Hannes Hentze

Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity

Using a murine model, we studied the effect of agonistic anti‐CD95 antibodies (aCD95) on sinusoidal lining cells and a potential protection by caspase inhibition. C3H/HeN mice were intravenously administered aCD95 (10 µg/mouse) or unspecific IgG (control) in the presence or absence of the caspase inhibitor z‐VAD‐fmk. Analysis of hepatic microcirculation using intravital fluorescence microscopy revealed severe (P<0.01) sinusoidal perfusion failure and reduced (P<0.05) phagocytic activity of Kupffer cells (KC) within 2 h. Transmission electron micrographs demonstrated loss of integrity of sinusoidal endothelial cells as early as 1 h after aCD95 application, whereas histological manifestation of hepatocellular apoptosis and hemorrhagic necrosis was most pronounced at 6 h. Blocking of caspase activity attenuated (P<0.01) both hepatic microvascular perfusion failure and KC dysfunction. Accordingly, full protection of the liver from apoptotic damage and intact microarchitecture was observed in histological sections after z‐VAD‐fmk treatment. Mortality rate was 40% 6 h after aCD95 administration, whereas all animals survived in the z‐VAD‐fmk group (P<0.05). The activation of caspases through CD95 may primarily lead to damage of sinusoidal endothelial cells and hepatic micro‐vascular perfusion failure. Moreover, reduced phagocytic capacity of KC may contribute to accumulation of toxic metabolites released by dying cells at the local site of inflammation, further aggravating liver injury.—Wanner, G. A., Mica, L., Wanner‐Schmid, E., Kolb, S. A., Hentze, H., Trentz, O., Ertel, W. Inhibition of caspase activity prevents CD95‐mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity. FASEB J. 13, 1239–1248 (1999)

Caspase-3 activity is present in cerebrospinal fluid from patients with traumatic brain injury

Loss of neurons after traumatic brain injury (TBI) might involve dysregulated apoptosis. Activation of caspase-3 is one hallmark of apoptosis. Therefore, caspase-3 activity (cleavage of DEVD-afc) was measured in cerebrospinal fluid (CSF) samples (n=113) from 27 patients with TBI at day 1 to 14 after trauma. Caspase-3 activity was detected in 31 (27.4%) CSF samples with highest values (> 5.5 microM/min) seen at day 2-5 after trauma. No caspase-3 activity was found in serum from patients or CSF from controls. The presence of activated caspase-3 in CSF suggests ongoing apoptotic processes during traumatic brain injury.

Kupffer Cell-Expressed Membrane-Bound TNF Mediates Melphalan Hepatotoxicity via Activation of Both TNF Receptors

Isolated hepatic perfusion of nonresectable liver cancer using the combination of TNF and melphalan can be associated with a treatment-related hepatotoxicity. We investigated whether, apart from TNF, also melphalan is cytotoxic in primary murine liver cells in vitro and investigated mediators, mode of cell death, and cell types involved. Melphalan induced a caspase-dependent apoptosis in hepatocytes, which was not seen in liver cell preparations depleted of Kupffer cells. Neutralization of TNF prevented melphalan-induced apoptosis and liver cells derived from mice genetically deficient in either TNFR 1 or 2, but not from lpr mice lacking a functional CD95 receptor, were completely resistant. Cell-cell contact between hepatocytes and Kupffer cells was required for apoptosis to occur. Melphalan increased membrane-bound but not secreted TNF in Kupffer cells and inhibited recombinant TNF-α converting enzyme in vitro. Melphalan induced also severe hepatotoxicity in the isolated recirculating perfused mouse liver from wild-type mice but not from TNFR 1 or 2 knockout mice. In conclusion, this study shows that melphalan elicits membrane TNF on Kupffer cells due to inhibition of TNF processing and thereby initiates apoptosis of hepatocytes via obligatory activation of both TNFRs. The identification of this novel mechanism allows a causal understanding of melphalan-induced hepatotoxicity.

Redox control of hepatic cell death

In necrotic liver failure like upon acetaminophen overdose, loss of the major intracellular thiol antioxidant glutathione was shown to be causal for hepatic dysfunction. In sharp contrast, fulminant apoptotic liver destruction upon overstimulation of the death receptors TNFR1 and CD95 was not associated with reduced hepatic glutathione levels. In view of the importance of the role of reactive oxygen intermediates versus antioxidants for apoptosis, we investigated the effect of phorone-induced enzymatic GSH depletion on the sensitivity of the liver towards CD95- or TNFR1-mediated hepatotoxicity. Our findings demonstrate in vivo that receptor-mediated hepatic apoptosis is disabled when glutathione is depleted, i.e. that an intact glutathione status is a critical determinant for the execution of apoptosis. In vitro, we did mechanistic studies in lymphoid cell lines and found that pro-caspase-8 at the CD95 death receptor and the mitochondrial activation of pro-caspase-9 are the enzyme targets that require sufficient intracellular reduced glutathione for their activation.

Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF‐mediated apoptosis

Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, α‐amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase‐3‐like protease activity, fragmentation of nuclear DNA, and late cytolysis. Hepatocytes derived from TNF receptor‐1 knockout mice were resistant to CPT/TNF‐induced apoptosis. CPT treatment completely abrogated the TNF‐induced NF‐kappa B activation, and mRNA expression of the antiapoptotic factors TNF‐receptor associated factor 2, FLICE‐inhibitory protein, and X‐linked inhibitor of apoptosis protein was also inhibited by CPT. The caspase inhibitors benzyloxycarbonyl‐Val‐Ala‐Asp‐(OMe)‐fluoromethylketone (zVAD‐fmk) and benzyloxycarbonyl‐Asp(OMe)‐Glu(OMe)‐Val‐Asp(OMe)‐chloromethylketone (zDEVD‐fmk), as well as depletion of intracellular ATP by fructose prevented CPT/TNF‐induced apoptosis. In vivo, CPT treatment sensitized mice to TNF‐induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies. (HEPATOLOGY 2004;39:1311–1320.)

Spontaneous in Contrast to Cd95-induced Neutrophil Apoptosis Is Independent of Caspase Activity

BACKGROUNDnThe influence of caspase inhibitors on spontaneous and on CD95-triggered apoptosis was investigated in neutrophils from healthy volunteers and compared with neutrophils from patients with severe sepsis.nnnMETHODSnTo further elucidate the mechanisms of neutrophil apoptosis, isolated neutrophils from healthy volunteers (n = 9) were either stimulated with the agonistic anti-CD95 antibody (100 ng/mL) or left unstimulated in the presence or absence of the caspase inhibitors zIETD-fmk (10 micromol/L), zDEVD-fmk (10 micromol/L), or zVAD-fmk (20 micromol/L). Apoptosis was determined by measuring DNA fragmentation and Annexin-V binding in FACS, and caspase-3-like activity by DEVD-afc cleavage assay. Results were compared with those from patients with severe sepsis (n = 15).nnnRESULTSnReduced spontaneous neutrophil apoptosis in patients with sepsis (-48.7%) was completely restored by incubation with agonistic anti-CD95 antibody (p < 0.05). Inhibition of caspases did not influence spontaneous neutrophil apoptosis in both groups. However, zVAD-fmk reduced anti-CD95 antibody-induced apoptosis in neutrophils from controls by -22.6% (p < 0.05) and in patients with sepsis by -43.1% (p < 0.05).nnnCONCLUSIONnThese results indicate that spontaneous in contrast to CD95-induced neutrophil apoptosis is independent of caspase activity.

Sepsis after severe injury interrupts caspase-dependent processing of interleukin-18.

BACKGROUNDnCysteine proteases (caspases) participate in the activation process of interleukin-18 (IL-18), a key cytokine for septic organ failure. This study evaluates the influence of caspase blockade on secretion of IL-18 into whole blood in patients with multiple injuries and in patients with severe sepsis.nnnMETHODSnHeparinized blood was collected from patients with multiple injuries, from septic patients, as well as from healthy humans. Whole blood was stimulated for 24 hours with lipopolysaccharide (LPS) or Staphylococcus aureus in the presence or absence of the caspase inhibitors Z-VAD and Z-DEVD. IL-18 was measured by enzyme-linked immunosorbent assay.nnnRESULTSnS. aureus Cowan strain differentially increased the release of IL-18 in the three study populations, whereas LPS was ineffective. Z-VAD and to a lesser degree Z-DEVD decreased (p < 0.05) S. aureus Cowan strain I-induced secretion of IL-18 into whole blood from control subjects and trauma patients. Caspase inhibitors did not influence release of IL-18 into whole blood from septic patients.nnnCONCLUSIONnSecretion of IL-18 into whole blood from healthy humans and trauma patients can be effectively controlled through blockade of caspase activity. In contrast, during sepsis, alternative mechanisms may regulate secretion of IL-18.

Endotoxin and Interferon-γ hemmen die Kaspasenaktivität neutrophiler Granulayten über Tyrosinkinase-abhängige Mechanismen

Die veminderte Apoptose von neutrophilen Granulozyten (PMN) wahrend der Sepsis fuhrt zu deren Akkumulation mit einer erhohten Freisetzung von zytotoxischen Mediatoren [1]. Intrazellulare Zystein-Proteasen (Kaspasen), die uber die Signaltransduktion aktiviert werden, sind fur die Regulation der PMN-Apoptose von groser Bedeutung [2]. Es war Ziel dieser Studie, die Kaspasenaktivitat in PMN and deren Regulation zu untersuchen.

Hemmung der Kaspase-Aktivität verhindert das Fas-vermittelte Mikrozirkulationsversagen der Leber und die Reduktion der Clearance-Funktion von Kupfferzellen (KC)

Die Aktivierung von Fas durch agonistische anti-Fas-Antikorper fuhrt im murinen Modell durch die Apoptose von Hepatozyten innerhalb von Stunden zu einer fulminaten Hepatitis mit akutem Leberversagen [1, 2]. Die Fas-induzierte Apoptose wird dabei uber eine intrazellulare Kaskade von Aspartat-spezifischen Cysteinproteasen (Kaspasen) vermittelt, die uber die transmembranare „death domain“von Fas aktiviert wird [3,4]. Jungste Studien zeigen, das neben Hepatozyten auch die sinusoidalen Zellen (Kupfferzellen (KC) and Endothelzellen) Fas exprimieren [5]. Es war deshalb Ziel dieser Studie, die Bedeutung der Fas-induzierten Aktivierung von Kaspasen fur die Mikrozirkulation der Leber und die Clearancefunktion von KC zu untersuchen.