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The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the 'zipper'-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3'-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages.

Due to loss of cell membrane integrity, necrotic cells passively release several cytosolic factors that can activate antigen presenting cells and other immune cells. In contrast, cells dying by apoptosis do not induce an inflammatory response. Here we show that necrotic cell death induced by several stimuli, such as TNF, anti-Fas or dsRNA, coincides with NF-kappaB-and p38MAPK-mediated upregulation and secretion of the pro-inflammatory cytokine IL-6. This event is greatly reduced or absent in conditions of apoptotic cell death induced by the same stimuli. This demonstrates that besides the capacity of necrotic cells to induce an inflammatory response due to leakage of cellular contents, necrotic dying cells themselves are involved in the expression and secretion of inflammatory cytokines. Moreover, inhibition of NF-kappaB and p38MAPK activation does not affect necrotic cell death in all conditions tested. This suggests that the activation of inflammatory pathways is distinct from the activation of necrotic cell death sensu strictu.

Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

In most cellular systems tumor necrosis factor (TNF) induces apoptotic cell death. However, in some particular cell lines, such as the L929sA fibrosarcoma, TNF induces necrotic cell death. This effect is not the result of an inability to die apoptotically, because triggering of Fas in L929sAhFas cells leads to apoptosis. Moreover, TNFR-1-induced necrosis can be reverted to apoptosis when cells are pretreated with geldanamycin, an Hsp90 inhibitor. In contrast, addition of caspase-inhibitors (zVAD-fmk) prevents Fas-induced apoptosis and switches it to necrosis. These results demonstrate that depending on the cellular context, the same stimulus can induce either apoptosis or necrosis. Apoptosis and necrosis are clearly distinguished by their morphology, although in the absence of phagocytosis, the late stage of apoptosis is associated with secondary necrotic cell death, which is hard to distinguish from necrotic cell death. Necrosis is described mostly in negative terms as cell death that is characterized by the absence of apoptotic parameters, such as caspase activation, cytochrome c release, and DNA fragmentation. Here we describe a selection of techniques used to distinguish both modes of TNFR-1-induced cell death, namely apoptotic or necrotic cell death.

Phylogenetic analysis clusters caspase-12 with the inflammatory caspases 1 and 11. We analyzed the expression of caspase-12 in mouse embryos, adult organs, and different cell types and tested the effect of interferons (IFNs) and other proinflammatory stimuli. Constitutive expression of the caspase-12 protein was restricted to certain cell types, such as epithelial cells, primary fibroblasts, and L929 fibrosarcoma cells. In fibroblasts and B16/B16 melanoma cells, caspase-12 expression is stimulated by IFN-gamma but not by IFN-alpha or -beta. The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA. These stimuli also induce caspase-1 and -11 but inhibit the expression of caspase-3 and -9. In contrast to caspase-1 and -11, no caspase-12 protein was detected in macrophages in any of these treatments. Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis. Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis. However, B16/B16 melanoma cells die when treated with the ER stress-inducing agent Tg whether they express caspase-12 or not.

In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.

Yersinia enterocolitica induces apoptosis in macrophages by injecting the plasmid-encoded YopP (YopJ in other Yersinia species). Recently it was reported that YopP/J is a member of an ubiquitin-like protein cysteine protease family and that the catalytic core of YopP/J is required for its inhibition of the MAPK and NF-kappaB pathways. Here we analyzed the YopP/J-induced apoptotic signaling pathway. YopP-mediated cell death could be inhibited by addition of the zVAD caspase inhibitor, but not by DEVD or YVAD. Generation of truncated Bid (tBid) was the first apoptosis-related event that we observed. The subsequent translocation of tBid to the mitochondria induced the release of cytochrome c, leading to the activation of procaspase-9 and the executioner procaspases-3 and -7. Inhibition of the postmitochondrial executioner caspases-3 and -7 did not affect Bid cleavage. Bid cleavage could not be observed in a yopP-deficient Y. enterocolitica strain, showing that this event requires YopP. Disruption of the catalytic core of YopP abolished the rapid generation of tBid, thereby hampering induction of apoptosis by Y. enterocolitica. This finding supports the idea that YopP/J induces apoptosis by directly acting on cell death pathways, rather than being the mere consequence of gene induction inhibition in combination with microbial stimulation of the macrophage.

Apoptosis and necrosis are two distinct forms of cell death. Caspases are indispensable as initiators and effectors of apoptotic cell death and are involved in many of the morphological and biochemical features of apoptosis. Major changes in mitochondrial membrane integrity and release of proapoptotic factors, such as cytochrome c from the mitochondrial intermembrane space, play an important sensor and amplifying role during apoptotic cell death. In vitro studies of cell death in cell lines have revealed that inhibition of the classical caspase-dependent apoptotic pathway leads in several cases to necrotic cell death. Thus, the same cell death stimulus can result either in apoptotic or necrotic cell death, depending on the availability of activated caspase. Therefore, death domain receptors may initiate an active caspase-independent necrotic signaling pathway. In this review, we describe what is known about the apoptotic and necrotic cell death pathways. Principal elements of necrosis include mitochondrial oxidative phosphorylation, reactive oxygen production, and non-caspase proteolytic cascades depending on serine proteases, calpains, or cathepsins.

Background: Ischemia followed by reperfusion (I/R) causes apoptosis, inflammation, and tissue damage leading to organ malfunction. Ischemic preconditioning can protect against such injury. This study investigates the contribution of the acute phase proteins alpha(1)-acid glycoprotein (AGP) and alpha(1)-antitrypsin (AAT) to the protective effect of ischemic preconditioning in the kidney., Methods and Results: Exogenous AGP and AAT inhibited apoptosis and inflammation after 45 minutes of renal I/R in a murine model. AGP and AAT administered at reperfusion prevented apoptosis at 2 hours and 24 hours, as evaluated by the presence of internucleosomal DNA cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and the determination of renal caspase-1- and caspase-3-like activity. AGP and AAT exerted anti-inflammatory effects, as reflected by reduced renal tumor necrosis factor-alpha expression and neutrophil influx after 24 hours. In general, these agents improved renal function. Similar effects were observed when AGP and AAT were administered 2 hours after reperfusion but to a lesser extent and without functional improvement. Moreover, I/R elicited an acute phase response, as reflected by elevated serum AGP and serum amyloid P (SAP) levels after 24 hours, and increased hepatic acute phase protein mRNA levels after 18 hours of renal reperfusion., Conclusions: We propose that the antiapoptotic and anti-inflammatory effects of AGP and AAT contribute to the delayed type of protection associated with ischemic preconditioning and other insults. This mechanism is potentially involved in the course of many clinical conditions associated with I/R injury. Moreover, exogenous administration of these proteins may provide new therapeutic means of treatment.

Rat/mouse T cell hybridoma-derived PC60 R55/R75 cells were used as a model to study the role of intracellular potassium in TNF-induced apoptosis and gene induction. A reduction of intracellular potassium with nigericin or valinomycin (ionophores), or ouabain (Na(+)/K(+)-ATPase inhibitor) sensitized PC60 R55/R75 cells to TNF-induced apoptosis. TNF-induced GM-CSF release in PC60 R55/R75 cells was enhanced by nigericin or ouabain. Similar results were obtained with human cervix carcinoma cells HeLaH21 exposed to TNF. These results suggest a role for intracellular potassium in TNF-induced apoptosis and gene induction., (Copyright 2000 Academic Press.)

Time kinetics of phosphatidyl serine (PS) exposure were compared to other apoptotic parameters following different apoptotic stimuli. Our data indicate that anti-Fas treatment of L929sAhFas cells results in rapid exposure of PS, which precedes decrease in mitochondrial transmembrane potential (DeltaPsi(m)) and release of cytochrome c, indicating that PS exposure occurs independently of these mitochondrial events. Also during TNF-, etoposide- or staurosporine-mediated apoptosis in PC60 RI/RII cells, PS-positive cells were observed before they had a decreased DeltaPsi(m). However, during growth factor depletion-induced death of 32D cells, both phenomena seemed to occur at the same time.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone++ +, tetrapeptide inhibitors of caspase-1- and caspase-3-like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas-induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor kappaB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

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-kappaB activation nor granulocyte/macrophage-CSF secretion. Possibly, additional factors different from TRAF2 are involved in TNF-mediated NF-kappaB activation.

The authors investigated the dependence on extracellular and intracellular free Ca2+ in the induction of apoptosis and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) by tumour necrosis factor (TNF) in a rat/mouse T cell hybridoma PC60 R55/R75, using the Ca2+ chelators EGTA and BAPTA/AM, respectively. TNF-induced apoptosis still occurred in the absence of free Ca2+, while GM-CSF production required the continuous presence of Ca2+. The latter was also true for GM-CSF production driven by interleukin 1 (IL-1). The dependence on Ca2+ in the induction of GM-CSF, but not of apoptosis, was further confirmed by the inhibition of TNF- or IL-1-induced cytokine production by cyclosporin A or FK506, drugs that block the Ca2+/calmodulin-dependent protein Ser/Thr phosphatase calcineurin. This differential requirement for Ca2+ illustrates the partial functional redundancy between TNF and IL-1, showing the activation of cytokine gene expression through a Ca(2+)-dependent activation of calcineurin, and a Ca(2+)-independent activation of apoptosis, exerted solely by TNF.

Emerging evidence suggests that multiple aspartate-specific cysteine proteases (caspases (CASPs)) play a crucial role in programmed cell death. Many cellular proteins have been identified as their substrates and serve as markers to assay the activation of CASPs during the death process. However, no substrate has yet been unambiguously identified as an effector molecule in apoptosis. PITSLRE kinases are a superfamily of Cdc2-like kinases that have been implicated in apoptotic signaling and tumorigenesis. In this paper we report that tumor necrosis factor (TNF)-mediated apoptosis is associated with a CrmA- and Bcl-2-inhibitable cleavage of PITSLRE kinases, indicating a role for CASPs. Testing of seven murine CASPs for their ability to cleave p110 PITSLRE kinase alpha2-1 in vitro revealed that only CASP-1 (ICE (interleukin-1beta-converting enzyme)) and CASP-3 (CPP32) were able to produce the same 43-kDa cleavage product as observed in cells undergoing TNF-induced apoptosis. Mutational analysis revealed that cleavage of p110 PITSLRE kinase alpha2-1 occurred at Asp393 within the sequence YVPDS, which is similar to that involved in the CASP-1-mediated cleavage of prointerleukin-1beta. TNF-induced proteolysis of PITSLRE kinases was still observed in fibroblasts from CASP-1(0/0) mice. These data implicate CASP-3 as a potentially important CASP family protease responsible for the cleavage of PITSLRE kinases during TNF-induced apoptosis.

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.

The rat/mouse T-cell hybridoma PC60 was transfected either with hTNF-R55 cDNA, hTNF-R75 cDNA, or both. Receptor-specific stimulation was achieved using agonistic monoclonal antibodies or receptor-specific muteins of hTNF. Either hTNF-R55 or hTNF-R75 could mediate the activation of NF-kappa B and the induction of GM-CSF, IL-6, and IFN-gamma. But only in cells carrying both hTNF-R55 and hTNF-R75, was TNF able to induce apoptosis. This apoptosis could be inhibited almost completely by cotransfection with human bcl-2 cDNA. Functional cooperation was observed between liganded and unliganded receptors for the induction of apoptosis. In vitro protein kinase activity was detected only in TNF-R75 immunoprecipitates from cells in which the receptor was signaling. Direct evidence was obtained for reactive oxygen intermediates of mitochondrial origin responsible for TNF-induced cytotoxicity in L929 cells.