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Activated caspase-1 is not a central mediator of inflammation in the course of ischemia-reperfusion. Daemen MA, Denecker G, van't Veer C, Wolfs TG, Vandenabeele P, Buurman WA. Department of General Surgery, University of Maastricht, The Netherlands. BACKGROUND: Upon transplantation, donor organs subjected to prolonged ischemia suffer from reperfusion injury. Recent observations suggest that caspase activation is involved in inducing the deleterious inflammatory reaction that mediates reperfusion injury. Release of cytokines like interleukin (IL)-1 and IL-18 may occur during apoptosis through activation of caspase-1/IL-1beta-converting enzyme. We hypothesized that caspase-1 activation is a key event in apoptosis/ caspase-dependent inflammation during the development of renal reperfusion injury. METHODS: Caspase-1-/-, caspase-1+/+ as well as Swiss mice were subjected to 45 min of renal ischemia and 24 hr of reperfusion. Animals were administered agents capable of neutralizing the pro-inflammatory activation products of caspase-1 (IL-1 receptor antagonist, anti-IL-1 receptor antibody, and anti-IL-18 antibody). The extent of renal functional deterioration, inflammation, and apoptosis were compared. RESULTS: No improvement in renal function as reflected by serum ureum and creatinine were found in caspase-1-/- mice as compared to wild type controls. Caspase-1-/- mice showed slightly attenuated renal inflammation as indicated by decreased renal neutrophil influx, but failed to show changes in intrarenal tumor necrosis factor-alpha production. Moreover, caspase-1-/- mice clearly exhibited reperfusion-induced apoptosis as reflected by renal terminal deoxynucleotidyltransferase histology and internucleosomal DNA cleavage. Treatment with IL-1 receptor antagonist, anti-IL-1 receptor antibody, or anti-IL-18 antibody minimally reduced renal functional deterioration, inflammation, and apoptosis. CONCLUSIONS: These findings suggest that activated caspase-1 and its inflammatory products are involved in, but not crucial to, the induction of inflammation after renal ischemia-reperfusion. Hence, apart from caspase-1, other (combinations of) activated caspases are likely to be more prominently involved in renal reperfusion injury

Lethal hepatitis can be induced by an agonistic anti-Fas Ab in normal mice or by TNF in mice sensitized to d-(+)-galactosamine or actinomycin D. In all three models, we found that apoptosis of hepatocytes is an early and necessary step to cause lethality. In the three models, we observed activation of the major executioner caspases-3 and -7. Two acute-phase proteins, α1-acid glycoprotein and α1-antitrypsin, differentially prevent lethality: α1-acid glycoprotein protects in both TNF models and not in the anti-Fas model, while α1-antitrypsin confers protection in the TNF/d-(+)-galactosamine model only. The protection is inversely correlated with activation of caspase-3 and caspase-7. The data suggest that activation of caspase-3 and -7 is essential in the in vivo induction of apoptosis leading to lethal hepatitis and that acute phase proteins are powerful inhibitors of apoptosis and caspase activation. Furthermore, Bcl-2 transgenic mice, expressing Bcl-2 specifically in hepatocytes, are protected against a lethal challenge with anti-Fas or with TNF/d-(+)-galactosamine, but not against TNF/actinomycin D. The acute-phase proteins might constitute an inducible anti-apoptotic protective system, which in pathology or disturbed homeostasis prevents excessive apoptosis.

TNF-R55 is the main receptor mediating TNF-induced cytotoxicity. However, in some cells TNF-R75 also signals cell death. In PC60 cells, the presence of both receptor types is required to induce apoptosis following either specific TNF-R55 or TNF-R75 triggering, pointing to a mechanism of receptor cooperation. In this study, we extend previous observations and show that TNF-R55 and TNF-R75 cooperation in the case of apoptosis in PC60 cells is bidirectional. We also demonstrate ligand-independent TNF-R55-mediated cooperation in TNF-R75-induced granulocyte/macrophage-CSF secretion, but not vice versa. To determine which part of the intracellular TNF-R75 sequence was responsible for the observed receptor cooperation in apoptosis, we introduced different TNF-R75 mutant constructs in PC60 cells already expressing TNF-R55. Our data indicate that an intact TNF-R-associated factors 1 and 2 (TRAF1/TRAF2)-binding domain is required for receptor cooperation. These findings suggest a role for the TRAF complex in TNF-R cooperation in the induction of cell death in PC60 cells. Nevertheless, introduction of a dominant negative (DN) TRAF2 molecule was not able to affect receptor cooperation. Remarkably, TRAF2-DN overexpression, which was found to inhibit the TNF-dependent recruitment of endogenous wild-type TRAF2 to the TNF-R75 signaling complex, could neither block TNF-R55- or TNF-R75-induced NF-κB activation nor granulocyte/macrophage-CSF secretion. Possibly, additional factors different from TRAF2 are involved in TNF-mediated NF-κB activation.

TNF-induced apoptosis, e.g. in murine PC60 cells, requires the TNF receptor p55 (TNF-R55) and the TNF receptor p75 (TNF-R75); the latter even does not have to be triggered. The intracellular domain of TNF-R55 can be activated in the cytosol by linking it to the trimeric CAT protein; induction of this fusion protein leads to a full TNF response. A new MAP kinase, p38, has been shown to be also activated by TNF. This activation is essential for gene induction, but not for cytotoxicity in L929 cells. TNF treatment of L929 leads to reactive oxygen formation in the mitochondria, resulting in cell death by necrosis. TNF treatment of many other cell types results in apoptosis, and this process involves activation of one or more ICE homologs (IHO). In the mouse, seven cysteine proteases of the IHO family have been cloned and partially characterized. One or more of these IHOs is involved in cell killing by proteolysis of critical substrate(s). One substrate, which may be a key effector molecule in the apoptotic process, is PITSLRE kinase.