receptor

PS = Phosphatidylserine

PTX3 = Long pentaxin 3

RIP1 = Receptor-interacting protein 1


ROS = Reactive oxygen species

TNFR1 = Tumor necrosis factor receptor-1

TLR = Toll-like receptor

TSP-1 = Thrombospondin-1

VDAC = Voltage-dependent anion channel

VEGF = Vascular endothelial growth factor

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Received: December 10, 2007 Revised: December 30, 2007 Accepted: January 10, 2008

Molecular Mechanisms and Pathophysiology of Necrotic Cell Death

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