The results presented here provide evidence for a novel function of caspase inhibitors in induction of necrotic-like cell death. Thus, both the vaccinia virus-encoded B13R protein and peptidyl caspase inhibitors can mediate necrotic killing of cells by TNF-α. Previous studies have demonstrated a key role for NF-κB in inhibiting TNF-α-induced apoptosis. Here we show that the caspase inhibitor z-VAD-fmk allows necrotic killing of fibroblasts by TNF-α but does not inhibit the activation of NF-κB. Thus, TNF-α cytotoxicity can be mediated by at least two mechanisms: inhibition of NF-κB results in death by apoptosis, while inhibition of caspase proteases results in death by necrosis. Our results demonstrate that TNF-α-induced necrotic killing can be used to eliminate cells infected with viruses encoding caspase inhibitors. Thus, the very presence of virus-encoded caspase inhibitors may render infected cells susceptible to TNF-α-induced necrotic cell death.
We have also shown that necrotic killing appears to be dependent on the generation of superoxides. Interestingly, superoxide production was not elevated during TNF-α-induced apoptosis of fibroblasts (data not shown), suggesting that superoxide production accompanies necrotic but not apoptotic cell death. In addition, TNF-α and z-VAD-fmk did not induce superoxide production in cells which are resistant to necrotic killing, such as HeLa (data not shown). In fact, vaccinia virus infection of HeLa cells can render them resistant to killing by TNF-α plus cycloheximide in a B13R-dependent manner (references 8
and data not shown). These results suggest that induction of necrosis by TNF-α and caspase inhibitors may occur only when superoxide generation takes place. Our results also demonstrate the involvement of the mitochondrial respiratory chain in superoxide production and induction of necrotic cell death. Thus, mitochondria may also play an important role in the regulation of necrotic cell death, in addition to their established function during apoptosis (12
). Interestingly, recent studies have shown that caspase proteases are also present in mitochondria (25
). It is intriguing to speculate that inhibition of these caspases may be responsible for increased production of ROSs by the mitochondrial respiratory chain and induction of necrotic killing by TNF-α.
Previous studies have demonstrated a key role for caspase proteases in the induction of apoptotic cell death. However, our results provide evidence for a novel function of caspase proteases in inhibition of necrotic cell death by TNF-α. Although caspases involved in inhibiting necrosis are not known, we have recently identified caspase 3 as a major caspase activated in RelA−/− 3T3 fibroblasts treated with TNF-α or RelA+/+ 3T3 fibroblasts treated with TNF-α plus actinomycin D (corresponding to P1 [unpublished results]). However, since both z-VAD-fmk and B13R (based on similarity to CrmA) are capable of interaction with many distinct caspase proteases (including caspase 3), their necrotic effects could be mediated by inhibition of multiple caspases. These may also include constitutively active caspases present at low levels in nonapoptotic cells, such as TNF-α-treated 3T3 fibroblasts.
A recent study has also demonstrated increased necrosis in L929 cells after caspase inhibition (39
). However, L929 cells also undergo necrosis in the presence of TNF-α alone (13
), while our results indicate that inhibition of necrosis by caspases is likely to be a more general phenomenon which can efficiently kill cells that are normally resistant to TNF-α. In this regard, our results are also distinct from those of previous studies, which have shown a switch from apoptosis to necrosis in the presence of caspase inhibitors but without affecting total killing of cells (16
). During the preparation of this paper, Khwaja and Tatton also reported that NIH 3T3 and U937 cells undergo necrotic killing in the presence of TNF-α and peptidyl caspase inhibitors (20
). These findings, and the results presented here, demonstrate a role for caspase inhibitors in killing of cells that are normally resistant to TNF-α.
We also show that caspase inhibitors can mediate cytotoxicity in dsRNA- and IFN-γ-treated cells. Similar to TNF-α, dsRNA and IFN-γ cytotoxicity was also significantly inhibited by antioxidant or mitochondrial respiratory-chain inhibitor treatment. These results suggest that a similar cytotoxic pathway is induced by TNF-α, dsRNA, and IFN-γ in the presence of caspase inhibitors. In this respect, it is interesting that viruses have devised strategies to subvert signaling by all three of these agents (17
). Thus, inhibition of these signaling pathways may not only inhibit the activation of antiviral gene expression (e.g., alpha/beta IFNs, PKR, adenylate synthase) but may also inhibit the killing of virus-infected cells. Similar to TNF-α, dsRNA, and IFN-γ, the Fas death receptor is an important mediator of host defense. More specifically, Fas expression on virus-infected cells mediates cytotoxicity through interaction with Fas ligand-expressing cytotoxic T lymphocytes (24
). Two studies have recently demonstrated the induction of necrotic killing by the Fas death receptor (18
). Vercammen et al. have shown that Fas induces the necrotic killing of L929 cells in the presence of caspase inhibitors (40
), while Kawahara et al. have demonstrated the induction of necrosis by Fas in cells devoid of caspase 8 (18
). These studies and the results presented here suggest that caspase inhibitor-mediated killing may be a general mechanism shared by multiple antiviral agents. Such killing may play an important role in host defense strategies against viral infections.