Here we report that gliotoxin causes death of cultured Kupffer cells via apoptosis and secondary necrosis. Previously, activated rat and human HSCs were found to undergo apoptosis and exclude trypan blue at 0.3–7.5 μM gliotoxin (1
) but necrosis was observed at concentration above 7.5 μM (2
). Hepatocytes are resistant to lower gliotoxin concentration but undergo necrosis at high concentration (50 μM). Apoptosis of Kupffer cells by lower concentration of gliotoxin (0.03–0.3 μM) indicates their greater sensitivity than HSCs and hepatocytes, and also peritoneal macrophages, which undergo apoptosis at higher (1 μM) concentration (9
Gliotoxin-induced apoptosis of Kupffer cells involved classical apoptotic pathway of mitochondrial cytochrome c release, caspase-3 activation and ATP depletion. ATP is required for apoptosis but its depletion below a threshold concentration leads to secondary necrosis (34
). Thus profound loss of ATP might be a mechanism of secondary necrosis of Kupffer cells at later times during gliotoxin treatment. Curiously, although gliotoxin stimulated caspace-3 activation, its inhibition exacerbated gliotoxoin-induced death of Kupffer cells. This finding contrasts the blockade of gliotoxin-induced nucleosomal ladder formation (but not morphological changes) in activated HSCs by caspase inhibitor Z-VAD.fmk (1
). In addition, serine protease inhibition by TPCK also exacerbated, even more strongly, the effect of gliotoxin. These unusual and unexpected findings do not have definitive explanation. It is likely that inhibition of caspase-3 activation or serine proteases may promote another pathway (necrotic) of gliotoxin-induced death. Indeed, preincubation of the cells with Z-DEVD.fmk or TPCK caused strong early increase in LDH release and uptake of trypan blue upon gliotoxin challenge.
We investigated the involvement of NFκB, MAPKs and ROS in gliotoxin-induced death of Kupffer cells. Gliotoxin inhibits TNF-α-inducible nuclear binding of NFκB but not constitutively activated NFκB in activated HSCs and promotes apoptosis (1
), probably by inhibiting TNF-α-induced activation of antiapoptotic genes (36
). In contrast, PDTC, an inhibitor of NFκB activation (40
) and an antioxidant (42
), blocked gliotoxin-induced death of activated HSCs (1
). Since NFκB promotes cell survival, its nuclear translocation by gliotoxin suggested adaptive stimulation of survival pathway, leading to the hypothesis that its inhibition should augment the toxic effect of gliotoxin. However, inability of PDTC to affect gliotoxin-induced death of Kupffer cells indicates that NFκB activation may not be related to this phenomenon or that the overwhelming activation of pro-death mechanisms overrides them. Interestingly, NFκB activation was shown to mediate elastase-induced apoptosis of Kupffer cells (23
Most evidence indicates that p38 MAPK inhibits cell proliferation (44
) and promotes apoptosis (46
); its activation was also shown to be associated with GdCl3
-induced apoptosis of Kupffer cells (49
). Activation of JNK was found to be associated with apoptosis, presumably by phosphorylation of antiapoptotic Bcl2 members (29
). There is also evidence for the role of ERK activation in apoptosis (50
). However, inhibition of these MAPK pathways did not prevent or inhibit gliotoxin-induced Kupffer cell death suggesting that their activation may be related to other responses.
Gliotoxin-induced apoptosis of peritoneal macrophages was reported to involve ROS (8
). Although 1.5–7.5 μM gliotoxin was found to cause apoptosis of culture-activated and immortalized HSCs respectively in the absence or at low level of oxidative stress (2
), higher concentrations induce oxidative stress and necrotic death of the immortalized HSCs (2
). Necrosis of hepatocytes induced by high concentrations of gliotoxin (>10 μM) was prevented by antioxidants catalase and superoxide dismutase (24
). However, gliotoxin did not stimulate SO and H2
generation in Kupffer cells, and several antioxidants did not prevent gliotoxin-induced death of these cells. Together, these observations indicate that the mechanism of gliotoxin-induced death of Kupffer cells(apoptotic and necrotic) is different from that for peritoneal macrophages and HSCs with respect to NFκB or ROS involvement, and is independent of MAPK activation.
Gliotoxin caused apoptosis of Kupffer cells as well as HSCs in the fibrotic rat liver; the effect was minimal in the normal liver. This may be because activated Kupffer cells and HSCs are more sensitive to gliotoxin, and the numbers of both cell types increase in cirrhosis (51
). The profound effect of gliotoxin observed on Kupffer cells isolated from normal liver as compared to the minimal effect in vivo
is interesting. A plausible explanation is that Kupffer cells may be activated in culture similar to those in vivo
during hepatic injury (52
). However, the activation does not seem to be complete as preincubation of the cells with endotoxin further sensitized Kupffer cells to gliotoxin-induced death. These observations contrast a recent study reporting comparable gliotoxin-induced apoptosis of Kupffer cells and HSCs in normal and cirrhotic livers (54
). The reason for this discrepancy may be that liver slices were incubated for 8h with gliotoxin in vitro
), while we and others (1
) investigated the effect of gliotoxin in vivo
. We also found significant gliotoxin-induced apoptosis of hepatocytes in the fibrotic liver but not the normal liver. Since hepatocytes undergo necrosis, but not apoptosis, upon treatment with high (>10 μM) concentration of gliotoxin in vitro
), these results suggest that hepatocytes are either modified during fibrosis to become sensitive to pro-apoptotic effect of gliotoxin or that gliotoxin may release proapoptotic stimuli from nonparenchymal cells that then cause apoptosis of hepatocytes.
In summary, our results show that gliotoxin is a nonspecific pro-death agent for HSCs, Kupffer cells and hepatocytes in the fibrotic liver. The results also demonstrate a novel death-augmenting effect (via necrosis) of caspase-3 and serine protease inhibition on gliotoxin-induced apoptosis of Kupffer cells. Considering the importance of macrophages in resolution of fibrosis (55
), removal of Kupffer cells might delay this process or even adversely affect the liver. Further research to investigate whether specific derivatives of gliotoxin cause apoptosis of activated HSCs selectively, excluding other liver cell types, is necessary for clinical application.