‘Necrosome’-induced inflammation:must cells die for it?David Wallach1, Andrew Kovalenko1 and Tae-Bong Kang1,2

1 Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel2 Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chung-Ju 380-701, Korea

Opinion

Necrosis, a form of death characterized by rupture of thecell membrane, is closely interlinked with inflammation.Cellular components released during necrotic death cantrigger inflammation. Conversely, inflammation oftenyields tissue damage and, as a consequence, cell death.Which occurs first – necrosis or inflammation – in specificin vivo situations is currently difficult to tell. A way out ofthis ‘chicken-and-egg’ conundrum may be found via therecent finding that both necrotic cell death and inflam-mation can be initiated by a distinct set of signalingproteins, the ‘necrosome’, that includes receptor-inter-acting protein (RIP)1, RIP3 and caspase-8. Further clari-fying the function of these signaling proteins shouldmake it possible to establish when they induce inflam-mation directly and when inflammation is caused bynecrotic cell death.

Necrosis and inflammationNecrosis, a term derived from the classical Greek wordnecros, meaning a corpse, has two distinct connotations.The older connotation refers to the death of groups of cellswithin the organism as a result of infection or injury [1].The more recent one denotes a particular form of death ofindividual cells, so-called necrotic cell death, which ischaracterized by rupture of the plasma membrane andrelease of cellular contents early in the death process[2]. This dual meaning of necrosis might be misleadingbecause there is only partial overlap between the twophenomena. One difference is that ‘in vivo necrosis’ incor-porates various forms of death, including both apoptoticand necrotic cell death. A more fundamental difference isthat induction of cell death in vivo, irrespective of the formof death, can be affected by other cellular processesthrough a variety of non-cell-autonomous mechanisms.

This article addresses recent studies concerning theinter-relationships of necrosis and inflammation. In thesestudies, in vitro and in vivo findings that implicated agroup of signaling proteins – receptor-inducing protein(RIP)1 and RIP3 (as inducing proteins) and caspase-8(as an inhibitor protein) – in the regulation of both necrosisand inflammation were interpreted as providing furtherevidence for a long-suspected role of necrotic cell death inthe initiation of inflammation [3–7]. Here, we argue theneed for caution in reaching such conclusions. This isbecause, first, these signaling proteins also seem capableof inducing inflammation independently of their ability to

Corresponding author: Wallach, D. (d.wallach@weizmann.ac.il)

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induce necrotic cell death, and second, when necrosis andinflammation occur side-by, in vivo necrosis might be aconsequence rather than a cause of inflammation.

Programmed necrosis (necroptosis) and its induction bythe ‘necrosome’Early studies of cell death focused mostly on apoptosis.During death of this type, the cell membrane remains intactuntil a relatively late stage, allowing dying cells to beengulfed by macrophages before the contents of the deadcells are released. The increasing amount of informationon the participating molecules and mechanisms is inter-preted as giving credence to the physiological significance ofdeath by apoptosis. Necrosis – death associated with earlyrupture of the cell membrane and release of the cellularconstituents – was believed to occur as a consequence ofaccidental damage, and to be ‘invariably associated with agross departure from physiological conditions’ [2].

The first clue that this might not be so can be traced toearly studies showing that the cytokine tumor necrosisfactor (TNF) and some other members of the TNF ligandfamily that are capable of inducing apoptotic cell death can,in some cells, induce necrotic cell death (reviewed in [8]).Whereas the apoptotic effect of TNF is mediated by cas-pases [9], induction of necrotic death by TNF or by someother members of the TNF family is enhanced by caspaseinhibitors [10,11]. Deficiency of one particular caspase,caspase-8, an enzyme required for the induction of apopto-tic cell death by TNF, has also been found to facilitate theinduction of necrosis [12,13]. By contrast, the proteinkinase RIP1 is required for the induction of necrotic death;a contribution that depends on its enzymatic function [12].siRNA screening has revealed the effects of many othercellular proteins on the induction of necrotic death bycytokines of the TNF family [3–5,14]. One of these,RIP3, a protein kinase related to RIP1, is as essential asRIP1 for the induction of necrosis, and like RIP1, contrib-utes to necrosis via its ability to phosphorylate some yetunknown target proteins [3–5]. In cells undergoing TNF-induced necrosis, caspase-8, RIP1 and RIP3 associate in acomplex that also contains some other signaling proteins,including FADD (Fas-associated protein with death do-main; Mort1), the adapter protein to which caspase-8binds. On the assumption that this complex mediatesthe induction of necrosis, it has been dubbed ‘necrosome’in some studies. In a few other studies, the term ‘ripopto-some’ was chosen instead, to denote the signaling com-plexes that contain RIP1 and initiates cell death (both

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necrosis and apoptosis). Our understanding of the hetero-geneity of the composition and functions of the complexesmediating the effects of these groups of proteins is still at aprimordial stage, and further knowledge might raise theneed to refine the terminology. Here, we use the termnecrosome to describe the concerted action of RIP1,RIP3 and caspase-8, with no intention of implying thatthis action is mediated by just one particular or well-characterized complex.

Several studies suggest that RIP1 kinase activity[assessed by determining the effect of necrostatin (Nec-1),a recently identified chemical blocker of this activity [15]],also contributes to necrosis induced by pathogenic agentssuch as viruses, cell detachment, T cell receptor stimulationand genotoxic stress, as well as other inducers [6,16–20]. It isnot yet understood how activation of these signaling mole-cules leads to necrosis [21]. The term ‘programmed necrosis’,or ‘necroptosis’, coined to describe this process, reflects thebelief – although real evidence is still missing – that it occursin a programmed manner, in which not only its initiation butalso the subsequent execution of cell death, are driven bydistinct and preordained mechanisms.

Components of the necrosome can mediate bothinflammation and necrosis in vivo

Several studies have shown that RIP1- and RIP3-kinaseactivities also possess inducing roles, and that caspase-8has an inhibitory role, in certain in vivo processes associ-ated with tissue damage and inflammation. This was firstindicated in studies in which injection of mice with Nec-1was found to protect them from tissue damage induced byischemic brain injury and myocardial infarction [22,23].Later, it was shown that knockout of Ripk3 (RIP3) alsoprotects mice from induction of tissue damage and inflam-mation (by the toxic agent cerulein [3,4] and by the patho-gens vaccinia virus [5] and cytomegalovirus [6]). Asmentioned above, induction of necroptosis in cultured cellsis antagonized by the proteolytic function of caspase-8.Suppression of caspase-8 function in vivo by expressionof an enzymatically inactive Casp8 (caspase-8) allele (alongwith an active one) results in widespread inflammation invarious internal organs as well as in the skin [24]. Deletionof Casp8 in the epidermis or certain other tissues also leadsto severe chronic inflammation [24,25]. Deletion of Ripk3(RIP3) prevents the induction of these inflammatory pro-cesses [7]. It thus appears that, similar to induction ofnecrosis by the necrosome in cell culture, the inflammationdue to arrest of caspase-8 function in vivo reflects impairedinhibition of RIP3 function.

Coordinated RIP1 and RIP3 function with the proteo-lytic activity of caspase-8, in a way that resembles theircombined action on necroptosis induction, also affects em-bryogenesis, apparently by controlling a crucial step inshaping of the yolk-sac capillaries. When this control isdisrupted by knockout of Casp8 (caspase-8) or Fadd, theembryos die at midgestation [26–30], and this can becircumvented by further deleting either RIP1 or RIP3.The dying embryos have been found to contain necroticcells. It has been suggested that these cells died by necrop-tosis and that their death was the cause of the embryonicdeath [7,13,31]. It is possible, however, that this necrotic

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cell death was simply a consequence of the death of theembryos themselves, an event that is harder to pinpoint intime than postnatal death.

The studies mentioned above clearly show that necro-some components control inflammation. They also suggesta role for these proteins in induction of cell death in vivo.They do not, however, provide clues about the causalrelation between these two functions of necrosome compo-nents in the mouse models studied.

Inflammation induced by necrosome componentsmight occur as a result of necrotic cell deathCertain intracellular compounds that serve vital functionswithin the intact cell may, when released from the cellduring its necrotic death, activate immune functions andprompt inflammation. This group of compounds, dubbed‘danger-associated molecular patterns’ (DAMPs) [32],encompasses various proteins including the chromatinregulator high mobility group box 1 (HMGB1), heat-shockproteins (e.g. HSP70, HSP60, and gp96), and calcium-binding proteins of the S100 group. Also included are somenon-proteinaceous cellular components such as ATP anduric acid. It is possible that certain compounds releasedfrom the dying cells act as DAMPs and that because of this,necroptosis in vivo can trigger inflammation.

Conversely, cell death mediated by necrosomecomponents in vivo might occur as a result of inducedinflammationWhen attempting to define the sequence of events by whichthe components of the necrosome mediate cell death andinflammation in vivo, it is important to take into accountthat in addition to inducing necrosis, the necrosome com-ponents initiate signaling pathways that may trigger in-flammation directly. The kinase function of RIP1 alsoparticipates in activation of the extracellular signal-regu-lated kinase and p38 mitogen-activated protein kinasecascades by TNF [33,34]. In addition, as recently described,RIP1 kinase activity might play a role both in activation ofJun N-terminal kinase 3 and induction of apoptotic celldeath as a consequence of extensive DNA damage [35]; italso participates in a distinct pathway of apoptosis induc-tion by TNF in Cellular Inhibitor of Apoptosis (cIAP)-suppressed cells [36]. Furthermore, RIP3 has been sug-gested to contribute to activation of both nuclear factor-kBand apoptosis [37–39] and to regulation of the phosphoi-nositide 3-kinase/Akt signaling axis [40]. Finally, caspase-8, besides blocking the induction of necroptosis, alsorestricts signaling for interferon regulatory factor-3 acti-vation by the Retinoic Acid-Inducible Gene-I (RIG-I) com-plex [41]. Moreover, the inhibitory effect of caspase-8 on thefunction of the RIG-I pathway and its restriction of necrosisinduction are thought to be mediated through cleavage ofRIP1 at the very same site [41]. These, as well as other yetunknown effects of RIP1, RIP3 and caspase-8, might en-able them to induce and regulate inflammation indepen-dently of their necrosis-inducing function.

An additional point to consider is that inflammationmight also be the cause rather than the consequence ofnecrosis. The circulatory changes underlying ‘redness,swelling, heat and pain’, the cardinal signs of inflammation,

Necroticcell death

Damage-associatedmolecularpatterns(DAMPs)

Inflammation

Inflammation

ROSWalling offComplementProteases…

Necrosis

Cellular necrosis prompts inflammation

Inflammation prompts necrosis

Caspase-8RIP1

RIP3

Caspase-8RIP1

RIP3

TRENDS in Immunology

Figure 1. Induction of necrosis and inflammation by necrosome components: a ‘chicken and egg’ conundrum. Illustrated are two possible sequences of events through

which the necrosome components may induce necrosis and inflammation. The three main known components of the necrosome – the protein kinases RIP1 and RIP3 and

the protease caspase-8 – are shown on the left. The red squares in the RIP1 and RIP3 diagrams correspond to a motif (RHIM–RIP homotypic interaction motif) through which

the two protein kinases bind to each other (they also bind through homotypic RHIM-motif interaction to some other proteins containing this motif, and such binding may

initiate their activation). Signaling by the kinase function of RIP3 depends often, but not always, on prior activation of the kinase function of RIP1. Caspase-8 inhibits this

signaling, at least in part by cleaving RIP1 and RIP3. As illustrated in the upper panel, activation of the kinase function of RIP1 and RIP3 (by TNF or other agents) can trigger

necrotic death of the cell in which this activation occurs. When necrosis occurs in vivo, DAMPs released from the dying cells might initiate inflammation. However, as

shown in the lower panel, induction of necrosis and inflammation by the necrosome components might also occur in the reverse order. RIP1 and RIP3 can signal for

activation of inflammatory genes. Inflammation, through a variety of mechanisms, including activation of reactive oxygen species (ROS), walling-off of the inflamed region,

activation of complement proteins, and activation of other proteases, often results in tissue necrosis.

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prepare the ground for the main function served by inflam-mation – elimination of the infectious or injurious agent thattriggered the inflammation (and then repair of the damage)[42]. Such elimination is often carried out by destruction ofcells and pathogens within the inflamed site. Cell destruc-tion during the inflammatory process is evident from thefrequent occurrence of necrotic regions in the inflamedtissue. These are enhanced when resolution of the inflam-mation is prevented, and their frequency depends on theintensity of the inflammation. The destruction is caused in avariety of ways [1,43,44]. These include walling off of thetissue (another hallmark of inflammation), causing restric-tion of nutrient and oxygen supply; they also include damageby complement proteins and by effects of inflammatorycytokines such as TNF. The earliest name given to TNF,necrosin, indeed reflected the impression that it plays amajor role in coordinating inflammation-related necrosis[45,46]. Polymorphonuclear leukocytes are thought to makea particularly prominent contribution to the damage,through release of oxygen radicals, proteases, and variouscytotoxic proteins (see, e.g. [47]).

In view of the apparent ability of the necrosome compo-nents to signal directly for inflammation, and becauseinflammation might result in necrosis, necrosis occurringas a result of activation of the necrosome components invivo might not necessarily be induced directly by thesesignaling proteins, but rather as a result of the inflamma-tion that the necrosome components induce.

How to define a causal relation between inflammationand necrosis in vivo?The necrosome components can directly signal both fornecrosis and for inflammation. Both processes can therefore

be induced by these signaling proteins in vivo. The fact thatinflammation can prompt necrosis and that necrosis caninitiate inflammation poses a ‘chicken and egg’ conundrum –

when is necrosis the initial effect of these proteins, and whenis the initial effect inflammation (Figure 1)?

Three kinds of molecular determinants can help usidentify a particular biological process and define its causalrelationship with other processes: (i) molecular featuresspecific to the process itself; (ii) specific inducers; and (iii)specific inhibitors. In some recent publications, the in-volvement of necrosome components in a process is mis-takenly taken to be a specific marker of necrosis. Datademonstrating inhibition of a process by Nec-1, a RIP1-kinase inhibitor, have been presented as evidence forinduction of programmed necrosis. Despite its name, how-ever, Nec-1 is not a specific blocker of necrosis. It willindiscriminately arrest whatever cellular changes can beelicited by the kinase function of RIP1 (and of at least twoother kinases – p21/Cdc42/Rac1-activated kinase 1 (PAK1)and cAMP-dependent protein kinase (PKAca) – that it isalso known to affect [35]). The contribution of RIP3 to aprocess, or its inhibition by caspase-8, has also been pre-sented as evidence for induction of programmed necrosis,even though both RIP3 and caspase-8 have other functions.

The truth of the matter is that we do not yet know of anymolecular parameter specific to necroptosis. This is truenot only in the case of signaling proteins that induce thisprocess, but also for the effector mechanisms that mediateit. For example, the generation of reactive oxygen specieswithin a cell might result in its death and is a suspectedeffector mechanism in necroptosis induction. However,when oxygen radicals are generated only to a limitedextent, for example, as an outcome of limited damage to

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the mitochondrial membrane, this might result in activa-tion of the NACHT, LRR and PYD domains-containingprotein 3 (NALP3) inflammasome and generation of in-flammatory cytokines such as interleukin-1b in a way thatdoes not incur cell death [48]. The generation of DAMPs inthe course of a pathological process is also not necessarilyindicative of necrosis. Various DAMPs are released notonly from dying cells, but also from live cells in response toinflammatory mediators and stress. This is true, for exam-ple, for the chromatin regulator HMGB1[49].

This limitation is not restricted to necrosis; it is also thecase with apoptosis. Despite the vast body of knowledge onthe mechanisms of apoptosis, when we observe it in vivo, itis not easy to tell what has caused it, or to determine itsfunctional consequences. It was once thought that it wouldbe possible to define apoptosis-specific inducers – ‘deathligands’ such as TNF, ‘death-receptors’ such as Fas, or‘death-signaling proteins’ such as FADD/Mort1 or cas-pase-8. It is now clear, however, that most (and possiblyall) of these molecules have other, non-apoptotic effects;thus, none of them can serve as an exclusive marker of anapoptosis-inducing mechanism or be applied to definetemporal relations between cell death and other in vivoprocesses [50].

To gain a better understanding of the causal relationsbetween different effects of the necrosome components invivo, we need to acquire broader molecular knowledge ofnecroptosis. We need to: (i) identify additional moleculesthat associate with necrosome components; (ii) character-ize the various post-transcriptional modifications of theseproteins (phosphorylation, ubiquitination, sumoylationand others); (iii) determine the specific components andprotein modifications that dictate induction of necrosis bythe necrosome, and which induce other effects of the necro-some; (iv) define the molecules that act downstream of thenecrosome to mediate necrosis induction, and those thatmediate alternative outcomes (such as induction of inflam-matory cytokines); and (v) define molecular markers thatcan be used to differentiate between different forms ofnecrosis – the necrosome-induced form, and forms thatwill turn out to be necrosome-independent.

We need molecular probes that will make it possible toidentify these specific molecular elements in tissues, aswell as inhibitors that will specifically block necrosis in-duction by the necrosome. Such molecular tools will makeit possible to define situations in which necrosis indeedoccurs as a direct consequence of necrosome function andnot as an event secondary to some other effects of thissignaling complex. These tools will also enable us to iden-tify situations in which these components induce inflam-mation directly, and in which necrosis, if observed at all,occurs as a consequence of the inflammation.

Concluding remarksThe finding that necrotic death, like apoptosis, can occur ina programmed manner represents a real paradigm shift inthe field of cell death. However, the excitement about thisconceptual development should not mislead us to overstatethe physiological significance of necrosis. After all, of thevarious ways by which the organism defends itself, killingof its own cells is the one in which it pays the heaviest price.

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Cell death would therefore be expected to occur only as alast resort in immune defense. Our enthusiasm over theability of specific signaling proteins to regulate necroticdeath should not divert our attention from other, non-deadly effects of these proteins.

The importance of gaining an accurate notion of the wayin which these proteins induce inflammation transcendsmere scientific interest. Inflammation plays a central roleboth in pathology and in defense. The finding that alteredfunction of the necrosome components (for example as aresult of caspase-8 deficiency) may trigger intense inflam-mation might pave the way to define new options fortherapy. Such options, however, can materialize only afterwe gain a valid notion of the sequence of events by whichthis inflammation is induced.

AcknowledgmentsWe thank Drs. Irun Cohen and Zvulun Elazar for their comments on themanuscript. Work cited from the authors’ laboratory was supported inpart by grants from Ares Trading SA, Switzerland, a Center of ExcellenceGrant from the Flight Attendant Medical Research Institute (FAMRI),the Kekst Family Center for Medical Genetics, and the Shapell FamilyCenter for Genetic Disorders Research at the Weizmann Institute ofScience. D.W. is the incumbent of the Joseph and Bessie FeinbergProfessorial Chair at the Weizmann Institute.

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