Going up in flames: necrotic cell injury and inflammatory diseases

Abstract

Recent evidence indicates that cell death can be induced through multiple mechanisms. Strikingly, the same death signal can often induce apoptotic as well as non-apoptotic cell death. For instance, inhibition of caspases often converts an apoptotic stimulus to one that causes necrosis. Because a dedicated molecular circuitry distinct from that controlling apoptosis is required for necrotic cell injury, terms such as "programmed necrosis" or "necroptosis" have been used to distinguish stimulus-dependent necrosis from those induced by non-specific traumas (e.g., heat shock) or secondary necrosis induced as a consequence of apoptosis. In several experimental models, programmed necrosis/necroptosis has been shown to be a crucial control point for pathogen- or injury-induced inflammation. In this review, we will discuss the molecular mechanisms that regulate programmed necrosis/necroptosis and its biological significance in pathogen infections, drug-induced cell injury, and trauma-induced tissue damage.

Fig. 1

Morphological distinctions of programmed necrosis. a Extensive intracellular vacuolation and rupture of plasma membrane in cells undergoing programmed necrosis. Mouse embryonic fibroblasts (MEFs) were infected with vaccinia virus and treated with TNF. Electron micrograph shows that the MEFs underwent necrosis marked by extensive formation of intracellular vacuoles (panel b) and dissolution of plasma membrane integrity (panel c). In panel a, an uninfected MEF was included for comparison. Scale bar 1 μm. b TNF-induced programmed necrosis does not result in chromosomal DNA fragmentation. Caspase-8 deficient (lanes 1–3) or wild-type (lanes 4–6) Jurkat cells were treated with FasL or TNF for 5 h as indicated. Chromosomal DNA was extracted from the cells and resolved by electrophoresis. The results show that DNA fragmentation is only observed in wild-type cells undergoing apoptosis, but is absent in caspase-8 deficient cells undergoing programmed necrosis. c Lack of nuclear condensation in programmed necrosis. Untreated wild-type Jurkat cells (panel a), TNF-treated caspase-8 deficient Jurkat cells (panel b), and FasL-treated wild-type Jurkat cells (panel c) were stained with Hoechst 33342. Nuclear morphology was imaged by epi-fluorescence microscopy. The results show that crescent-shaped nuclear condensation was only observed in apoptotic cells, but not necrotic cells. d Necrotic cells exhibit concomitant positive staining for Annexin V and PI. Wild-type untreated Jurkat cells (panel a), TNF-treated caspase-8 deficient Jurkat cells (panel b) or FasL-treated wild-type Jurkat cells were stained with Annexin V and PI and analyzed by flow cytometry. The majority of necrotic cells are positive for both markers, whereas the majority of apoptotic cells exhibit only Annexin V staining

Fig. 2

Domain structures of RIP1 and RIP3, the two crucial kinases for programmed necrosis. The kinase and RHIM domains of RIP1 and RIP3 are required for death cytokine (TNF, FasL, and TRAIL)-induced programmed necrosis. Evidence indicates that the kinase activity of RIP1 is also required for assembly of an alternative caspase-8 activating complex in response to apoptosis induced by TNF and IAP antagonist [33]. In contrast, cleavage by caspase-8 at D324 (for RIP1) [45] and D328 (for RIP3) [46] releases the kinase domains from the RIP kinases and likely prevents the phosphorylation and activation of downstream substrates. Polyubiquitination of RIP1 at K377 inhibits TNF-induced apoptosis, possibly by blocking the transition of the receptor-associated complex to the cytoplasmic death signaling complexes [41]. DD death domain

Fig. 3

Regulation of programmed necrosis by ubiquitination, phosphorylation, and caspase cleavage. TNF-induced programmed necrosis is regulated at multiple steps involving positive (indicated by red arrows) and negative (indicated by black arrows) mechanisms. TNF-R2 signaling enhances TNF-R1 mediated programmed necrosis through a poorly defined mechanism [32, 43]. Upon binding to TNF-R1, RIP1 becomes modified by polyubiquitination at K377. Polyubiquitinated RIP1 binds to NEMO, the regulatory subunit of NF-κB, to promote NF-κB activation. NF-κB activation counters the death signals by inducing pro-survival genes. The plasma membrane-associated receptor signaling complex containing polyubiquitinated RIP1 migrates to the cytoplasm where the receptor falls off the complex and RIP1 becomes deubiquitinated. The deubiquitinating enzymes A20 or CYLD may facilitate this reaction. In the presence of caspase-8 inhibition, RIP1 and RIP3 interact with each other via the RHIM to form the pro-necrotic signaling complex. This interaction is further stabilized by phosphorylation of both kinases. However, active caspase-8 cleaves RIP1 and possibly RIP3 to blunt the pro-necrotic complex. The active caspase-8 complex can go onto cleave additional substrates, culminating in cell death by apoptosis

Fig. 4

RIP3-dependent programmed necrosis controls the outcome of vaccinia virus and MCMV infections. a TNF-induced, RIP3-dependent programmed necrosis is an important innate inflammatory response against vaccinia virus infection. TNF and other inflammatory cytokines are rapidly induced early during vaccinia virus infection. TNF-induced apoptosis is inhibited by the virally encoded caspase inhibitor Spi2, which skews the response towards RIP3-dependent programmed necrosis. The death of the infected cells limits the viral factory and contributes to the clearance of the virus. In addition, programmed necrosis might facilitate cross-priming of dendritic cells as the plasma membrane ruptures. This might result in further production of inflammatory cytokines and the recruitment of innate immune effector cells to the site of infection. b Inhibition of RIP3-dependent programmed necrosis is crucial for productive infection by MCMV. Infection with MCMV containing a mutant M45 leads to RIP3-dependent programmed necrosis, premature termination of the viral replication cycle and failure for the virus to establish productive infection. Inactivation of RIP3 by siRNA or infection in RIP3^−/− cells restored productive infection by the mutant virus similar to that observed with wild-type MCMV

Fig. 5

Hypothetical model of programmed necrosis as a sensor for different forms of cellular insults. Programmed necrosis can be induced by diverse insults from pathogens, cytotoxic drugs, and physical trauma. As RIP1/RIP3-dependent programmed necrosis occurs, cellular “danger-associated molecular patterns (DAMPs)” are released into the tissue milieu. The released DAMPs can cause inflammation through activation of various DAMP receptors such as Toll-like receptors (TLRs) or the inflammasomes [57]