benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Sepsis

Caspase-9 is believed to play an essential role in sepsis-induced lymphocyte apoptosis. The aim of this study was therefore to evaluate its contribution within the caspase-dependent apoptosis pathway in a murine model of polymicrobial sepsis. Local injections of Z-LEHD-fmk, a specific caspase-9 inhibitor, into thymi of septic mice led to the complete inhibition of caspase-9, decreased apoptosis of resident tissue cells, and, in addition, reduced further downstream caspase-3 activity. In contrast to its systemic administration, only local injections improved the overall survival of septic mice. However, local injections of a pancaspase inhibitor (Z-VAD-fmk) did not improve survival, although caspase-3 activity was reduced to a similar degree as by the administration of Z-LEHD-fmk. These results indicate that local apoptosis of lymphatic tissue in polymicrobial sepsis is processed dependent of caspase-9 and suggests alternative caspase-dependent beneficial effects, which may determine a positive outcome.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 9; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytokines; Dose-Response Relationship, Drug; Female; Lymphocytes; Mice; Mice, Inbred C57BL; Oligopeptides; Sepsis; Survival Rate; Thymus Gland

Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK-treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Apoptosis; Caspase Inhibitors; Female; HMGB1 Protein; Male; Mice; Mice, Inbred BALB C; Sepsis

Inflammatory mediators delay neutrophil apoptosis, which contributes to the persistence of inflammation. The mechanisms responsible for this delay and resistance to Fas antibody-induced apoptosis are unknown but are dependent on protein synthesis. These proteins have been shown to inhibit caspase activity central to the induction of apoptosis. The inhibitors of apoptosis proteins have been shown to inhibit caspase activity and prevent apoptosis in a number of cellular systems. We hypothesize that the regulation of neutrophil apoptosis is dependent on the expression of the IAPs. c-IAP-1, c-IAP-2, and XIAP are expressed in the neutrophil at both the mRNA and protein level, but their relative protein expression is low compared with other cell types. The in vitro aging of human neutrophils results in their induction of apoptosis, which is associated with the loss of c-IAP-1 expression. The pancaspase inhibitor (zVAD-FMK) and LPS, which delay spontaneous apoptosis, also prevented this loss of c-IAP-1. Gene chip microarrays have shown that LPS increases c-IAP-1 and c-IAP-2 mRNA expression in neutrophils. However, this does not correspond to an increase in protein. Neutrophils from septic patients with delayed apoptosis show an increase in XIAP, with no change in cIAP-1 or cIAP-2 mRNA, demonstrating that different mechanisms contribute to the delay in neutrophil apoptosis. This study demonstrates that the loss of IAP expression may facilitate the induction of neutrophil apoptosis, and preventing this loss of IAP expression may represent a more significant contribution to delayed apoptosis rather than an increase in their expression.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Cells, Cultured; Cysteine Proteinase Inhibitors; Gene Expression Profiling; Humans; Inflammation; Inhibitor of Apoptosis Proteins; Lipopolysaccharides; Neutrophils; Polymerase Chain Reaction; Proteins; Reference Values; Sepsis; X-Linked Inhibitor of Apoptosis Protein

Activation-induced cell death in macrophages has been observed, but the mechanism remains largely unknown. Activation-induced cell death in macrophages can be independent from caspases, and the death of activated macrophages can even be triggered by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD). Here, we show that this type of macrophage death can occur in the septic mouse model and that toll-like receptor (TLR)-2 or TLR4 signaling is required in this process. We conclude that Nur77 is involved in the macrophage death because Nur77 expression correlates with cell death, and cell death is reduced significantly in Nur77-deficient macrophages. The extracellular signal-regulated kinase pathway, which is downstream of TLR2 or TLR4, and myocyte-specific enhancer binding factor 2 (MEF2) transcription factor activity, which is up-regulated by zVAD, are required for Nur77 induction and macrophage death. Reporter gene analysis suggests that Nap, Ets, Rce, and Sp1 sites in the Nur77 promoter are regulated by TLR4 signaling and that MEF2 sites in the Nur77 promoter are regulated by zVAD treatment. MEF2 transcription factors are constitutively expressed and degraded in macrophages, and zVAD increases MEF2 transcription factor activity by preventing the proteolytic cleavage and degradation of MEF2 proteins. This paper delineates the dual signaling pathways that are required for Nur77 induction in macrophages and demonstrates a role of Nur77 in caspase-independent cell death.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Caspases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Female; Lipopolysaccharides; Macrophage Activation; Macrophages; MAP Kinase Signaling System; MEF2 Transcription Factors; Membrane Glycoproteins; Mice; Mice, Knockout; Molecular Sequence Data; Myogenic Regulatory Factors; Nuclear Receptor Subfamily 4, Group A, Member 1; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Sepsis; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Transcription Factors

Despite intensive therapy, severe septic shock is commonly associated with myocardial dysfunction and death in humans. No new therapies have proven efficiency against cardiovascular alterations in sepsis. Here, we addressed the question of a beneficial effect of pharmacological inhibition of caspases on myocardial dysfunction following endotoxin treatment. Hearts from rats treated with endotoxin (10 mg/kg, intravenously) were isolated 4 h posttreatment for analysis. Assessment of myocardial contractility ex vivo and detection of apoptosis were performed. Hearts from endotoxin-treated rats displayed multiple caspase activities and also typical apoptosis pattern as detected by TUNEL, DNA fragmentation assays, and cytochrome c release as compared with control rats. z-VAD.fmk (3 mg/kg, intravenously), a broad spectrum caspase inhibitor (but not the irrelevant peptide z-FA.fmk), in coinjection with endotoxin, not only reduced caspase activities and nuclear apoptosis but also completely prevented endotoxin-induced myocardial dysfunction evaluated 4 h and even 14 h after endotoxin challenge. These data indicate that caspase activation plays an important role in myocardial cell dysfunction. Moreover, these results suggest that inhibitors of caspases may have important therapeutic applications in sepsis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Endotoxins; Heart; Male; Myocardium; Rats; Rats, Sprague-Dawley; Sepsis

The 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.. To 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).. Reduced 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).. These results indicate that spontaneous in contrast to CD95-induced neutrophil apoptosis is independent of caspase activity.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Case-Control Studies; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; fas Receptor; Humans; Neutrophils; Sepsis

The interleukins (IL)-1beta and IL-18 represent potent players in the proinflammatory cytokine cascade. Their activation is regulated predominantly through the IL-1-converting enzyme (ICE)/caspase-1. The role of caspases in the secretion of IL-1beta and IL-18, as well as in the release of the secondary-induced cytokines IL-12 and interferon (IFN)-gamma in whole blood from septic patients compared to healthy controls, was studied. Inhibition of caspase activity by Z-VAD significantly reduced lipopolysaccharide (LPS) and Staphylococcus aureus (SAC) induced release of mature IL-1beta in septic patients and controls. In contrast, in whole blood from septic patients significantly elevated basal level of IL-18 were found, which could neither be further increased by LPS or SAC, nor be inhibited by Z-VAD. Release of IL-12 p40 was significantly lower in septic patients compared to controls and was not affected by Z-VAD. Despite high levels of IL-18, IFN-gamma was not detected in whole blood from septic patients even after stimulation with SAC or LPS. Thus, during sepsis, caspases participate in the processing of IL-1beta, whereas maturation of IL-18 during sepsis appears to be independent of caspases. The lack of IFN-gamma release seen in septic patients could be attributed to low IL-12 release rather than to diminished IL-18 release.

Topics: Adult; Amino Acid Chloromethyl Ketones; Case-Control Studies; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytokines; Female; Humans; Interferon-gamma; Interleukin-1; Interleukin-12; Interleukin-18; Lipopolysaccharides; Male; Middle Aged; Sepsis; Staphylococcus aureus