Microbes and Infection 11 (2009) 1050e1062

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Major cell death pathways at a glance

Linde Duprez a,b, Ellen Wirawan a,b, Tom Vanden Berghe a,b, Peter Vandenabeele a,b,*
a

VIB, Department for Molecular Biomedical Research, Unit for Molecular Signaling and Cell Death, Technologiepark 927, B-9052 Ghent (Zwijnaarde),
Belgium
b
Ghent University, Department of Biomedical Molecular Biology, Unit for Molecular Signaling and Cell Death, Technologiepark 927, B-9052 Ghent
(Zwijnaarde), Belgium
Available online 4 September 2009

Abstract
Cell death is a crucial process during development, homeostasis and immune regulation of multicellular organisms, and its dysregulation is
associated with numerous pathologies. Cell death is often induced upon pathogen infection as part of the defense mechanism, and pathogens
have evolved strategies to modulate host cell death. In this review, we will discuss the molecular mechanisms and physiological relevance of four
major types of programmed cell death, namely apoptosis, necrosis, autophagic cell death and pyroptosis.
2009 Elsevier Masson SAS. All rights reserved.
Keywords: Apoptosis; Necrosis; Autophagy; Pyroptosis

1. Introduction
Different types of cell death are often defined by morphological criteria, and are classified as apoptotic, necrotic,

autophagic or associated with mitotic catastrophe. Additionally, cell death is defined based on enzymological criteria,
including the involvement of different classes of proteases
(caspases, calpains, cathepsins and transglutaminases) and

Abbreviations: AICD, activation-induced cell death; AIM2, absent in melanoma 2; Ambra-1, activating molecule in Beclin-1-regulated autophagy; ANT,
adenine nucleotide translocator; Apaf-1, apoptosis protease activating factor-1; ASC, apoptotic speck protein containing a CARD; Bcl-2, B-cell lymphoma-2; BH3,
Bcl-2-homology 3; Bif-1, Bax interacting factor-1; CARD, caspase activation and recruitment domain; Cardinal, CARD inhibitor of NF-kB activating ligands;
cFLIP, cellular FLICE-like inhibitory protein; cIAP, cellular inhibitor of apoptosis protein; CypD, cyclophilin D; Cyt c, cytochrome c; DAMP, danger/damageassociated molecular pattern; DED, death effector domain; DISC, death-inducing signaling complex; Diablo, direct IAP binding protein with low pI; DIF-1,
differentiation-inducing factor-1; ER, endoplasmic reticulum; FADD, Fas-associated death domain; FIP200, focal adhesion kinase family interacting protein of
200 kD; HIN-200 protein, hematopoietic interferon-inducible nuclear protein with a 200-amino-acid repeat; HIV-1, human immunodeficiency virus-1; HtrA2, high
temperature requirement protein A2; IAP, inhibitor of apoptosis protein; ICE, interleukin-1 converting enzyme-b; IL-18, interleukin-18; IL-1b, interleukin-1b;
Ipaf, ICE-protease activating factor; I/R, ischemia/reperfusion; JNK, c-Jun N-terminal kinase; LC3, microtubule-associated protein light chain 3; LPC, lysophosphatidylcholine; LPS, lipopolysaccharide; LRR, leucine-rich repeat; MAPK, mitogen activated protein kinase; MEF, mouse embryonic fibroblast; MPT,
mitochondrial permeability transition; MPTP, mitochondrial permeability transition pore; mTOR, mammalian target of rapamycin; NACHT, nucleotide binding
and oligomerization domain; Nalp, NACHT/LRR/PYD-containing protein; Nec-1, necrostatin-1; NF-kB, nuclear factor kB; NLR, NOD-like receptor; nNOS,
neuronal nitric oxide synthase; PAMP, pathogen-associated molecular pattern; PAR, poly(ADP-ribose); PARP-1, poly(ADP-ribose) polymerase-1; PE, phosphatidylethanolamine; PI3P, phosphatidylinositol-3-phosphate; PI3KC3, phosphatidylinositol 3-kinase class 3; Pycard, PYD and CARD domain containing protein;
PRR, pathogen recognition receptor; PS, phosphatidylserine; PYD, pyrin effector domain; RIP, receptor interacting protein; RLR, RIG-I-like receptor; RNAi, RNA
interference; ROS, reactive oxygen species; Smac, second mitochondria-derived activator of caspase; TLR, toll-like receptor; TNF, tumor necrosis factor; TNFR,
tumor necrosis factor receptor; TRADD, TNFR-associated death domain; TRAF2, TNF receptor-associated factor 2; TRAIL, TNF-related apoptosis-inducing
ligand; TRAIL-R, TRAIL-receptor; ULK, UNC-51-like kinase; UVRAG, UV radiation resistance-associated gene; VPS34, vacuolar protein sorting 34; XIAP, Xlinked inhibitor of apoptosis protein; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone.
* Corresponding author. VIB, Department for Molecular Biomedical Research, Unit for Molecular Signaling and Cell Death, Technologiepark 927, B-9052
Ghent (Zwijnaarde), Belgium. Tel.: 32 9 3313763; fax: 32 9 3313609.
E-mail address: peter.vandenabeele@dmbr.vib-ugent.be (P. Vandenabeele).
1286-4579/$ - see front matter 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.micinf.2009.08.013

L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

nucleases, on functional aspects (programmed or accidental,

physiological or pathological) or on immunological characteristics (immunogenic or non-immunogenic) [1]. The present
paradigm is that caspase-dependent apoptosis is the predominant cell death pathway, but (1) that caspase-independent
mechanisms can cooperate with (or substitute for) caspases in
the execution of lethal signaling pathways, (2) that the major
necrotic cell death pathway is mediated through the serine/
threonine kinases receptor interacting protein 1 (RIP1) and 3
(RIP3), (3) that signaling involved in caspase-1-mediated cell
death (termed pyroptosis) differs from classical caspasedependent apoptosis, and (4) that autophagic cell death is
a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. In this review, we will
discuss the four major forms of programmed cell death at the
molecular, cellular and physiological levels.
2. Apoptotic cell death
In 1972, the term apoptosis was used for the first time to
describe a form of cell death associated with specific
morphological features. Since then, apoptosis has been
extensively studied and underlying signaling events are now
well characterized. Morphologically, apoptosis is associated
with cell shrinkage, membrane blebbing, and chromatin
condensation. It is a cell-intrinsic programmed suicide
mechanism that results in the controlled breakdown of the cell
into apoptotic bodies, which are subsequently recognized and
engulfed by surrounding cells and phagocytes. Two main
evolutionarily conserved protein families are involved in
apoptosis, namely the Bcl-2 family of proteins, which control
mitochondrial integrity [2], and the cysteinyl aspartate-specific
proteases or caspases, which mediate the execution phase of
apoptosis [3].
2.1. Molecular mechanisms of apoptosis
In humans, twelve caspases belonging to the apoptotic and
the inflammatory subfamilies of caspases have been described.
Apoptotic caspases can be further subdivided into initiator
(caspase-2, -8, -9, -10) and executioner (caspase-3, -6, -7)
caspases. All caspases are expressed as inactive proenzymes
consisting of an N-terminal prodomain of variable length,
followed by a large subunit (p20) and a small C-terminal
subunit (p10). The long prodomain of the initiator caspases
contains proteineprotein interaction motifs belonging to the
death domain superfamily, namely death effector domains
(DEDs) and caspase activation and recruitment domains
(CARDs). Initiator caspases, present in the cell as inactive
monomers, are recruited to platform molecules via these
proteineprotein interaction domains and are subsequently
activated by oligomerization and proximity-induced autoproteolysis [4]. Short prodomain caspases exist as preformed
dimers in the cell and their enzymatic activity requires
proteolytic maturation by the action of upstream caspases [4].
In mammalian cells, caspases are activated by the intrinsic
or the extrinsic apoptotic pathway (Fig. 1). The intrinsic

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pathway is activated by various stimuli, such as DNA damage

and cytotoxic insults, and acts through the mitochondria,
which are controlled by the Bcl-2 family of proteins [2]. In
homeostatic conditions, the anti-apoptotic Bcl-2 family
members maintain mitochondrial integrity by preventing the
pro-apoptotic multidomain Bcl-2 family members Bax and
Bak from causing mitochondrial damage. During cellular
stress, Bcl-2-homology 3 (BH3)-only proteins are activated
and antagonize the anti-apoptotic Bcl-2 family members.
Consequently, the inhibition of Bax and/or Bak is relieved,
leading to their oligomerization and formation of a channel
through which cytochrome c (cyt c) is released into the
cytosol. Then, cyt c associates with Apaf-1 and ATP, forming
a platform for recruitment and activation of procaspase-9, also
known as the apoptosome [5]. Active caspase-9 cleaves and
activates the downstream executioner caspases-3, -6 and -7,
which are crucial for the execution of apoptotic cell death. In
addition, other pro-apoptotic proteins released from the
mitochondria contribute to the cellular suicide mechanism. For
example, Smac/Diablo antagonizes the action of inhibitor of
apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 [6].
Binding of Smac/Diablo to XIAP relieves its inhibitory
interaction with caspase-9, -3 and -7. cIAP1 and cIAP2, unlike
XIAP, do not directly inhibit caspases. Instead, they interact
with tumor necrosis factor receptor (TNFR)-associated factor
2 (TRAF2), which leads to their recruitment to TNFR1. There,
they contribute to TNF-induced NF-kB activation by mediating the ubiquitination of RIP1 [6]. The NF-kB-mediated
expression of anti-apoptotic genes might account for the antiapoptotic function of cIAP1 and cIAP2.
The extrinsic pathway of apoptosis is induced upon stimulation of death receptors belonging to the TNFR family, such
as TNFR, Fas and TRAIL-R. Signaling by these receptors can
induce a variety of cellular responses, including proliferation,
differentiation and cell death. Apoptosis is induced by the
formation of a death-inducing signaling complex (DISC). In
this complex, Fas-associated death domain (FADD) recruits
the initiator caspases-8 and/or -10 via homotypic death
domain interactions [7]. In contrast to signaling induced by
Fas and TRAIL-R, TNFR1 aggregation leads to the sequential
formation of two complexes [8]. Complex I consists of
TNFR1, TNFR-associated death domain (TRADD), TRAF2,
RIP1, cIAP1 and cIAP2 and is formed at the plasma
membrane. These proteins are important mediators of TNFinduced activation of NF-kB and MAPKs. Endocytosis of
TNFR1 is followed by the formation of complex II, which is
analogous to the receptor-proximal DISC induced by FasL and
TRAIL and includes TRADD, FADD, and caspase-8 and/or
-10. Activation of caspase-8 and -10 leads to activation of the
downstream executioner caspases. In addition, caspase-8mediated cleavage of the BH3-only protein Bid amplifies the
death receptor-induced cell death program by activating the
mitochondrial pathway of apoptosis. Recently, it was found
that TNF induces one of two distinct caspase-8 activation
pathways, depending on the cellular condition [9]. One
pathway is RIP1 independent and is primarily inhibited by
cFLIP, a protease-dead caspase-8 homologue that competes

L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

1052
e.g. TNF

RIP1

Cytosol

TRADD

e.g. TNFR1

2 Intracellular stress signals

Complex I

TRAF2

Ubiquitination

cIAP1,2

BH3-only

Complex II - DISC
DED
DED
FADD
TRADD DED

p20

p10

DED

p20

p10

DED

Bid

DED

Bcl-2,
Bcl-XL

tBid

Procaspase-8,10
Bax/Bak
Cyt c

NF-kB
Active caspase-8,10

Apaf-1
CARD

p20
p20

Apoptosome

p10
p10

Procaspase-3,6,7

Active
caspase-3,6,7

p20

p10

Procaspase-9

Pro-apoptotic
Pro-apoptotic
Pro-apoptotic
proteins
proteins
proteins
e.g.
Active caspase-9

Anti-apoptotic
genes

Substrate cleavage

Smac/Diablo
Smac/Diablo
Smac/Diablo

Nucleus

Apoptotic cell death

Fig. 1. Schematic representation of extrinsic and intrinsic apoptotic signaling. Stimulation of e.g. TNFR1 initiates the extrinsic pathway of apoptosis (1). Upon
TNF stimulation, complex I is formed, resulting in NF-kB activation and subsequent transcription of anti-apoptotic genes. After endocytosis of TNFR1, complex II
is formed, in which caspase-8 is recruited and activated. Subsequent activation of the executioner caspases leads to cleavage of their substrates and to cell death.
The intrinsic pathway of apoptosis (2) is activated upon intracellular stress signals at the mitochondrial level. Activation of Bax and Bak induces the release of
several mitochondrial apoptotic mediators. The subsequent formation of the apoptosome results in the activation of caspase-9. In addition, caspase-8-mediated
cleavage of Bid amplifies the extrinsic pathway of apoptosis by activating the mitochondrial pathway. See text for detailed sequence of events.

for caspase-8 binding to FADD. The other pathway was

discovered in experiments using treatment with TNF and
Smac mimetics and is absolutely dependent on kinase active
RIP1. Smac mimetics induce the autodegradation of cIAP1
and cIAP2, leading to release of RIP1 from the receptor
complex to form a caspase-8 activating platform consisting of
RIP1, FADD, and caspase-8 [9].
2.2. Physiological relevance of apoptosis
During development, apoptotic cell death has an important
role in organ and tissue remodeling, e.g. in development of the
lens, in shaping of the inner ear, in cardiac morphogenesis, in
muscle development, and in removal of interdigital webs [10].
Furthermore, establishment of both the nervous and the
immune systems are characterized by initial overproduction of
cells and subsequent elimination of excess cells by apoptosis
[11]. Apoptosis is crucial in maintaining homeostasis in the
adult organism, as in post-lactational mammary gland

regression, ovarian follicular atresia and post-ovulatory

regression, and in terminating an immune response by eliminating of activated immune cells [11]. Because apoptotic cells
are recognized and taken up by phagocytes before they lose
plasma membrane integrity, it is generally believed that
apoptotic cell death is immunologically silent and does not
provoke inflammation [12].
Apoptosis of host cells during bacterial or viral infection
functions as a defense mechanism by destroying the site of
pathogen replication. Pathogens have therefore evolved
several ways to prevent apoptosis, e.g. by protecting the
mitochondria and preventing cyt c release, by activating cell
survival pathways, or by preventing caspase activation [13,14].
Conversely, pathogens may also benefit from apoptosis, either
by inducing apoptosis of infected cells, thereby favoring
systemic infection, or by killing uninfected immune cells,
thereby evading the host defense [13,14].
Dysregulation of apoptosis can result in the development of
various pathologies. Insufficient apoptosis can lead to the

L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

development of cancer and autoimmune diseases. Excessive or

inappropriate apoptosis, on the other hand, contributes to the
injury that accompanies several diseases, such as sepsis,
stroke, myocardial infarction, ischemia, neurodegenerative
diseases, and diabetes.
3. Necrotic cell death
For a long time, necrosis has been considered an accidental
and uncontrolled form of cell death lacking underlying
signaling events. This might be true for cell death resulting
from severe physical damage, such as hyperthermia or detergent-induced cytolysis. However, accumulating evidence
supports the existence of caspase-independent cell death
pathways that can function even in a strictly regulated developmental context, such as interdigital cell death [15]. Necrotic
cell death is characterized by cytoplasmic and organelle
swelling, followed by the loss of cell membrane integrity and
release of the cellular contents into the surrounding extracellular space.
3.1. Molecular mechanisms of necrosis
Over the past decade, it has become evident that in certain
conditions, necrosis is the result of a strictly regulated

1053

interplay of signaling events, which are initiated by a diverse

range of stimuli (Fig. 2). In most cell lines, death receptor
ligands activate apoptosis rather than necrosis as the default
cell death pathway. However, if caspase activation in this
pathway is hampered, necrotic cell death might ensue instead,
acting as a kind of back-up cell death pathway. zVAD-fmk is
frequently used as a potent inhibitor of caspases, but off-target
effects can also contribute to caspase-independent cell death.
For example, zVAD-fmk binds and blocks the adenine
nucleotide translocator (ANT), inhibits other proteases such as
cathepsins, and generates the highly toxic fluoroacetate due to
metabolic conversion of the fluoromethylketone group [16,17].
FADD remains a crucial adaptor protein in Fas- and
TRAIL-R-induced necrosis, but the importance of FADD in
TNF-induced necrosis is controversial [18,19]. Recently, with
the generation of the TRADD knockout mouse, it was
demonstrated that TRADD is essential for TNF-induced
necrosis in MEF cells [20]. RIP1 is a crucial initiator of death
receptor-mediated necrosis [21] and the term necroptosis was
introduced to designate programmed necrosis that depends on
RIP1 [22]. The kinase activity of RIP1 is dispensable for the
activation of NF-kB and MAPKs, but is required for necroptosis [19,22,23]. Necrostatin-1 (Nec-1) was identified as
a small molecule inhibitor of necroptosis [22], and more
recently, the RIP1 kinase activity was found to be the target of

e.g. TNF
LPS

RIP1

dsRNA

RIP3

TRAF2

FADD
TRADD

TRADD

RIP1

Cytosol

E.g. Shigella

TLR-4

e.g. TNFR1

TLR-3

Endosome

RIP1

Nalp-3

Pro-survival

NF- kB

MAPK

ROS
Ca2+
Phospholipases
Calpains
Cathepsins

AP-1

NO

PARP-1

Ceramide

TRAF2
RIP1

Nucleus

Necrotic cell death

DNA damage

Fig. 2. Schematic representation of necrotic signaling. Stimulation of e.g. TNFR1 leads to the activation of RIP1, which induces a pro-survival pathway by
activating transcription factors, such as NF-kB and AP-1. In addition, RIP1 interacts with RIP3 and both are crucial initiators of death receptor-induced necrotic
signaling. Through RIP1 kinase activity, a wide range of necrotic mediators are activated, such as ROS, calcium, calpains, cathepsins, phospholipases, NO and
ceramide. The same mediators can be activated by DNA damage or by triggering of TLR-3, TLR-4 and Nalp-3. See text for detailed sequence of events.

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L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

Nec-1 [24]. Furthermore, recent studies identified RIP3 as

a crucial upstream activating kinase that regulates RIP1dependent necroptosis [25e27]. TNF treatment induced the
formation of a RIP1eRIP3 pro-necrotic complex and the
kinase activity of both RIP1 and RIP3 was crucial for stable
complex formation and subsequent induction of necrosis.
During death receptor-induced apoptosis, RIP1 and RIP3 are
cleaved by caspase-8, which suppresses their anti-apoptotic
and/or pro-necrotic properties [28,29].
Besides death receptor-mediated necrosis, triggering of
pathogen recognition receptors (PRRs) can also lead to
necrotic cell death. Receptors of this family include the
transmembrane toll-like receptors (TLRs), the cytosolic
NOD-like receptors (NLRs) and the RIG-I-like receptors
(RLRs). They all recognize pathogen-associated molecular
patterns (PAMPs) found in bacteria or viruses, such as LPS,
flagellin and double-stranded RNA (dsRNA), and stimulation
of these receptors leads to the activation of innate immunity
and/or cell death. In Jurkat cells and L929 cells, the recognition of synthetic dsRNA by TLR3 induces necrotic cell
death, which was suggested to be RIP1-dependent [30].
TLR4 is expressed on macrophages and monocytes and is
critical for the recognition of LPS from Gram-negative
bacteria. Impeding caspase-8 activation switches TLR4induced cell death from apoptosis to RIP1-dependent
necrosis [31]. Pathogen-induced activation of NLRs results
most commonly in caspase-1-dependent cell death or
pyroptosis (see below). However, a recent report showed that
the NLR member Nalp-3 mediates necrotic cell death of
macrophages infected with Shigella flexneri at high multiplicity of infection [32]. RLR-induced activation of NF-kB
and production of type I interferons are both dependent on
FADD, RIP1 and TRADD [33,34]. Whether these proteins
are also involved in RLR-induced cell death is unknown.
Extensive DNA damage causes hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) and leads to necrotic
cell death [35]. When DNA damage is moderate, PARP-1
participates in DNA repair processes. However, excessive
PARP-1 activation causes depletion of NAD by catalyzing
the hydrolysis of NAD into nicotinamide and poly(ADPribose) (PAR), leading to ATP depletion, irreversible cellular
energy failure, and necrotic cell death. PARP-1-mediated cell
death requires the activation of RIP1 and TRAF2 [36].
However, the mechanism by which these molecules sense the
activation of PARP-1 has not been elucidated.
Many mediators are involved in the execution phase of
necrotic cell death, including reactive oxygen species (ROS),
calcium (Ca2), calpains, cathepsins, phospholipases, and
ceramide [37]. Oxidative stress leads to damage of cellular
macromolecules, including DNA, proteins, and lipids. As
discussed earlier, excessive DNA damage results in hyperactivation of PARP-1 and necrotic cell death. Modification of
proteins by ROS leads to loss of the normal functions of
proteins and enhances their susceptibility to proteolytic
degradation. Other targets of ROS are the polyunsaturated
fatty acid residues in the membrane phospholipids, which are
extremely sensitive to oxidation. In mitochondria, lipid

peroxidation affects vital mitochondrial functions. In addition,

it destabilizes the plasma membrane and intracellular
membranes of endoplasmic reticulum and lysosomes, leading
to intracellular leakage of Ca2 and lysosomal proteases,
respectively. Among the different ROS, hydrogen peroxide
(H2O2) plays a particularly important role because it diffuses
freely across cellular membranes and can interact with iron in
the Fenton reaction [37]. This reaction is favored in the
lysosomes, because they are rich in free iron and do not
contain H2O2-detoxifying enzymes. The resulting highly
reactive hydroxyl radicals are among the most potent inducers
of lipid peroxidation.
Ca2 overload of mitochondria causes mitochondrial
permeability transition (MPT) by the opening of large
nonselective pores (the so called mitochondrial permeability
transition pores, MPTPs) connecting the cytosol with the
mitochondrial matrix [38]. MPT is accompanied by mitochondrial inner membrane depolarization, uncoupling of
oxidative phosphorylation, matrix swelling, and outer mitochondrial membrane rupture [38]. If most mitochondria of the
cell are disrupted, and glycolytic sources of ATP are inadequate, the cell becomes profoundly ATP-depleted. Cyclophilin
D (CypD) might have an important role in MPT, as inhibition
of CypD renders cells resistant to MPT, and CypD-deficient
mice are more resistant to ischemic injury than wild type mice
[39,40]. Besides affecting mitochondrial respiration, Ca2
overload can activate phospholipases, proteases and neuronal
nitric oxide synthase (nNOS), all of which contribute to the
execution phase of necrotic cell death. For example, calpains
are activated by elevated Ca2 levels, which then cleave the
Na/Ca2 antiporter in the plasma membrane, resulting in
a sustained Ca2 overload. Strong activation of calpains may
also contribute to the release of cathepsins in the cytosol by
causing lysosomal membrane permeabilization, as proposed in
the calpainecathepsin hypothesis by Yamashima and
colleagues [41].
3.2. Physiological relevance of necrosis
Although apoptosis is indispensable for tissue remodeling
during embryogenesis, necrosis can, at least in some conditions, substitute for apoptosis to eliminate unwanted cells. For
example, removal of interdigital cells during the development
of digits in the presence of the caspase inhibitor zVAD-fmk or
in Apaf-1/ mice occurs by a caspase-independent necroticlike process [15]. Necrosis is also involved in physiologically
relevant signaling processes, such as ovulation, the death of
chondrocytes associated with the longitudinal growth of
bones, and cellular turnover in the small and large intestines
[42]. In addition, necrotic cell death participates in activationinduced cell death (AICD) of T lymphocytes, which is an
important mechanism for reducing T cell numbers after an
immune response [19]. However, it is important to point out
that in most of the above-mentioned pathways, necrotic cell
death is always observed together with apoptosis or in the
presence of caspase inhibitors, suggesting that it functions as
a back-up mechanism and is never the sole cell death pathway.

L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

Necrotic cell death is often associated with pathological

conditions. Necrosis has been observed during ischemia/
reperfusion (I/R), which can lead to injury of organs, including
heart, brain, liver, kidney, and intestine [43]. Necrotic cell
death also contributes to excitotoxicity, which may be
involved in stroke, traumatic brain injury, and neurodegenerative disorders [44]. More specifically, using Nec-1, it was
shown that RIP1-dependent necrotic cell death or necroptosis
contributes to a wide range of pathological cell death events,
such as ischemic brain injury [22] and myocardial infarction
[45]. Furthermore, RIP3/ mice failed to initiate vaccinia
virus-induced tissue necrosis and inflammation, resulting in
much more viral replication and mortality [26]. Several other
reports also illustrate the occurrence of necrotic cell death
during infection by other pathogens, such as Shigella, HIV-1,
West Nile virus, and Coxsackievirus B [37]. In addition,
patients carrying a disease-associated mutation in Nalp-3 show
excessive necrotic-like cell death with features similar to the
Shigella flexneri-induced Nalp-3-dependent necrosis [32].
In contrast to apoptosis, the recognition and uptake of
necrotic cells by macropinocytosis is slower, less efficient and
occurs only after the loss of plasma membrane integrity [46].
As a result, necrotic cells initiate a proinflammatory response
by the passive release of DAMPs (danger/damage-associated
molecular patterns) [47]. In addition, necrotic cells actively
secrete inflammatory cytokines due to the activation of NF-kB
and MAPKs [48].
4. Autophagic cell death
Autophagy is an evolutionarily conserved catabolic
pathway that allows eukaryotes to degrade and recycle cellular
components. Proteins and organelles are sequestered in
specialized double-membrane vesicles, designated autophagosomes, which are typical of autophagic cells. Basal levels of
autophagy ensure the maintenance of intracellular homeostasis, but in addition, many studies have revealed its diverse
functions in important cellular processes, such as cellular
stress, differentiation, development, longevity and immune
defense. Although a pro-survival role for autophagy is wellestablished, frequently debated is whether or not autophagy
has a causative role in cell death. The presence of autophagic
vacuoles in dying cells has led to the introduction of autophagic cell death, although autophagy often accompanies
rather than causes cell death. It is plausible though that
massive autophagic activity could result in cellular demise. In
addition, several interconnections exist between autophagy
and apoptotic or necrotic cell death [49].
4.1. Molecular mechanisms of autophagy
Several types of autophagy have been described, including
chaperone-mediated autophagy, microautophagy and macroautophagy. Macroautophagy (hereafter referred to as autophagy) typically occurs in severe stress conditions and is therefore
the best characterized (Fig. 3). Macroautophagy functions by de
novo formation of specialized vacuoles, the autophagosomes, in

1055

which the proteins are sequestered before being targeted to the

lysosomes. So far, 30 autophagy-related (atg) genes have been
identified in yeast, and several mammalian homologues have
been isolated and functionally characterized [50].
The classical pathway of autophagic signaling acts through
mTOR (mammalian target of rapamycin), a protein kinase that
is important in controlling translation and cell-cycle progression and in negative regulation of autophagy. When mTOR is
inhibited in response to starvation or treatment with rapamycin, the ULK-Atg13-FIP200 complex is activated, leading to
the induction of autophagosome formation [51]. The synthesis
of autophagic vacuoles requires vesicle nucleation, which is
initiated by the assembly of another complex, the PI3KC3
(Vps34)-complex. Within this complex, Beclin-1 (Atg6)
serves as a platform for binding PI3KC3 (Vps34), UVRAG,
Bif-1 and Ambra-1, all of which positively regulate PI3KC3
activity [52]. Interestingly, Bcl-2, amongst other anti-apoptotic
Bcl-2 family members, represses autophagy by binding of
Beclin-1 and thereby abrogating autophagic signaling [53].
During starvation, Bcl-2 becomes phosphorylated, which
releases Beclin-1 and stimulates autophagy [54]. Activation of
the PI3KC3 (Vps34)-complex finally results in the generation
of PI3P. Consequently, other Atg proteins that mediate vesicle
membrane elongation are recruited. Two ubiquitin-like
conjugation systems are implicated in this process. During
a first conjugation step, an Atg12eAtg5eAtg16(L) multimer
complex that could be involved in vesicle curvature is formed
[55]. During a second conjugation step, LC3 (Atg8) is lipidated by binding to phosphatidylethanolamine (PE). In
contrast to the cytoplasmic localization of LC3, LC3ePE
(mostly referred to as LC3-II) specifically binds to the autophagic membranes, and for that reason it is generally used as
an autophagic marker. Finally, the autophagosome fuses with
a lysosome, releasing its autophagic content into the lysosomal
lumen for degradation by hydrolases.
The outcome of autophagic signaling mostly reflects its
pro-survival function. In homeostasis, autophagy exerts its
housekeeping role in cytoplasmic and protein turnover, while
during stress, cells are protected from dying by elimination of
harmful organelles and protein aggregates. However, several
studies suggest a role for autophagy in cell death [56]. In
cells in which apoptotic signaling is perturbed, a necrotic-like
cell death is often induced as a back-up cell death mechanism, which can be associated with autophagy, as monitored
by LC3-II accumulation. For example, treatment of
apoptosis-deficient Bax/Bak double knockout MEF cells with
DNA-damaging or ER-stress-inducing agents causes nonapoptotic cell death, which depends on the presence of
Beclin-1 and Atg5 [57]. Another interesting study shows the
involvement of FADD and caspase-8 in controlling autophagic signaling in proliferating T cells [58]. T cells lacking
FADD or caspase-8 undergo extensive autophagy and
succumb to RIP1-dependent necrotic cell death, but inhibition of autophagy rescued the T cells. Moreover, treatment
with Nec-1 repressed LC3-II accumulation and prevented T
cells from dying [58]. Similarly, in L929 cells, caspase
inhibition with zVAD-fmk or caspase-8 RNAi also resulted in

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L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

2
1 Nutrients
Growth factors
Rapamycin

mTOR

ULK
Atg13
Induction

ULK-Atg13-FIP200
complex

FIP200

Bif-1
UVRAG

AMBRA-1

Beclin-1
Bcl-2

PI3KC3

Nucleation

Atg12

PI

PI3KC3
complex

PI3P

LC3
Atg5
Atg16

+
LC3-II

Elongation

Autophagosome formation
Fig. 3. Schematic representation of autophagic signaling. In the presence of nutrients and growth factors (1) mTOR is activated, leading to the inhibition of
autophagy. Nutrient or growth factor deprivation (2), or treatment with rapamycin leads to inhibition of mTOR and the activation of the ULKeAtg13eFIP200
complex, which induces autophagosome formation. First, vesicle nucleation occurs, which requires the assembly of the PI3KC3 complex. Subsequent generation of
PI3P leads to the recruitment of other Atg proteins and vesicle membrane elongation. During this process, an Atg12eAtg5eAtg16(L) multimer complex is formed,
and LC3 is lipidated (LC3-II). Bcl-2 is capable of inhibiting autophagy through direct binding of Beclin-1. See text for detailed sequence of events.

a RIP1-dependent autophagic cell death [59]. Cell death was

caused by severe ROS accumulation and cell damage.
Surprisingly, elevated ROS after zVAD-fmk treatment was
dependent on autophagy, which selectively degraded catalase, a major key enzyme in the anti-oxidant defense mechanism [60]. However, another group recently reported
compelling data supporting a pro-survival instead of a cell
killing role for autophagy in zVAD-fmk-induced cell death
[61]. Rapamycin, a potent inducer of autophagy, blocked
zVAD-fmk-induced cell death, whereas chloroquine, a lysosomal enzyme inhibitor, greatly sensitized L929 cells for this

form of cell death. The authors suggested that zVAD-fmk, in

addition to inhibiting caspases, also blocks cathepsins,
resulting in decreased autophagosomal degradation and
hence the appearance of typical autophagic hallmarks.

4.2. Physiological relevance of cell death associated with

autophagy
Autophagic cell death has been described primarily in
developmental processes that require extensive cell destruction

L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

and elimination. During metamorphosis in Drosophila, death

of salivary gland cells is associated with upregulation of atg
genes and induction of autophagy, though this is accompanied
with caspase activation and the appearance of typical apoptotic
features, including DNA fragmentation [62]. However, studies
of Atg8 mutant flies or flies overexpressing dominant negative
Atg1 support the hypothesis that autophagy is needed for
proper salivary gland degradation. Moreover, transgenic
overexpression of Atg1 in salivary glands is sufficient to
induce autophagic cell death, which is independent of caspase
activation [63].
The protist, Dictyostelium, does not contain any caspase
genes, implying that cell death in this organism is by default
caspase-independent [64]. It has been shown that the autophagy-like cell death occurs in monolayer cultures of Dictyostelium cells subjected to starvation and differentiationinducing factor DIF-1, mimicking the development of fruiting
bodies. These fruiting bodies contain a mass of spores supported by a stalk composed of extensively vacuolated dead
cells. However, atg1 gene disruption blocks autophagic
vacuolization, but does not suppress cell death [65], suggesting that cell death occurs independently of autophagic
signaling or that redundant cell death pathways exist. The nonvacuolar cell death in Atg1 mutants possesses classical characteristics of necrotic cell death [66].
Increasing evidence suggests a role for autophagy in cell
death during ischemia/reperfusion (I/R). For example, Atg7deficiency in neurons protects against neonatal hypoxic/
ischemic brain injury [67]. Similarly, inhibition of autophagy
blocks cardiac myocyte death after I/R and the size of
myocardial infarction is significantly decreased in Beclin-1/
mice [68]. Autophagy might also contribute to virus-induced
cell death. For example, HIV-1 induces apoptotic cell death in
uninfected bystander CD4 T lymphocytes. However, cell
death is associated with typical autophagic features and inhibition of autophagy significantly blocks T cell death [69].
Autophagy has also been implicated in another aspect of
cell death, namely apoptotic cell clearance. Both Atg5/ and
Beclin-1/ cells fail to express phosphatidylserine (PS)
(eat-me signal) and secrete less lysophosphatidylcholine
(LPC) (come-and-get-me signal) [70]. Interestingly, both
PS and LPC exposure require ATP, suggesting that autophagy
contributes to the production of engulfment signals by maintaining cellular ATP levels. Because effective engulfment also
requires PS exposure on the phagocytes [71], it is possible that
intact autophagy is also needed for the proper phagocytic
function of the engulfment cells.

5. Pyroptosis
Pyroptosis is a more recently recognized form of regulated
cell death with morphological and biochemical properties
distinct from necrosis and apoptosis [72]. Pyroptosis has been
described in monocytes, macrophages and dendritic cells
infected with a range of microbial pathogens, such as Salmonella, Francisella and Legionella, and is uniquely dependent on

1057

caspase-1 [73]. In addition, non-infectious stimuli, such as

DAMPs, can induce pyroptosis in non-macrophage cells.
5.1. Molecular mechanisms of pyroptosis
Caspase-1, previously known as Interleukin-1 (IL-1b) Converting Enzyme (ICE), was the first mammalian caspase to be
identified. As a member of the inflammatory caspases, it is not
involved in apoptotic cell death [74]; vice versa, the apoptotic
caspases usually do not contribute to pyroptosis [75]. Like all
caspases, caspase-1 is present in the cytosol as an inactive
zymogen. In analogy to activation of caspase-9 in the apoptosome, caspase-1 is activated in a complex called the inflammasome. This molecular platform includes NLR family members
that recruit caspase-1 through adaptor molecules, such as ASC/
Pycard and is formed through homotypic interactions between
these inflammasome components (Fig. 4). Until now, four
inflammasomes have been characterized and named after their
NLR (Nalp-1, Nalp-3 and Ipaf) or HIN-200 protein (AIM2)
[73,76]. Assembly of the inflammasome occurs when NLRs are
triggered by intracellular bacterial, viral or host danger signals.
For example, Nalp-1 recognizes cytosolic delivery of Bacillus
anthracis lethal toxin, Ipaf recognizes cytosolic flagellin, and
Nalp-3 responds to multiple DAMPs and PAMPs [73] (Fig. 4).
Most NLRs consist of three distinct domains: an N-terminal
CARD domain or pyrin effector domain (PYD), a central
nucleotide binding and oligomerization domain (NACHT), and
several C-terminal leucine-rich repeats (LRRs). In addition,
Nalp-1 has a C-terminal extension that harbors a CARD domain.
In contrast to human Nalp-1, the mouse orthologue Nalp-1b does
not contain an N-terminal PYD domain. Upon stimulation, NLRs
undergo oligomerization through homotypic NACHT domain
interactions. Subsequently, the NLRs associate with the adaptor
protein ASC through homotypic PYD interactions. In addition,
Nalp-3 associates with the adaptor Cardinal in its inflammasome.
These adaptor molecules then recruit caspase-1 through CARDe
CARD interactions, resulting in its oligomerization and proximity-induced activation.
Recently, the AIM2 inflammasome was identified [76].
Through its HIN domain, AIM2 can directly bind to dsDNA,
resulting in the activation of caspase-1 and maturation of proIL-1b. The source of the cytoplasmic dsDNA appears unimportant for AIM2 activation because viral, bacterial, mammalian and synthetic dsDNA could all activate caspase-1 [76].
Double stranded DNA-dependent cell death depends on AIM2,
ASC and caspase-1 and shows features of pyroptosis [77].
Active caspase-1 is the central executor of pyroptotic cell
death and acts mainly by inducing the formation of discretely
sized ion-permeable pores in the plasma membrane, as
analyzed by the use of membrane impermeable dyes and
osmoprotection experiments [78]. The resulting osmotic
pressure leads to water influx, cell swelling and ultimately cell
lysis. Furthermore, caspase-1 activation initiates an inflammatory response by the cleavage of the proinflammatory
cytokines pro-IL-1b and pro-IL-18, which are released by the
cell upon their activation [79]. However, this inflammatory
response is not required for the execution of cell death [80].

L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

1058

DAMPs:
MSU, PPD,
silica, alum
asbestos
amyloid-

Bacillus anthracis
lethal toxin,
MDP

Flagellin

PAMPs:
LPS,
peptidoglycan,
viral and bacterial
RNA, DNA

dsDNA

Cytosol

Ipaf
inflammasome

Nalp-1b
inflammasome

Nalp-3
inflammasome

AIM2
inflammasome
Cytosolic dsDNA

LRRs

Nalp-3

Nalp-1b

CARD

p20
p20

ASC

p10
p10

p10
p10

Caspase-1

Caspase-1
p10
p10

Caspase-1

Caspase-1

p20
p20

ASC
p20
p20

CARD

ASC

FIIND

PYD

ASC?

AIM2

p10
p10

NACHT

p20
p20

Ipaf

Active caspase-1

Cleavage of
other substrates:
Pro-IL-1
Pro-IL-18

IL-1
IL-18

Pore formation
in plasma membrane

chaperones
cytoskeletal proteins
translational machinery proteins
glycolytic proteins
caspase-7
...

Inflammation
Pyroptotic cell death
Fig. 4. Schematic representation of pyroptotic signaling. During pyroptosis, recognition of bacterial, viral or host danger signals induces the formation of different
inflammasomes by triggering oligomerization of Nalp-1b, Nalp-3, Ipaf or AIM2. Via the adaptor ASC, caspase-1 is recruited and activated, leading to pore
formation in the plasma membrane and concomitant pyroptotic cell death. In addition, caspase-1-mediated maturation of IL-1b and IL-18 results in the induction of
an inflammatory response. Furthermore, caspase-1 activation leads to the cleavage of a range of other substrates. See text for detailed sequence of events. (MDP,
muramyl dipeptide; MSU, monosodium urate; PPD, calcium pyrophosphate dehydrate).

Although caspase-1 activation is inherently associated with an

inflammatory response, it is still unclear whether it is inevitably linked to pyroptotic cell death. Which molecular
mechanisms are implicated in the bifurcation between caspase-1-dependent inflammation and caspase-1-mediated
pyroptosis is also an open question.

The digestome of caspase-1 comprises more than 40 caspase-1 substrates, among which are chaperones, cytoskeletal
and translation machinery proteins, proteins involved in
immunity, and a series of unexpected proteins along the
glycolysis pathway [81]. Whether these caspase-1-dependent
cleavage events contribute to pyroptosis is unknown. In

L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

addition, by using a proteomics approach to search for novel

caspase-1 targets, caspase-7 was identified as a caspase-1
substrate [75]. Also, it was reported that caspase-7 processing
induced by Salmonella or Legionella infection required
inflammasome signaling [75,82]. Caspase-7/ macrophages
infected with Legionella showed defective delivery of the
organism to the lysosome, delayed cell death and substantial
Legionella replication [82]. However, a role for caspase-7 in
pyroptosis should not be generalized, as caspase-7/
macrophages infected with Salmonella were not protected
from cell death [75].
Cells dying by pyroptosis have biochemical and morphological features of both apoptotic and necrotic cells [73].
Pyroptotic cells lose their mitochondrial membrane potential
and plasma membrane integrity and release their cytoplasmic
contents into the extracellular milieu. As in apoptosis,
pyroptotic cells undergo DNA fragmentation and nuclear
condensation. However, this caspase-1-dependent nucleasemediated cleavage of DNA does not exhibit the oligonucleosomal fragmentation pattern characteristic of apoptosis [83].
In addition, the DNA damage and concomitant PARP-1 activation associated with pyroptotic cell death are not required
for cell lysis to occur [78].
5.2. Physiological relevance of pyroptosis
As a cellular suicide program, pyroptosis is part of the host
defense system for fighting off pathogens. Death of the host
cell destroys the pathogenic niche, thereby limiting microbial
replication and exposing the pathogen to other antimicrobial
mechanisms. On the other hand, host cell death can be beneficial for certain pathogens, as in the elimination of immune
cells. Pathogens have therefore evolved strategies to efficiently
inhibit or to induce pyroptotic host cell death. For example,
poxviruses encode PYD-containing proteins that interact with
ASC in the inflammasome, thereby inhibiting caspase-1 activation and promoting infection [84].
Because of its dependence on caspase-1 activity, pyroptosis
is associated with the initiation of a proinflammatory response,
which is further amplified by the release of the cytoplasmic
content upon cell lysis. Since NLR-mediated activation of
caspase-1 affects several cellular pathways, it is difficult to
distinguish the precise role of caspase-1 in the cell death
process itself. Caspase-1-deficient mice are more susceptible
than wild type mice to infection, for example by Salmonella,
and these mice are more susceptible than IL-1b and IL-18
double knockout mice [85]. On the other hand, caspase-1deficiency confers protection against Escherichia coli-induced
sepsis, again in contrast to wild type mice and IL-1b and IL-18
double knockout mice [80]. These observations suggest that
the phenotype associated with caspase-1-deficiency is not only
due to lack of these proinflammatory cytokines but that
pyroptosis itself or other caspase-1-dependent events are
involved in the control of infection.
Inappropriate or overwhelming activation of caspase-1 is
associated with the pathogenesis of several diseases, such as
myocardial infarction, cerebral ischemia, neurodegenerative

1059

diseases, inflammatory bowel disease and endotoxic shock

[73]. These diseases are all characterized by inflammation and
cell death. Caspase-1-deficiency, or its pharmacological inhibition, results in protection against inflammation and cell
death associated with these diseases [86]. These studies
suggest that the level of caspase-1 activation after recognition
of danger signals plays a role in the host response [73]. Low
levels of caspase-1 initiate cell survival responses, control
intracellular bacterial growth and stimulate inflammatory
cytokine production. In contrast, higher levels of caspase-1
may lead to the induction of pyroptosis, and inappropriate or
overwhelming caspase-1 activation contributes to several
inflammatory conditions.
6. Concluding remarks
It has become clear over the past decade that apoptosis is not
the only cell death program available to mammalian organisms
for removal of unwanted cells. Apoptotic cell death is mediated
by caspases, which are responsible for its typical morphological
features. Accidental necrosis results from physicochemical
damage without involvement of underlying signaling events, but
necrosis can also be the result of a programmed interplay of
signaling events leading to plasma membrane rupture. Apoptosis
and necrosis differ in their inflammatory outcome. Whereas
apoptosis is immunologically silent, necrosis results in the release
of the cytoplasmic contents with consequent inflammatory
responses. Autophagic cell death can have characteristics of both
apoptotic and necrotic cell death. Whether autophagy represents
a distinct form of cell death or rather triggers or accompanies
another form of cell death is debatable and needs further investigation. Pyroptosis is part of the host defense system to fight off
pathogens and has morphological and biochemical properties
distinct from necrosis and apoptosis. Because pyroptosis is
associated with caspase-1-mediated maturation of pro-IL-1b and
pro-IL-18 and ultimately results in release of the cytoplasmic
content, it also elicits an immune response. As discussed
throughout this review, all four forms of programmed cell death
are involved in several pathological conditions. Better understanding of signaling events critical in these cell death pathways
would not only give insight into disease pathogenesis, but could
also lead to the development of new therapeutic strategies for
treatment of cell death-related diseases.
Acknowledgements
We thank A. Bredan for editing the manuscript. This
research has been supported by Flanders Institute for
Biotechnology (VIB), European grants (EC Marie Curie
Training and Mobility Program, FP6 ApopTrain, MRTN-CT035624; EC RTD Integrated Project, FP6 Epistem, LSHB-CT2005-019067; EC RTD Integrated Project, Apo-Sys,
FP7-200767), Belgian grants (Interuniversity Attraction Poles,
IAP 6/18), Flemish grants (Fonds Wetenschappelijke Onderzoek Vlaanderen, 3G.0218.06), and Ghent University grants
(BOF-GOA - 12.0505.02 and 01GC0205). Peter Vandenabeele
is full professor at Ghent University and holder of

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L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

a Methusalem grant from the Flemish Government, Tom

Vanden Berghe is supported by an FWO postdoctoral fellowship, Ellen Wirawan is supported by GOA project (01GC0205)
and Linde Duprez obtained a predoctoral fellowship from the
FWO.

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